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Structure-based Drug Design

by Veerapandian, Pandi.
New York Marcel Dekker, Inc., 1997.
ISBN: 0824798694
eBook ISBN: 0585157448
Subject: Drugs--Design. Drugs--Structure-activity
relationships. Drugs--Conformation. Drug Design.
Structure-Activity Relationship.
Language: English
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Table of Contents
Structure-Based Drug Design
Preface
Contents
Contributors
1 Inhibitors of HIV-1 Protease
2 Structural Studies of HIV-1 Reverse
Transcriptase and Implications for
Drug Design
3 Retroviral Integrase: Structure as a
Foundation for Drug Design
4 Bradykinin Receptor Antagonists
5 Design of Purine Nucleoside
Phosphorylase Inhibitors
6 Structural Implications in the Design
of Matrix-Metalloproteinase Inhibitors
7 StructureFunction Relationships in
Hydroxysteroid Dehydrogenases
8 Design of ATP Competitive Specific
Inhibitors of Protein Kinases Using
Template Modeling
9 Structural Studies of Aldose
Reductase Inhibition
10 Structure-Based Design of
Thrombin Inhibitors
11 Design of Antithrombotic Agents
Directed at Factor Xa
12 Polypeptide Modulators of Sodium
Channel Function as a Basis for the
Development of Novel Cardiac...
13 Rational Design of Renin Inhibitors
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14 Structural Aspects in the Inhibitor

Design of Catechol OMethyltransferase
15 Antitrypanosomiasis Drug
Development Based on Structures of
Glycolytic Enzymes
16 Progress in the Design of
Immunomodulators Based on the
Structure of Interleukin-1
17 Structure and Functional Studies of
Interferon: A Solid Foundation for
Rational Drug Design
18 The Design of Anti-Influenza Virus
Drugs from the X-ray Molecular
Structure of Influenza Virus Ne...
19 Rhinoviral Capsid-Binding
Inhibitors: Structural Basis for
Understanding Rhinoviral Biology and
f...
20 The Integration of Structure-Based
Design and Directed Combinatorial
Chemistry for New Pharmaceut...
21 Structure-Based Combinatorial
Ligand Design
22 Peptidomimetic and Nonpeptide
Drug Discovery: Impact of StructureBased Drug Design
Index
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1
Inhibitors of HIV-1 Protease
Krzysztof Appelt
Agouron Pharmaceuticals, Inc., San Diego, California
I. Introduction
Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired
immunodeficiency syndrome (AIDS), perhaps the largest and most powerful consortium of scientists
ever assembled to tackle a single disease has been brought to bear on the problem of AIDS and its
treatment. From an unprecedented wealth of information regarding the molecular biology and virology
of HIV collected in recent years, it became possible to identify numerous intervention points in the viral
life cycle that could be exploited in the development of drugs for AIDS therapy (for reviews see
Reference 1, 2, and 3). Among these, the virally-encoded enzymes, in particular reverse transcriptase
and protease, have emerged as the most popular targets. A separate chapter of this book is dedicated to
the description of reverse transcriptase and its inhibitors [4]. For the purpose of introduction only, it
should be noted that nucleoside inhibitors of reverse transcriptase (AZT, ddI, ddC, d4T, and 3TC) have
been widely used in clinical practice since 1987. Since then it has become apparent that this class of
agents, while slowing progression of disease in HIV-infected patients, is limited in both activity and the
duration of the clinical responses produced. Therefore in the search for better anti-HIV agents, the focus
of effort was expanded to include the search for clinically useful inhibitors of a second viral enzyme,
namely the protease. In contrast to reverse transcriptase, for which activity is required prior to the
integration of viral genetic information into the host cell chromosomes, the viral protease plays a key
role late in the virus life cycle and inhibitors of this enzyme display equal anti-viral activity in chronic
and acute infection models in vitro [5].
The HIV protease (HIV PR) is encoded by the 5' portion of the retroviral pol gene, which encodes all
replicative enzymes. Viral structural proteins (p24,
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p17, p9, and p7) and replicative enzymes (protease, reverse transcriptase/ RnaseH, and integrase) are
translated as either polyprotein P55-GAG, or a larger frameshift product P160-GAG-POL. In the
process of virus assembly these polyproteins are proteolytically cleaved by the protease and this
processing step, both in its timing and accuracy, is essential for the formation of infectious particles of
HIV [6]. It was also shown early on that the inactivation of HIV PR, either by chemical inhibition or
certain mutations, leads to the production of immature, noninfectious viral particles [7,8].
Structurally HIV PR is a 99-amino-acid protein translated initially as a central part of the P160-GAGPOL polyprotein precursor. The autocatalytic processing from the 160 kDa precursor is poorly
understood, but most likely occurs during the process of budding of pre-formed viral particles from the
host cell [9]. After release from the precursor polyprotein, HIV PR forms a homodimer and acts in trans
to correctly process GAG and GAG-POL polyproteinsa process required for formation of the viral
capsid and nucleoprotein core.
Retroviral proteases such as HIV PR are the latest additions to the wellstudied family of aspartic
proteases. This family of enzymes, which includes, among others, proteases such as pepsin, renin, and
cathepsins D and E, has been intensely studied in the past, and the knowledge gained from studies of
these enzymes allowed early inferences as to the structure and function of the dimeric HIV PR.
Moreover, the intensive effort over the past two decades to make inhibitors of human renin provided
impetus for the early design of inhibitors of HIV PR. In fact, some of the renin inhibitors have turned
out to be effective inhibitors of retroviral aspartic proteases as well and have served as the starting point
for drug design. As a result of this many early inhibitors of HIV PR were peptidyl in nature and the best
known example of such compounds is Ro31-8959, better known as saquinavir, a hydroxyethylaminecontaining mimetic of a hexapeptide substrate [10]. This potent inhibitor of HIV PR was discovered
using a substrate-based rational approach to drug design and displays extremely high in vitro activity
against clinical isolates and laboratory strains of HIV. Saquinavir has been recently approved by the
FDA for the treatment of AIDS in combination with nucleoside inhibitors of reverse transcriptase, and
the discovery of this compound was the first breakthrough and the starting point for many other
innovative designs.
Determination of the crystal structures of HIV PR gave new impetus to the design of novel inhibitors.
One measure of the intensity with which new inhibitors were designed or discovered is the total number
of crystal structures of inhibitory complexes, currently exceeding 250, that have been determined over
the past 5 years. Very detailed crystallographic analysis combined with extensive biochemical
characterization and site-specific mutagenesis studies made HIV PR perhaps the best characterized
enzyme to date.
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Based on the avalanche of papers describing the structure-based design of various HIV PR inhibitors, it
would be reasonable to assume that, with the exception of saquinavir, all other HIV PR inhibitors that
entered the stage of preclinical or clinical development were discovered using the elements of a structurebased approach. From the long list of more than 30 inhibitors considered as clinical candidates [11],
currently there are three compounds (saquinavir, ritonavir, and indinavir) already approved by the FDA
as anti-HIV drugs. Many factors that are requisite for in vivo activity in AIDS patients can only be
predicted a priori in a very general sense. For instance, erratic oral bioavailability in humans, first-pass
metabolism, binding to plasma proteins or tissue distribution may disqualify a perfect in vitro inhibitor
of HIV replication and such properties can be very poorly predicted by any process of drug design. A
potential answer to these problems is the parallel design of several chemically distinct compounds that
may have similar in vitro activity but significantly different in vivo properties. The application of protein
structure-based design offers such possibilities and in this text the discovery and optimization of
different series of potent inhibitors of HIV PR will be discussed. In order to familiarize the reader with
the architecture of HIV PR and the properties of its active site, the first paragraphs are devoted to the
detailed description of the x-ray structures of the enzyme followed by several examples of inhibitors in a
bound conformation.
A. Three-Dimensional Structure of HIV PR
Retroviral proteases such as HIV PR were tentatively assigned to the aspartic protease family on the
basis of putative active-site sequence homology [12]. Mammalian aspartic proteases are bilobal, singlechain enzymes in which each lobe (or domain) contributes an aspartic acid residue to the active site [13].
The active site itself is formed at the interface on the N- and C-terminal domains and exhibits
approximate two-fold symmetry. Since the retroviral proteases are only about one-third the size of the
two-domain eukaryotic enzymes, they were hypothesized to function as dimers in which each monomer
contributes a single aspartic acid to the active site [14]. Obligate homodimeric proteases, in addition to
providing a regulatory mechanism to control activation of the enzyme, represent the most efficient use
of genetic information which, in retroviruses, is naturally parsimonious.
The crystal structures of HIV PR confirmed the predicted dimeric character of the enzyme [15,16]
(Figure 1). In all published crystallographic investigations of the unliganded form of the enzyme, the
monomers are related to each other by crystallographic two-fold symmetry and are necessarily identical.
The general topology of the HIV PR monomer is similar to that of a single-domain
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Figure 1
Stereo view of the -carbon backbone of HIV PR dimer. (a) The apoenzyme
with flaps in the open conformation. (b) Inhibited form of HIV PR with flaps in a
closed conformation. For clarity, the inhibitor is removed from the active site.
pepsin-like aspartic protease and consists of antiparallel -strands and a short, two-turn -helix
connected by loops of varying length. The dimer interface is formed by an antiparallel -sheet
comprising two strands from each monomer. The hydrophobic residues from those -strands and two
symmetry-related -helices form the core of the dimer. The dimer is further stabilized by a net of
hydrogen bonds involving the residues around the catalytic aspartic acids. The active site is formed by
the dimer interface and is composed of equivalent contributions of residues from each monomer. The
substrate-binding cleft is bound on one side by the active site aspartic acid (Asp25/25') and on the other
side by a pair of two-fold related, antiparallel -hairpin structures, commonly referred to as flaps.
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The conserved active-site residues (Asp25, Thr26, and Gly27 from both monomers) form a symmetrical
and highly hydrogen-bonded arrangement virtually identical to that described for pepsin [17]. The two
aspartates are nearly coplanar with the inner carboxylate oxygens hydrogen bonded to the amide
hydrogens of Gly27/27'. This designation (e.g. Gly 27/27') will be used throughout this text to indicate
equivalent residues of the dimer. The two threonines are inaccessible to solvent and are hydrogenbonded to the main-chain amide groups of the other monomer, forming a rigid network called a
fireman's grip [17]. As in the case of the structures of eukaryotic pepsins, there is electron density for
a water molecule bound between the two carboxylates of the active-site aspartates.
In the structure of the apo-form of HIV PR, the flaps from both monomers are related by
crystallographic two-fold symmetry and can be considered as being in an open conformation. In the
structures of related proteases from Rous Sarcoma Virus and HIV-2, the flaps are either
crystallographically disordered or in a partly closed conformation [18]. This suggests that, in solution, in
the absence of ligands, the flaps are rather flexible and that the stable conformation of the flaps observed
in the crystal structure of the apo-enzyme of HIV PR could be considered to result from kinetic trapping
during the crystallization process.
In the apo-form of HIV PR, the active site residues are located at the bottom of a rather shallow groove.
Upon binding an inhibitor, the protease undergoes significant structural changes, particularly apparent in
the flap region. As a result, a tunnel-like site is formed, which runs diagonally across the dimer
interface. The tunnel has a volume of approximately 1140 3 and is 23 long. Because of the dimeric
nature of HIV PR, the active site has approximate two-fold symmetry with the dyad axis intersecting the
plane of the catalytic aspartates. Along the active site tunnel, starting from the central aspartates, there
are distinct subsites S1, S2, S3, and S4, and corresponding symmetry related subsites S1', S2' S3', and
S4' (Figure 2). It should be noted that in this chapter, the convention of Schechter and Burger [19] will
be used to describe enzyme specificity subsites (S1, S1', etc.) and the corresponding side chains of
inhibitors (P1, P1', etc.). The boundaries of the subsites are formed by residues from both monomers of
HIV PR. All subsites, with the exception of S4/S4', which are exposed to solvent, are bounded by mostly
aliphatic side chains and have hydrophobic character. The borders of the S1/S1' subsites are formed by
the side chains of Ile23/23', Ile50/50', Ile84/84', Pro81/81', the carbon of Thr80/80', carboxylates of the
active site Asp25/25', and the carbonyl oxygens of Gly27/27'. The S2/S2' subsites are bounded by
Val32/32', Ile50/50', Ile47/47', Leu76/76', Ala28/28', and the carboxylates of Asp30/30'. The S3/S3',
subsites are partly exposed to solvent and are bordered by the side chains of Leu23/23', Val82/82',
Pro81/81', and the guanidinium groups of Arg8/8', which form a salt bridge with the carboxylates of
Asp29/29'. Most of
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Figure 2
Schematic representation of the specificity subsites of the HIV PR active site with bound
peptidic inhibitor JG-365. Amino acids forming the boundaries of the particular
subsites are shown.
the hydrogen bond donor and acceptor functional groups of the active site are located in an approximate
plane that lies along the long axis of the tunnel and is somewhat perpendicular to the plane of the
subsites. The hydrogen-bonding functionalities include the carboxylates of the catalytic aspartates, the
carbonyl oxygens of Gly27 and Gly48, the amide nitrogens of Asp29' and Gly48, the carboxylate of
Asp29', and the dimer symmetry-related groups on the other side of the active site. Additional groups
capable of forming hydrogen bonds with ligands are located in the outer part of the S2/S2' subsite and
include the amide nitrogens and the carboxylates of Asp30/30'. There are five conserved water
molecules in the active site of HIV PR. Four of the waters are symmetrically distributed in the S3/S3'
subsites and one, hereafter called Wat301, is located near the two-fold axis of the dimer and, in the
presence of most inhibitors, is approximately tetrahedrally coordinated by the hydrogen bonds formed
between carbonyl oxygens of the ligand(s) and the amide nitrogens of Ile50/50' of the flaps. In the
ligand-bound form of HIV PR, Wat301 is completely inaccessible to solvent, and it has been speculated
that its functional substitution could be energetically favorable [18] or at least may lead to discovery of
novel nonpeptidic inhibitors [20]. Thus, there are 18 hydrogen bond donors or acceptors in
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the active site of HIV PR-16 that could form hydrogen bonds directly and two in which the interaction is
mediated by the conserved Wat301. The total solvent accessible surface area of the eight subsites of the
HIV PR active site is approximately 1150 2. Because of the large number of groups with hydrogen-bondforming potential, 450 2 of the surface has a polar character, and the nonpolar area of the subsites is
slightly larger, approximately 700 2.
B. Structural Flexibility of HIV PR
In the process of viral assembly, HIV PR specifically cleaves nine cleavage sites on GAG and GAG-POL
polypeptides [21]. Examination of the amino acid composition of the recognized substrate sites (Table 1)
indicates their hydrophobic character and significant sequence variability. The loose specificity of HIV PR
most likely reflects its functions in a world of reduced complexity within the confines of the budding
virion. The length of the viral protein precursors (approximately 1500 amino acids) reduces the number of
potential sequences the protease must discriminate from in selecting its nine cleavage sites. Therefore,
HIV PR and other retroviral proteases are not enzymes that have evolved to carry out a single reaction at a
rapid rate, but rather enzymes with minimum specificity required to cleave the viral precursors in a
specific and orderly manner.
The loose specificity requirements demonstrated by effective binding and catalytic processing of all nine
sequences, albeit at different rates [22], was the
Table 1 The Sequences of the Proteolytic Processing Sites of HIV-1
HIV-1 PR
Cleavage sites
Scissile bond
P17/P24
P24/P2
P2/P7
P7/P1
P1/P6
TF/PR
PR/RT
RT/RN
RN/IN
P5
P4
P3
P2
P1
P1'
P2'
P3'
P4'
P5'
Schechter-Berger
notation
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TFtransframe, PRprotease, RTreverse transcriptase, RHRNAse H, INintegrase. The location of the

processing sites in HIV-1 were determined by protein sequencing of HIV-1 virion proteins.
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first indication that the recognition subsites of the HIV PR can display flexibility upon binding of
substrates or inhibitors. Early crystal structures of the HIV PR apo-enzyme and complexes with peptidic
inhibitors showed several conformations of the active site forming flaps, which include the residues
Met46/46' to Ile54/54' [15,16]. Increased availability of coordinates of HIV PR complexed with various
inhibitors and crystallized in different crystallographic space groups allowed for more rigorous
examination of domain movements and structural changes in the active site.
The alignment of several crystal structures of HIV PR in a common frame of reference, which most
commonly includes the region around the symmetryrelated active site triad Asp25/25'-Thr26/26'Gly27/27', will highlight those regions of the backbone where significant displacement occurs upon
accommodating the individual inhibitors. Examination of the aligned structures, which included
examples of all classes of inhibitors, indicated only small variation of the backbone and limited
movements in the two binding loops, comprising residues Leu76-Ile84 from both monomers. The loops
form the outer walls of subsites S1/S1' and S3/S3' with inward-facing hydrophobic side chains of
isoleucines and valines. The flexibility of these loops, which in some cases can move outward by as
much as 2.5 , has a significant impact on the volume of the specificity subsites, which in turn can
accommodate corresponding P1/P1' and P3/P3' moieties of various sizes. Interestingly, the predominant
resistance-causing mutations are located on the same loops and involve changes in residues Val82/82'
and Ile84/84' (see below). It should be noted, that while the alignment of several crystal structures
provides information about the flexible regions, the extent of flexibility of the residues around the HIV
PR active site can be limited by crystal packing forces and may represent a crystallographic artifact. In
all characterized crystal forms of HIV PR [23] the loops 7684 and 4656 participate in crystal lattice
formation and the particular conformation of these loops can be driven by crystallization conditions or
interactions with other molecules related by the crystallographic symmetry.
C. Inhibitors of HIV PR
In general, inhibitors of HIV PR can be divided into three distinct groups. The first group includes
peptidic inhibitors that utilize various transition-state dipeptide analogs such as statine, hydroxyethylene,
and hydroxyethylamine incorporated into peptidic frameworks of differing lengths. Several crystal
structures of this type of inhibitor complexed with HIV PR were solved and the structural information
provided a wealth of information as to the minimum size of inhibitors, geometry of hydrogen bonds
formed within the active site, and the structural flexibility of the subsites (for reviews see Reference 18
and 23). The second and perhaps largest group of HIV PR inhibitors includes peptidomimetic
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compounds that utilize similar transition-state analogs and retain at least one peptide bond with a side
chain corresponding to a naturally occurring amino acid. Several compounds from this group have
excellent pharmacokinetic and antiviral properties and, in fact, all three HIV PR inhibitors approved for
clinical use (saquinavir, ritonavir, and indinavir) belong to this class of compounds. The last and the
smallest group of HIV PR inhibitors has a distinct nonpeptidic character. Compounds from this class
were discovered either by screening libraries of existing compounds or by structure-based de novo
design. Illustrative examples of inhibitors belonging to all three classes and a brief description of the
discovery of selected compounds are presented below.
D. Peptidic Inhibitors of HIV PR
The concept of peptidic inhibitors of HIV PR can be exemplified by the crystal structure of the statinecontaining peptidic compound AG1002 (Figure 3) [23]. In AG1002, the statine moiety replaces the
scissile dipeptide while the flanking amino acids were derived from the natural substrate cleaved by HIV
PR. The inhibitor binds to the active site in an extended conformation with the central hydroxyl group of
the statine moiety forming hydrogen bonds with the active-site aspartic acids 25/25'. The peptidic
backbone and the side chains of the
Figure 3
Stereo view of the peptidic inhibitor AG1002 bound to the active site of HIV PR.
The distribution of the specificity subsites S and S' is similar to that shown in
Figure 2. The boundaries of the HIV PR active site are indicated by the dotted
surface.
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inhibitor form 16 hydrogen bonds and occupy subsites from S4 to S1, S2', and S3'. The carbonyl
oxygens of P2 and P1' accept two hydrogen bonds from the flap water Wat301, which in effect is nearly
tetrahedrally coordinated. Due to the structural nature of statine, which lacks the P1' side chain, the S1'
pocket remains unoccupied. The S1 subsite is only partially filled by the P1 side chain of leucine. The
P2 and P2' side chains of asparagine and glutamine form hydrogen bonds with Asp30' and 30, while the
aliphatic carbons of both side chains make several hydrophobic contacts in the S2 and S2' pockets
respectively. Despite the large number of hydrogen bonds formed within the HIV PR active site,
AG1002 has rather low inhibitory potency with a binding constant of 0.55 M The low binding constant
most likely reflects the absence of the P1' group, the free energy required for desolvation of the
hydrophilic side chains, and the charged N- and C-termini as well as entropic effects caused by the
flexible nature of the heptapeptide.
Other interesting examples of peptidic inhibitors are compounds utilizing other transition-state analogs,
e.g. reduced amide-containing hexapeptide MVT-101 [24], hydroxyethylene-containing octapeptide U85548e [25], and hydroxyethylamine-containing heptapeptide JG-365 [26]. All these compounds bind to
the active site of HIV PR in a similar extended conformation and the small differences in the geometry
of hydrogen bonds formed with HIV PR can be attributed to the different character and length of the
transition-state analogs. The chemical structures and inhibition constants of these inhibitors are
summarized in Table 2. Note that the inhibition constants cited throughout this chapter and in Tables 2,
3, and 6 were determined in different laboratoriesoften using significantly different assay
conditionsand therefore might not be meaningfully comparable.
Due to their substantial size and peptidic nature, inhibitors from this class were not suitable for clinical
application. Nevertheless, the structural information derived from many crystal structures of peptidic
inhibitors bound to the HIV PR active site was critical for subsequent modeling and design of the next
generation of peptidomimetic and nonpeptidic inhibitors of HIV PR.
E. Peptidomimetic Inhibitors of HIV PR
Design and Structure of Ro-31-8959 (Saquinavir)
The strategy of designing saquinavir was based on the transition-state mimetic concept, an approach that
has been used successfully in the design of potent inhibitors of renin and other aspartic proteases [10].
From the variety of nonscissile transition-state analogs of a dipeptide, the hydroxyethylamine mimetic
was selected because it most readily accommodates the amino acid moiety characteristic of the Phe-Pro
and Tyr-Pro cleavage sequence of the
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retroviral substrates. In the first step of design, the dipeptide analog consisting of
Phe[CH(OH)CH2N]Pro was used to determine the minimum sequence required for potent inhibition.
From this study a compound was selected that included benzyloxycarbonyl at the N-terminal side of the
inhibitor followed by the P2 asparagine, the hydroxyethylamine isostere with side chains of
phenylalanine and proline in the P1 and P1' positions respectively and the NH-t-butyl group at the Cterminal part. In the following design, the side chain of proline was consequently modified to a
piperidine and finally to a decahydroisoquino-
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line moiety, and the N-terminal benzyloxycarbonyl group was replaced by the quinoline-2-carbonyl. The
resulting compound, Ro-31-8959, was one of the first peptidomimetic inhibitors with very high antiviral
potency and became a benchmark for further design of HIV PR inhibitors [10].
The high-resolution crystal structure of saquinavir bound to the active site of HIV PR was solved in
many laboratories [23,27]. The incorporation of decahydroisoquinoline moiety, which can be considered
as a conformationally restrained mimic of cyclohexylalanine, has some important consequences. First,
the length of the C-terminal part of the inhibitor has been restricted to the P2' residue which, in
saquinavir, consists of a NH-t-butyl group. Second, it restrained the conformational freedom of the
otherwise peptidic backbone, minimizing the entropic penalty to the free energy of binding. In the
crystal structure of saquinavir with HIV PR (Figure 4), the decahydroisoquinoline in the preferred chairchair conformation, makes extended hydrophobic contacts in the S1' subsite. The bond between the
methylene carbon and the nitrogen of decahydroisoquinoline is in the low-energy equatorial
conformation and the nitrogen, even if protonated, is not in a position to form a hydrogen bond with the
active-site residues. The central hydroxyl group is in the R(syn) conformation and is within the
hydrogen-bond-forming distance with both carboxylates
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Figure 4
Stereo view of the peptidomimetic inhibitor Ro 31-8959 (saquinavir) bound
to the active site of HIV PR. The distribution of the specificity subsites S and
S' is identical to that shown in Figure 2. Note the stacking interaction
between the quinoline moiety and the P1 side chain of phenylalanine.
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of the active-site aspartates. Similar to Ag1002 and other peptidic inhibitors, the carbonyl oxygens of the
P2 and P1' amides are within hydrogen-bonding distance of the flap water molecule; however, the
geometry of the second hydrogen bond is distorted due to the additional spacing between both carbonyl
groups. The nitrogen of the t-butylamide is displaced from the normal P2' position by approximately 1.8
and, as a result, cannot form a direct hydrogen bond with the carbonyl oxygen of Gly27. Instead the tbutylamide nitrogen interacts via highly ordered water molecules with the amide nitrogen of Asp29 and
the carbonyl oxygen of Gly27. The aliphatic t-butyl moiety occupies the S2' subsite and the position of
the backbone in this region prohibits any further extension into the S3' pocket. The P1 and P2 side
chains of phenylalanine and asparagine, respectively, occupy the corresponding subsites and have a
similar conformation to the equivalent groups observed in peptidic inhibitors. In the crystal structure, the
N-terminal quinoline-2-carboxylate is moved to the side and, as a result, the carbonyl oxygen forms
hydrogen bonds with the ordered water molecule and with the amide nitrogen of Asp29'. The quinoline
ring is in a low-energy conformation with respect to the preceding carbonyl oxygen, which places the
aromatic nitrogen in unfavorable close contact (3.3 ) to the carbonyl oxygen of the flap Gly48.
Because of the absence of any further contacts with the HIV PR active site residues, the contribution of
the quinoline moiety to the free energy of binding remains unclear. Perhaps in solution, a stacking
interaction of the P1 phenyl ring and the aromatic quinoline restricts the conformational freedom of Ro31-8959, in effect diminishing the free-energy loss due to the entropic and desolvation effects.
Saquinavir, despite its distinct peptidomimetic character is a very potent inhibitor of HIV PR with an
inhibition constant of 0.9 nM and an antiviral IC50 in vitro of 0.020 M [10]. Although it suffers from a
low oral bioavailability (510% in humans), it became an important starting point for the design of
second generation, less-or nonpeptidic inhibitors. Saquinavir became the first HIV PR inhibitor
approved by the FDA for treatment of AIDS.
Design and Structure of ABT-538 (Ritonavir)
An interesting concept for designing specific HIV PR peptidomimetic inhibitors with internal two-fold
symmetry was first formulated by John Erickson and his colleagues from Abbott Laboratories [28].
They reasoned that if HIV PR incorporates symmetry into its active site structure, compounds that
mimic this symmetry might be novel, more specific, and potent inhibitors and, furthermore, due to the
bidirectionality of peptide bonds, might be sufficiently less peptidic in character and pharmacologically
superior to the classical peptide-based compounds. The crystal structure of one of the first compounds
from this series (A74704) verified the assumption of symmetrical binding conformation in the
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active site of HIV PR. The inhibitor consists of the central diamino alcohol moiety with symmetrically
distributed phenylalanine side chains and two flanking, Cbz-blocked, valine residues. Except for the
asymmetric hydroxyl group, A74704 binds to the active site in a symmetric mode and the inconsistent
distribution of the terminal Cbz groups is most likely caused by crystal lattice contacts and may not
reflect the binding mode in solution [28].
The design of symmetrical inhibitors was further extended to include a series of diamino, diol core units,
in which the C2 axis bisects the bond connecting the two hydroxy-bearing carbon atoms [29]. Such
inhibitors consistently showed greater potency than A74704, but the relative potencies of the diols
differed for different diastereomers, and they did not exhibit a uniform dependence on the
stereochemistry at the hydroxymethyl position. Surprisingly, high-resolution crystal structures of HIV
PR with all possible diol diastereomers, (S,S, R,R and R,S) revealed that most of the inhibitors bind in a
clearly asymmetric fashion placing only one of the diol hydroxyl groups on the C2 axis dissecting the
active site of HIV PR and the catalytic carboxylates of Asp25/25'. The asymmetric placement of diols
causes translation of inhibitors along the long axis of the active site and, as a result, the midpoint of the
compounds is displaced by up to 0.9 from the two-fold axis of the HIV PR. Nevertheless, the dihedral
angles of the symmetry-related bonds are in most cases within 10, and the inhibitors maintain overall
symmetry in the bound conformation [23,29].
The ABT-538 design was a direct consequence of the pioneering work with peptidomimetic compounds
with the internal C2 symmetry [30]. Since the high-resolution crystal structures of a family of diolcontaining compounds indicated that in most cases only one of the diol hydroxyls interacts with the
catalytic aspartic acids 25/25', in subsequent designs the noninteracting hydroxyl group was replaced by
a hydrogen. This substitution reduced the free energy penalty required for desolvation of the hydroxyl
group and increased the inhibitory potency without perturbing the binding mode of the compounds [30].
In the further search for related inhibitors with improved oral bioavailability, the focus of effort
concentrated on the effect that molecular size, aqueous solubility, and hydrogen-bonding capability has
on pharmacokinetic behavior. This study resulted in a smaller compound, A-80987, in which the P2'
side chain of valine was eliminated and the terminal 2-pyridinyl group was replaced by 3-pyridinyl
moiety that makes van der Waals contacts in the S2' subsite and forms a hydrogen bond with the amide
nitrogen of Asp30 [31]. The pharmacokinetic properties of A-80987 were significantly improved over
larger, symmetrical compounds from this series and, at the same time, the high antiviral activity typical
for these inhibitors was largely unaffected. In subsequent optimization, which focused on the metabolic
stability of these inhibitors in vivo, the electron-rich and oxidation-prone pyridinyl groups were replaced
by thiazoles.
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Thiazoles are less electron-rich isosteres of pyridines and therefore it was speculated that compounds
with such substitution may have improved metabolic stability [30]. The modeling of A-82200 in which
the N-terminal pyridinyl group was substituted by a 4-thiazolyl moiety indicated that the 5-membered
ring binds in the S3 subsite and can be further derivatized at the 2 position by an isopropyl group. The
isopropyl functionality makes van der Waals contacts with Val82 and fills the hydrophobic part of the
S3 subsite in nearly optimal fashion.
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The resulting compound, ABT-538 (Table 3), binds to the active site of HIV PR in an extended
conformation. The central, asymmetric hydroxyl group is within hydrogen-bonding distance of the
catalytic aspartates 25/25', and the P1/P1' phenylalanine side chains are symmetrically distributed in the
corresponding subsites. The nitrogens of the symmetric amide bonds on both sides of the central
aminoalcohol are barely within the hydrogen-bonding distance of the carbonyl oxygens of Gly27/27'
(3.4 ) and the carbonyl oxygens of these amide bonds participate in the tetrahydral coordination of the
flap water molecule Wat301. On the N-terminal side of the compound, the P2 side chain of valine fills
the S2 subsite and the terminal 2-isopropyl-4-thiazolyl makes hydrohobic contacts with the residues in
the S3 pocket and has a stacking interaction with the P1 phenylalanine. On the C-terminal side, the 5thiazole is positioned to interact within the S2' subsite, and the nitrogen on the 5-membered ring is
within hydrogen-bonding distance of the amide of Asp30'.
Despite two peptide bonds present in ABT-538, this compound has substantial oral availability in
humans and a very high antiviral activity in vivo [30]. Recently, ABT-538, better known as ritonavir,
has been approved by the FDA for treatment of AIDS in combination with inhibitors of the reverse
transcriptase.
Design and Structure of L-735,524 (Indinavir)
Indinavir is another example of very potent peptidomimetic compound discovered using the elements
the crystal structure-based design [32] and SAR (structure activity relationship). The starting point for
the design was a series of compounds containing the hydroxyethylene isostere of a scissile dipeptide
[33]. An example of compounds from these series is L-685,434, which consists of a tert-butylcarbamate
group forming the P2 moiety, symmetrically distributed phenylalanine side chains in the P1/P1', and the
indanol group in the P2' portion of the inhibitor. Although very potent, the optimized molecules from
this series lacked aqueous solubility and an acceptable pharmacokinetic profile [32]. The Merck group
hypothesized that incorporation of a basic amine-containing functionality, such as the
decahydroisoquinoline group of saquinavir, into the backbone of L-685,434 series might improve the
solubility and bioavailability of this type of compound. Also the replacement of the P2/P1
functionalities, the tert-butylamide and phenylalanine side chain by the decahydroisoquinoline tertbutylamide, would generate a novel class of hydroxylaminepentanamide isostere with potentially
improved metabolic stability in vivo. An additional strong argument for using decahydroisoquinoline as
an isostere of P1/P2 moieties was the restricted conformational freedom of the enclosed-into-a-ring
basic amine, which should decrease the entropy change upon binding to HIV PR in a similar fashion to
that observed in saquinavir. In
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the resulting chimeric inhibitor the central hydroxyl group forms hydrogen bonds with the catalytic
aspartic acids 25/25' and the hydrophobic side chains of the P1/P1' decahydroisoquinoline and
phenylalanine respectively are separated from the central hydroxyl-bearing carbon by the methylene
linkers forming a pseudosymmetrical arrangement. In the subsequent optimization of inhibitors from
this novel series, a smaller piperazine group was substituted for the decahydroisoquinoline group, which
offered a possibility to expand from the N4 position to the partially lipophilic S3 subsite. One of the first
compounds from the piperazine series possessed a benzyloxycarbonyl moiety attached to the piperazine
ring and the additional hydrophobic interaction in the S3 subsite was reflected by substantial increase in
both intrinsic potency and in the ability to inhibit viral spread in infected cells in vitro. Finally, the
replacement of the benzyloxycarbonyl group by the 3-pyridylmethyl moiety (Table 3) provided both
lipohilicity for binding to the HIV PR active site and a weakly basic nitrogen that increased aqueous
solubility and oral bioavailability. The crystal structure of L-735,524 (indinavir) bound to the active site
of HIV PR [34] indicates that the 3-pyridylmethyl group attached to the N4 position of the piperazine
ring makes hydrophobic contacts with the residues in the S3 and S1 pockets and the tert-butyl moiety
fills the S2 subsite in the fashion previously observed in the structure of saquinavir. The positions of the
P2 and P1' carbonyls maintain the proper alignment to form hydrogen bonds with the flap water
Wat301. The terminal indanol group of indinavir occupies the S2' subsite with the hydroxyl group
within hydrogen-bonding distance of the amide nitrogen of Asp29.
The high aqueous solubility and largely nonpeptidic character of indinavir may be responsible for the
good oral bioavailability, respectable pharmacokinetic profile, and high antiviral activity observed with
this compound. Similar to saquinavir and ritonavir, indinavir has been recently approved by the FDA for
treatment of AIDS.
F. Nonpeptidic Inhibitors of HIV PR
The nonpeptidic inhibitors of HIV PR can be divided into two subclasses. Compounds that belong to the
first group maintain the general binding mode of the peptidomimetic inhibitors including formation of
the key hydrogen bonds with the active site residues. An example of such nonpeptidic inhibitors of HIV
PR is AG1343 (nelfinavir). The second group of nonpeptidic HIV PR inhibitors includes compounds
with a binding mode significantly different from that described for the peptidomimetic compounds.
Most inhibitors in this latter class were initially discovered by screening natural-products libraries or by
structurebased de novo design. The most interesting examples of the nonpeptidic inhibitors from this
group are the independently discovered but structurally
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related 4-hydroxypyrans and 4-hydroxycoumarins, the cyclic urea-based DMP323 series, and AG1284.
Design and Structure of AG1343 (Nelfinavir)
Analysis of the crystal structure of saquinavir with HIV PR indicated that while the nonpeptidic
components of the ligand, namely the decahydroisoquinoline and the t-butylamide moieties fill the S1'
and S2' subsites nearly optimally, the N-terminal portion offered the possibility for remodeling, aimed at
the elimination of the peptidic character. Also, the contribution of the quinoline to the binding affinity to
HIV PR was difficult to rationalize. Since the removal of quinoline resulted in a nearly 1000-fold loss in
binding constant, it was concluded that the stacking interactions of the P1 phenyl ring and the P3
aromatic moiety of quinoline are necessary for the conformational stability of Ro 31-8959. In an attempt
to redesign the N-terminal part of the ligand, the nonpeptidic portions of the P1' and P2' were maintained
but for reasons of synthetic accessibility, the decahydroisoquinoline moiety was replaced by an orthosubstituted benzylamide [35]. Crystallographic analysis of both compounds showed that saquinovir and
the modified LY289612 bind essentially identically to the active site of HIV PR and their inhibition
constants and antiviral activity were very similar (Table 4 and Table 3).
In the first attempt to functionally substitute the P2 side chain of asparagine, the isophthalic-acidcontaining compound was modeled and the low-energy conformation of the aromatic ring, required for
binding in the S2 subsite, was stabilized by a tertiary carboxamide in the P3 region of the inhibitor [36].
The analysis of the binding mode and interactions of the isophthalic ring in the S2 subsite indicated a
lipophilic pocket deep on the border between the S2 and S1' subsites, which could be conveniently filled
with a methyl group extending from the 2 position of the ring. The resulting compound II in Table 4 lost
most of the peptidomimetic character of LY289612 but retained its inhibitory potency.
In an independent line of design, the relationship between the P1 phenylalanine side chain and the P3
quinoline was investigated. In the crystal structure of saquinavir bound to the active site of HIV PR
(Figure 4), the aromatic ring of the P1 phenylalanine makes several van der Waals contacts with
residues forming the S1 subsite. Computer modeling indicated that an extension of the phenylalanine
side chain to phenethyl (homophenylalanine) would lead to prohibitive close contacts of the phenyl ring
with the aliphatic side chains of HIV PR. On the other hand, replacement of the -carbon of the
homophenylalanine by sulphur, which has a more acute C-S-C bond angle, would direct the aromatic
ring into the neighbouring S3 subsite without changing the desired lipophilic nature of the P1 side chain.
The increased area of hydrophobic interactions in the S1 and S3 subsites by compounds with the Sphenylcysteine and
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S-naphthylcysteine derived side chains in P1 resulted in a substantial increase in the inhibition constants
[37]. The increase in the binding affinity to the low picomolar range in enzyme inhibition assay, allowed
for subsequent truncation of the P3 quinoline moiety. The final compound from this miniseries
(compound III in Table 4) consisted of the ortho-substituted benzamide in the P1' and P2', Snaphthylcysteine in P1 and asparagine in P2. Despite reduced molecular weight, the inhibition constant
of this compound for HIV PR was comparable to LY289612.
The observation that a larger, nonpeptidic moiety in the P1 could eliminate the need for the P3 side
chain led to hybrid molecules that incorporated ring structures as the P2 component and maintained the
P1 S-naphthylcysteine side chain of compound III. In this miniseries several bicyclic functionalities
were modeled as the P2 substituents and one example, compound IV utilizing a tetrahydroquinoline
group, is shown in Table 4 [38]. In subsequent modeling, it was noticed that the P2 bicyclic functionality
might be replaced by 2,3-disubstituted phenyl rings. In particular, a methyl substitution in position 2
would increase the area of hydrophobic interaction in a manner previously observed in the isophthal
series. Addition of a hydrophilic functionality attached at position 3 could increase the solubility of the
compound and contribute to the binding constant by forming a hydrogen bond with the carboxylate
oxygen of Asp30. A compound with a 2-methyl-3-hydroxy substitution pattern was synthesized and
showed an improved inhibition constant of 3 nM in the HIV PR enzyme assay (Table 4). The crystal
structure of compound V with HIV PR was solved and indicated the predicted binding mode with the
possibility of a stacking interaction between the P2 phenyl and the P1 thio-naphthyl groups and the
expected hydrophobic and hydrogen-bonding interactions of the P2 moiety with the protein side chains
in the corresponding specificity pocket [38].
As with the optimized compounds from other series, compound V suffered from low aqueous solubility.
The replacement of the P1' aryl group by the basic amine-containing decahydroisoquinoline
dramatically increased the solubility and allowed for truncation of the P1 S-naphthylcysteine side chain
to S-phenylcysteine without any loss of inhibitory activity. The resulting compound VI, AG1343 or
nelfinavir, has an inhibition constant of 1.9 nM in the HIV PR enzyme assay and respectable antiviral
activity with an IC90 of 60 nM [39]. The nonpeptidic character, pH-dependent solubility profile, and the
small molecular weight of nelfinavir may contribute to its good pharmacokinetic profile in humans
[40,41]. Currently, this compound is being tested and is in the advanced phase of clinical trials.
The crystal structure of nelfinavir bound to the active site of HIV PR is shown in Figure 5. The general
binding mode of this compound, in particular the path of the backbone, is similar to the binding mode of
peptidyl inhibitors. Nevertheless, the lack of any peptide bonds utilizing naturally occurring amino
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Figure 5
Stereo view AG1343 (nelfinavir) bound to the active site of HIV PR.
acids qualifies nelfinavir to be a member of the group of nonpeptidic inhibitors of HIV PR. The unique,
and perhaps crucial hydrogen-bonding interaction of the P2 hydroxyl group with the carboxylate oxygen
of Asp30, combined with the smaller area of hydrophobic contacts in the S1 and S3 specificity subsites
are the principal differences from other clinically active HIV PR inhibitors and may contribute to a
distinct resistance pattern and point to additional utility of nelfinavir in the treatment of AIDS.
Design and Structure of DMP323
A cyclic urea-containing HIV PR inhibitor, DMP323, was discovered using de novo structure-based
design principles. Similar to the concept of Erickson and his co-workers from Abbott Laboratories, the
group from DuPont-Merck attempted to take advantage of the two-fold symmetry of HIV PR in
designing compounds that maintained the interaction of the diol with the catalytic aspartic acids 25/25'
and at the same time were able to functionally displace the ubiquitous flap water molecule Wat301.
They hypothesized that incorporation of the binding features of this structural water molecule into an
inhibitor would be beneficial because of the entropic gain due to its displacement and because the
conversion of a flexible linear inhibitor into a rigid, cyclic structure with restricted conformation should
provide an additional, positive entropic effect. In the initial design, a cyclohexanone with the ketone
oxygen as the structural
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water mimic was used and in subsequent synthetic targets the cyclohexanone ring was enlarged to a 7membered ring to incorporate a diol functionality. This target was further modified to a cyclic urea,
which can be symmetrically substituted from both nitrogens without creating unnecessary stereocenters.
The crystal structures of about 10 cyclic-urea-based inhibitors with HIV PR were solved [42]. In all
cases, the C2 symmetric inhibitors bind to the HIV PR active site with the diad symmetry axes of the
protease and the compounds being nearly coincident. The 7-membered ring of the inhibitors is roughly
perpendicular to the plane of the catalytic aspartates 25/25' and both hydroxyl groups of the diol are
positioned to interact with their carboxylates. The carbonyl oxygen of the inhibitors accepts hydrogen
bonds from backbone amides of symmetrically distributed residues Ile50/50' of the flap. In the structure
of DMP323, symmetrically substituted moieties of hydroxymethylbenzyls and phenylalanines extend
towards the S2/S2' and S1/S1' subsites respectively and are involved in van der Waals interactions with
the hydrophobic residues of these pockets [42].
The interaction of DMP323 with the residues of HIV PR are restricted to the central four specificity
subsites of the active site. Despite this limited area of hydrophobic interaction and hydrogen bonding
restricted to the central cyclic urea functionality, DMP323 is a very potent inhibitor of HIV PR with
good antiviral activity in vitro (Table 5). The limited solubility of this compound was perhaps
responsible for erratic oral availability in humans, and after short trials, DMP323 was withdrawn from
the clinical investigation. Nevertheless, the discovery of this class of compounds represents a very
interesting and, by now, classical example of de novo structure-based drug design.
Design and Structure of AG1284
Another compound discovered by the application of de novo structure-based design is AG1284 [43]. In
the initial design of a lead compound, the nonpeptidic hydroxyethyl-t-butylbenzylamide portion of
LY289612 occupying the S1' and S2' subsites was retained as a starting module. In attempting to fill
the pockets related by the dimer two-fold symmetry it was discovered that, by extending a two-carbon
fragment from the central hydroxyl carbon, the S1 subsite could be accessed by an aromatic ring. The
ring was oriented orthogonal to the observed P1 phenyl group of the classical inhibitors and this allowed
further extension off the ortho position towards the S2 subsite. In order to maintain the critical hydrogen
bond to the flap water Wat301, in the initial compounds an acylated amino group was used, replaced in
subsequent designs by a benzamide functionality. In this model, the geometry of the hydrogen bonds to
the flap water was somewhat perturbed, and the nitrogens of the t-butyl amides on both sides of the
compound were in a position to interact favorably with solvent,
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potentially lowering the desolvation penalty. The absence of hydrogen-bonding interactions with the
carbonyl oxygens of Gly27/27' was viewed as a positive factor, since the accumulated structural and
mechanistic information suggested that formation of these hydrogen bonds may not be energetically
favorable [23]. The compound was synthesized as a racemic mixture of two enantiomers of the central
hydroxymethyl group and had the inhibition constant of 24 M. Despite the modest binding constant of
compound II (Table 6) and very low water solubility, the co-crystal structure with HIV PR was solved at
2.3 resolution, providing a critical starting point for further design. The inhibitor was found to bind
largely as anticipated with the two aromatic rings occupying the S1 and S1' subsites and the two
benzamide carbonyls forming hydrogen bonds to the flap water Wat301. Both benzamide nitrogens
interact via a string of highly ordered water molecules with the amide nitrogens of Asp29/29'. The
crystal structure of the complex indicated that the S enantiomer was the more active component of the
racemic mixture and this was confirmed by stereoselective synthesis of subsequent compounds [44].
In the subsequent designs, the ortho-substituted benzyl rings were consecutively replaced by larger
naphthyl groups that occupied more of the S1S3 and S1'S3' subsites. The increased area of
hydrophobic interactions with the residues in these subsites was reflected in a substantial improvement
in the
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binding constant and also in reduced aqueous solubility. Also, due to the very tight fit of both naphthyl
moieties in the S1 and S1' subsites, subsequent design targeting the S3/S3' subsites proved to be difficult
and synthetically challenging [44]. In the search for a simpler solution, the di-tertiary amides were
designed using the crystal structure of compound II (Table 6) as a starting model. Branching from the
amide nitrogens provided an interesting possibility to access S2S3/S2'S3' subsites while
simultaneously increasing the solubility and stability of the compounds. In the first design, the
hydroxyethyl moieties were fused to the amide nitrogens and the hydroxyl groups were intended to form
hydrogen bonds with the amide nitrogens of Asp29/29' (compound III in Table 6). The addition of both
hydroxyethyl groups resulted in a rather significant increase in the binding constant, and the racemic
mixture had the Ki of 1.1M. When the crystal structure of compound III complexed with HIV PR was
solved at 2.2 resolution, it was observed that the inhibitor had undergone an inversion in binding
mode relative to the secondary amide series. The phenyl groups of compound III occupied the S2/S2'
subsites, switching positions with the t-butyl groups, which were in turn occupying the S1/S1' pockets
(Figure 6). Due to this change in binding mode, the R enantiomer would be expected to be preferred
relative to S. The final position of the hydroxyethyl moieties was less effected by the change, and both
hydroxyls were within hydrogen-bonding distance from the amide nitrogens of Asp 29/29'. In the S2/S2'
pockets, the phenyl groups occupied only a fraction of subsites, but the interaction was strengthened by
highly ordered water molecules involved in electrostatic interaction with the aromatic rings and by
forming hydrogen bonds to Asp30/30'. Interestingly the position of the hydrogen bonds with respect to
the flap water was significantly disturbed in the new binding mode, and the conserved Wat301 was no
longer tetrahydrally coordinated [43,45].
This unanticipated change in binding mode presented a potential for new avenues of design different
from those of the secondary amides. The ability to design into neighboring subsites depends to a large
extent on the positions of bond vectors suitable for substitution in the bound conformation of a given
inhibitor. These vectors in the crystallographically discovered new binding mode of compound III were
positioned ideally to access unfilled space in the S3/S3' pockets. The discovery of this new conformation
of compound III highlighted the power of crystallographic feedback in the process of inhibitor design
and, without this structural information, further design in this series would have been severely impeded.
Inspection of the crystal structure of compound III bound to the active site of HIV PR revealed
lipophilic cavities extending off the S1/S1' subsites adjacent to the t-butyl groups of the benzamidine
moiety. The cavities are bordered by flexible loops around Pro81/81' and previous crystallographic
studies indicated that both loops can move back by up to 2.5 , extending the size and
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Figure 6
Change of the binding mode of compound III observed during
iterative design of AG1284. (a) Crystallographically determined
binding mode of compound II. Pseudosymmetrically distributed aryl groups
are bound in the S1 and S1' specificity subsites. (b) Crystallographically
determined binding mode of compound III. Note the inversion of the binding
mode. The ortho-substituted benzyl groups bind in a pseudosymmetric
fashion in the S2 and S2' subsites.
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volume of the active site. With this in mind, the dimethylbenzyl group was attached to compound III and
the additional phenyl ring was accommodated well in the lipophilic pocket of the S1'/S3' sides. As the
S1 pocket was not fully occupied, a Monte Carlo-based De Novo Ligand-Generating program
(MCDNLG) [46] was used to identify other amide substituents that might fill this subsite more
effectively. From several moieties identified by the MCDNLG program, a larger cyclopentylethyl group
showing very good shape complementarity to the S1/S3 subsite was selected for synthesis. In addition,
due to the asymmetrical nature of this compound, additional space was identified at the bottom of the
S2' pocket that was conveniently filled with either a methyl or a chlorine group on the 5 position of the
benzamidine ring. The inhibition constant of the resulting compound (compound IV in the Table 6) was
0.008 M, which represents approximately a 2500-fold improvement over the first compound from this
series.
The crystal structure of compound IV or AG1284 complexed with HIV-1 PR was solved, revealing
excellent complementarity between the ligand and protein. The ligand forms only 4 hydrogen bonds
with either protein functional groups or ordered water molecules, in contrast to the nine hydrogen bonds
formed by peptidomimetic LY289612, despite their similar binding affinities. The nonpeptidic character
of AG1284 may have contributed to good oral bioavailability and pharmacokinetics in three animal
species [43].
Despite very good inhibitory potency on the enzyme level, AG1284 has rather modest antiviral activity
in vitro (Table 6). The reason for this discrepancy is unclear but could be related to the low water
solubility and higher affinity for membranes, which may effect cell partitioning. A similar lack of
correlation between the potency of enzyme inhibition and antiviral activity has been previously observed
with other HIV PR inhibitors [11].
Hydroxypyrans and Hydroxycoumarins
The lead compounds for the 4-hydroxypyran and 4-hydroxycoumarin series were discovered in
biological screens as low potency inhibitors of HIV PR [4749]. Successful structure solution of both
lead compounds with HIV PR enabled rapid optimization of their enzyme inhibitory potencies and antiHIV activities, and one of these compounds, U96988, has already entered Phase I clinical testing
[49,50]. The binding mode of this type of inhibitor differs substantially from the classical
peptidomimetic compounds and is somewhat similar to de novo-designed compounds from the cyclic
urea series. In the case of 4-hydroxycoumarin, the two oxygen atoms of the lactone functionality are
positioned within hydrogen-bonding distance of the two NH amides of Ile50/50' on the flap, replacing
the ubiquitous water molecule Wat301. The 4-hydroxyl group (Table 5) is located within hydrogenbonding distance of the two catalytic
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aspartic acid residues Asp25/25' and this hydrogen-bonding network of the 4-hydroxycoumarin defines
the essential pharmacophore of this new class of inhibitors. In the structure of U96988, this
pharmacophore is pseudosymmetrically subsituted by an ethyl and a phenyl group at the C-3a and an
ethyl and a benzyl group at the C-6a positions. These four substituents extend into the central core of
S2/S2' subsites, where they make van der Waals contacts with the hydrophobic residues of the active site
[49]. With a molecular weight of 362 U96988 is the smallest inhibitor of HIV PR in clinical testing. It
suffers from rather low antiviral activity (ED90 of ~ 10 M)but can be considered as the first in a series
of this promising class of nonpeptidic HIV PR inhibitors.
II. Structural Basis of Resistance of HIV PR Inhibitors
The dimeric character and the two-fold symmetry of the active site, in which the monomers contribute
equivalent residues to symmetrically distributed specificity subsites, led to early speculations that HIV
PR may be less susceptible to resistance than, for example, reverse transcriptase. In the case of retroviral
proteases, a single base mutation in the viral genome corresponds to two changes in the threedimensional structure and two structurally identical changes in the active site could result in an enzyme
with a drastically modified specificity profile and impaired catalytic activity. Identification of HIV PR
variants in cell-culture experiments clearly indicated, however, that this class of drugs is not immune to
the challenge of viral resistance. It should be stressed that HIV, unlike other human viruses, is
characterized by a dynamic viral turnover in the steady state [51,52]. The rapid replication rate coupled
with the lengthy duration of infection will favor the emergence of resistant mutants to targeted antiviral
agents [53].
The accumulated data from cell-culture sequential-passage experiments with several structurally
different inhibitors and from the resistant variants identified during clinical exposure to four HIV PRtargeting drugs indicate a very complex pattern of mutations in the structure of HIV PR. In contrast to
mutations in the reverse transcriptase, which frequently cause multihundredfold resistance [54], single
base changes in the HIV PR gene (i.e., two identical substitutions per protease dimer) lead in most cases
to 510-fold decrease in the antiviral potency of a given drug [11]. It has been shown for the most
clinically studied HIV PR inhibitors, such as indinavir and ritonavir, that the clinical manifestation of
resistance (increase in the viral load and decrease in the CD4 count) requires the simultaneous
appearance of several mutations [55,56]. For example the resistant HIV strain isolated from patients
exposed for 40 weeks to indinavir carried mutations at residues 10/10'L > R, 46/46'M > I, 63/63'L > P,
82/82'V > T, and 84/84'I > V [59,60]. However, the combination of these five
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Figure 7
Cartoon representation of the HIV PR dimer. The sites of primary
resistance-causing mutations in the active site are indicated. For clarity, the names of the
residues are shown for one monomer only.
mutations (ten assuming the dimeric nature of HIV PR) changed the susceptibility of the resistant strain
to indinavir by only eight-fold if compared to the wild type HIV.
The resistance-causing mutations are localized in a few hot spots in the structure of HIV PR and can
be divided into two groups. The first group consists of the primary mutations located directly in the
active site and includes changes at residues Val82/82', Ile84/84', somewhat less frequently at Gly48/48'
and, in the case of nelfinavir, Asp30/30' (Figure 7). Residues 82/82' and 84/84' are located on the
flexible loops that form the outer walls of the S3/S3' and S1/S1' subsites, respectively. In the resistant
variants, valine 82/82' is most frequently substituted by the smaller side chain of alanine or the larger
side chains of phenylalanine or isoleucine [57,58]. The change in position 82/82' is usually accompanied
by a substitution of Ile84/84', most commonly to the smaller amino acids alanine or valine [57]. From
the clinically tested compounds, ritonavir and indinavir, which were optimized to form strong
hydrophobic interactions with the side chain of Val82/82' in the S3 subsite, suffer most significantly
from any change at this position. On the other hand, the antiviral activities of saquinavir, and nelfinavir,
which do not form any interaction in the S3/S3' subsite are not affected by mutations at Val82/82' and
are only marginally cross resistant to changes involving Ile84/84' [57,58,62]. The resistance-causing
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mutation of Asp30/30' to asparagine seems to be specific for nelfinavir and was initially observed in cellculture sequential passage experiments [62]. Recently, the same phenotypic change was confirmed as
the predominant mutation in the resistant variants appearing in AIDS patients exposed to low doses of
this HIV PR inhibitor [64]. The molecular basis of resistance involving this mutation can be rationalized
as follows: in the crystal structure of nelfinavir with the wild type HIV PR, the 3-hydroxyl group of the
2,3-substituted phenyl group is within hydrogen-bonding distance of the carboxylate oxygen of Asp30 in
the S2 subsite. Due to the expected coulombic character of this interaction, the hydrogen bond formed
with the negatively charged carboxylate of Asp30 would be expected to be a relatively strong one. The
change of the negatively charged carboxylate of Asp30/30' to the amide oxygen of the asparagine side
chain should reduced the strength of this interaction. Apparently the loss in the enthalpic contribution to
the free energy of binding is only partially balanced by the entropic gain caused by the difference in
desolvation of a charged vs. neutral side chain of the receptor, leading to decreased binding affinity of
nelfinavir and eventually to viral resistance.
An additional resistance-causing mutation that qualifies as a primary mutation involves the change of
Gly48/48' to valine. This particular mutation seems to be specific for saquinavir and was observed both
in cell-culture sequential passage experiments and in AIDS patients exposed to this inhibitor [61,65].
Located on the lower strands of the active-site forming flaps, Gly48/48' can be considered a part of the
S4/S4' subsites. The replacement of the glycine hydrogen by the rigid side chain of valine has most
likely a dual effect: first it has a direct impact on the interaction of the quinoline moiety of saquinavir
with the active site of HIV PR, and second it may change the mobility of the flaps, which in turn will
effect the binding kinetics of the natural substrates or inhibitors. Although none of the other clinically
tested inhibitors form any interaction with this part of the flap, the HIV variants with mutation of
Gly48/48' seem to be cross-resistant to all compounds, which is reflected by a 35-fold reduction of
their antiviral activity [55,62].
While the effect of primary mutations on reduced binding affinities of inhibitors can be at least partially
explained in view of the accumulated structural data, the function of secondary, or compensatory
mutations in the resistant HIV PR is difficult to rationalize as yet. The predominant compensatory
mutations observed in the resistant variants involve residues Leu63/63', Ala71/71', Met46/46',
Asn88/88', Leu10/10', and Leu90/90' (Figure 8) [60,63]. Changes of these residues alone do not confer
viral resistance, but their appearance increases the viability of the virus carrying the primary mutations
in the active site of protease. All these residues are located far away from the active site of HIV PR do
not participate in any apparent way in the inhibitor binding and it seems unlikely that they form a longrange interaction with the natural
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Figure 8
Cartoon representation of the HIV PR dimer. The sites of compensatory
mutations are indicated.
substrate. Also, the reported compensatory mutations are conservative in nature and have no effect on
the overall distribution of atomic charges on the surface of HIV PR.
Sequence polymorphism at the Leu63/63' position, located on the surface at the base of HIV PR, has
been observed in clinical isolates of the virus not exposed to any HIV PR inhibitors. Variations of
Ala71/71', where the side chains are buried very close to Leu63/63', are less commonly found in clinical
isolates. After a prolonged challenge by HIV PR inhibitors, Leu63/63' changes to proline and Ala71/71'
to valine.
The side chains of Met46/46' are fully exposed to solvent and these residues are located on the hairpins that form the active side flaps. It has been speculated that the compensatory change of
Met46/46' to isoleucine or phenylalanine may affect the dynamics of the flap movement, which in turn
could influence the rates of catalytic activity of HIV PR impeded by the primary mutations in the active
site [58].
Any changes to Asn88/88' and Leu90/90', buried in the body of HIV PR, most likely affect the structural
stability of the enzyme. The side chains of Asn88/88' form buried hydrogen bonds and replacement of
this residue by aspartic acid or serine not only eliminates some of these bonds but also introduces
unfavorable interactions in the core of HIV PR. Similarly, Leu90/90' is buried in a tight hydrophobic
space close to the fireman's grip motif, which
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involves the catalytic asparates 25/25'. The structural effect of a mutation of Leu90/90' to the larger
methionine is rather difficult to predict since it can either rigidify or destabilize the HIV PR dimer or it
may have an effect on the catalytic efficiency of the resistant enzyme.
The complicated pattern of HIV resistance to protease inhibitors, in particular the appearance of
compensatory mutations that alone do not confer any resistance, suggests that the key to understanding
the basis of decreased susceptibility of the virus to a given drug is the kinetics of specific processing of
the GAG and GAG-POL polyproteins. The reduction in sensitivity of a mutant HIV PR towards any
inhibitor can be conveniently reflected by the ratio of Ki mutant/Ki wild type. However, this reduced
inhibitor sensitivity is only one component that distinguishes mutant-form from wild-type proteases. For
virus encoding of a mutant HIV PR to be viable, the mutant protease must be capable of a minimal
(although not yet quantified) level of enzymatic activity towards all substrates required for maturation of
the virions. This proteolytic efficiency is reflected in the specificity constants (Kcat/Km) as determined for
mutant and wild type HIV PRs. In order to rationalise these potentially conflicting relationships between
enzymes, substrates, and inhibitors, Gulnik and his colleagues [66] introduced the term Vitality
Factor, in which
In order for the Vitality Factor to be predictive for the level of resistance expected for a particular drug
or combination of drugs for a given resistant strain of HIV, the determination of the specificity constants
(Kcat/Km for mutants) must be repeated for all nine known substrates processed by HIV PR. The
inhibition constants of a given compound should not depend on the substrate, but the Kcat/Km ratios do
and therefore vitality values will differ for different substrates. It will be expected that the mean for all
nine vitality values will be predictive for the change in antiviral activity for a particular compound.
Although those data will be derived from in vitro experiments and are clearly not without some
limitations, they may help in understanding the molecular basis of resistance and may contribute some
value to possible multidrug strategies for the clinical management of AIDS.
III. Perspective
HIV PR inhibitors with acceptable oral availability and pharmacokinetic properties offer great promise
for the treatment of HIV infection and AIDS. Efficacy studies of indinavir, ritonavir, or nelfinavir using
plasma viral RNA as a marker have demonstrated up to three log reductions in RNA copy numbers that
are
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Page 33
sustained in many patients [6769]. In contrast, nucleoside antiretroviral therapy that targets reverse
transcriptase rarely results in more than one log reduction of viral RNA, indicating fairly poor inhibition
of viral replication by this class of compounds. One of the reasons for the apparent greater in vivo
antiviral activity of HIV PR inhibitors could be the mechanistic difference of the two enzymes and their
respective activities in the viral life cycle. However, growing evidence of retroviral resistance to
protease inhibitors remains a concern. The availability of several chemically distinct HIV PR inhibitors,
including the second generation of compounds currently under preclinical development, offers a
possibility of combining two or more drugs that share little cross-resistance. Also, it seems reasonable to
evaluate these compounds in combination with various nucleoside and nonnucleoside reverse
transcriptase inhibitors. Early clinical data from such combination therapy indicates reduction of
retroviral RNA in plasma to levels lower than the currently available limit of detection [70]. This is the
first indication that the application of well-chosen combination therapy can place AIDS patients in
prolonged virologic and clinical remission.
Undoubtedly, protein crystallography and other elements of structure-based drug design were widely
applied in the discovery of HIV PR inhibitors. It will be prudent to assume that, in the absence of
structural feedback, rapid discovery of several chemically different and potent inhibitors of HIV PR
would have been severely impeded if not even impossible. However, structure-based drug design still
remains a new and developing technology. Further success of this drug discovery technique largely
depends on the development of methods of computational chemistry. Several computational approaches
such as ALADDIN [71], DOCK [72], and MCDNLG [46] have been applied with a limited degree of
success in a search for novel inhibitors of HIV PR and these methods will be developed further. The
most difficult and challenging computational task required for full implementation of structure-based
drug design involves assigning a priority to designed compounds before their synthesis, by computation
of the absolute free energy of binding or by prediction of the relative difference in the binding constants
of chemically related compounds. While the former approach is technically very difficult, due to the size
of configurational space that must be sampled and the limited accuracy of the force field that describes
atomic interactions in the molecular system [73], the latter approach has had some successes [74,75].
Nevertheless, owing to the various assumptions and approximations that underlie these techniques, such
methods are useful only as order-of-magnitude estimates [74]. Further improvements of these methods
heavily depend on the availability of structural and thermodynamic data for several closely related
compounds that could be used to calculate parameters required for the implementation of such
thermodynamic-integration cycles. The large number of high-resolution crystal structures of HIV PR
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complexed with various inhibitors offers a unique opportunity for the development of such
computational methods if the structural data can be coupled with thermodynamic measurements of
inhibitor-protein binding. These include direct measurements by microcalorimetry of the association
constant, K, and in addition the enthalpy, entropy, heat capacity, and stoichiometry of binding. The
combination of such thermodynamic and structural data will lead to a more precise understanding of the
factors that influence binding and, ultimately, will lead to new general design principles that can be
applied to drug discovery in the area of AIDS as well as other challenging diseases.
Acknowledgments
I wish to thank all my co-workers from Agouron who contributed to these studies, in particular J.
Davies, S. Reich, M. Melnick, V. Kalish, A. Patick, L. Musick, and B-W. Wu. Steven Kaldor from Ely
Lilly is acknowledged for his contribution in designing AG1343 (nelfinavir). I would like to thank
Richard Ogden for critical reading of the manuscript and D. Olson for expert assistance in preparing the
manuscript.
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2
Structural Studies of HIV-1 Reverse Transcriptase and Implications for
Drug Design
Jianping Ding, Kalyan Das, Yu Hsiou,
Wanyi Zhang, and Edward Arnold
Center for Advanced Biotechnology and Medicine, and Rutgers University,
Piscataway, New Jersey
Prem N. S. Yadav
University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey
Stephen H. Hughes
ABL-Basic Research Program, NCI-Frederick Cancer Research and
Development Center, Frederick, Maryland
I. Introduction
Like all other retroviruses, human immunodeficiency virus type 1 (HIV-1) contains the multifunctional
enzyme reverse transcriptase (RT). Retroviral RTs have a DNA polymerase activity that can use either
an RNA or a DNA template and an RNase H activity. HIV-1 RT is essential for the conversion of singlestranded viral RNA into a linear double-stranded DNA that is subsequently integrated into the host cell
chromosomes [14]. In this conversion process HIV-1 RT catalyzes the incorporation of approximately
20,000 nucleotides. Chemotherapeutic agents have been identified that target virtually all stages of the
HIV-1 replication cycle (see review [5]). Since both the polymerase and RNase H activities of HIV-1
RT are essential, inhibiting either step blocks viral replication. Therefore, HIV-1 RT is an important
target for the treatment of AIDS. Two major classes of antiviral agents that inhibit HIV-1 RT
polymerization have been identified; these are nucleoside RT inhibitors (NRTIs) (Figure la) and
nonnucleoside RT inhibitors (NNRTIs) (Figure 1b). Nucleoside analogs, such as 3'-azido-2',3'dideoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxycytidine (ddC), 2',3'-dideoxy-3'thiacyti-
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Document
Figure 1
(a) Chemical structures of representative nucleoside analog
inhibitors of HIV-1 RT. AZT: 3'-azido-2',3'-dideoxythymidine; d4T:
2',3'-didehydro-2',3'-dideoxythymidine; ddI: 2',3'-dideoxyinosine;
ddC: 2',3'-dideoxycytidine; 3TC: 2',3'-dideoxy-3'-thiacytidine;
PMEA: 9-(2-phosphonylmethoxylethyl)adenine.
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(b) Chemical structures of representative nonnucleoside inhibitors of HIV-1 RT.

Nevirapine: 11 -cyclopropyl-5,11 -dihydro-4-methyl-6H-dipyrido (3,2-b:2'3'-e)(1,4)
diazepin-6-one; -APA: anilinophenylacetamine; TIBO:
tetrahydroimidazo-(4,5,1-jk)
(1,4)-benzo-diazepin-2(1H)-one and thione;
pyridinones; HEPT: 1-{(2-hydroxyethoxy)
methyl}-6-(phenylthio)thymine;
BHAP: bis(heteroaryl)piperazine; TSAO:
{2',5'-bis-O-(tert-butyldimethylsilyl)}-3'
-spiro-5''-(4''-amino-1",2"-oxathiole)-2",
2"-dioxide; L-737,126: 5-chloro3-(phenylsulfonyl)indole-2-carboxamide; TBA:
1-(2',6'-difluoro-phenyl)-1H,3H-thiozolo -(3,4-a) -benzimidazole;
quinoxaline S-2720: 6-chloro-3,3-dimethyl-4-(isopropenyloxycarbonyl)
-3-4-dihydroquinoxalin-2(1H)-thione.
Document
dine (3TC), and 2',3'-didehydro-2',3'-dideoxythymidine (d4T), have been widely used in the treatment of
HIV-1 infections [57]. However, the effectiveness of these drugs is limited by their cytotoxicity and the
rapid emergence of drug-resistant viral strains [5,812]. Nonnucleoside inhibitors, e.g., the HEPT
derivatives [13], TIBO derivatives [14], nevirapine [15], pyridinones [16], BHAP derivatives [17], TBA
derivatives [18,19], TSAO derivatives [20], -APA [21], and quinoxalines (HBY) [22,23], are potent
inhibitors of HIV-1 RT (see reviews [5,11,12]). While these inhibitors differ considerably in chemical
structure, all of them are quite specific for HIV-1 RT and inhibit neither HIV-2 RT nor variety of
cellular polymerases. Challenging a virus with these drugs
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Figure
Continued
rapidly selects viral strains containing drug-resistance mutations [9,24,25]. HIV-1 viral variants are
known whose RT is resistant to all of the currently available drugs/inhibitors (see reviews
[5,9,11,12,26,27]). In some cases, drug-resistant variants can be selected in very short periods of time
[9], a consequence of the high viral load and rapid turnover of viral populations in infected individuals
[2830]. A better understanding of how these viral variants confer resistance should provide insight into
the limitations of their genetic flexibility. In the past few years, substantial progress has been made in
understanding the three-dimensional structure of HIV-1 RT. This paper will discuss the recent
biochemical, genetic, and clinical data of HIV-1 RT in the context of the crystal structure of HIV-1 RT
and prospects for development of more effective inhibitors of HIV-1 replication.
II. Three-Dimensional Structures of HIV-1 RT
Three-dimensional crystal structures of HIV-1 RT have been determined both for the unliganded form of
the protein and for complexes with either template-primer substrate or nonnucleoside inhibitors (Figure
2 and Table 1). Structures of HIV-1 RT have been determined in complexes with a series of NNRTIs,
including nevirapine [3133], 1051U91 (a nevirapine analog) [33], -APA R95845 [34], -APA
R90385 [33], HEPT [33], 8-Cl TIBO (R86183) [35], and 9-Cl TIBO (R82913) [36,37]. The structure of
HIV-1 RT in a ternary complex with a 19-mer/18-mer double-stranded DNA (dsDNA) template-primer
and an antibody Fab fragment has been described [38]. In addition, structures of unliganded HIV-1 RT
have also been solved in multiple crystal forms [3943]. The structure of a polypeptide corresponding to
the fingers and palm subdomains of the HIV-1 RT polymerase domain has also been determined [44].
HIV-1 RT is an asymmetric heterodimer consisting of the p66 (66 kDa) and p51 (51 kDa) subunits. The
N-terminal 440 residues of the p66 subunit constitute the polymerase domain and the C-terminal 120
residues of p66 form the RNase H domain; the p51 subunit has the same amino acid sequence as the
polymerase domain of the p66 subunit [1,2]. The polymerase domain of the p66 subunit has been
likened to a human right hand. On this basis the subdomains of both p66 and p51 have been designated
as fingers, palm, thumb, and connection (Figure 2) [31,38]. In the p66 subunit, the fingers, palm, and
thumb subdomains form a large cleft that can accommodate the DNA substrate. The polymerase active
site, which contains three strictly conserved aspartic acid residues (Asp110, Asp185, and Asp186), is
located in the DNA-binding cleft and is part of the p66 palm subdomain (Figure 3) [31,38]. In the p51
subunit, however, the thumb is rotated away from the fingers and the connection subdomain is folded
over onto the palm subdomain between the fingers and thumb subdomains. As a
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Table 1 Crystal Structures of HIV-1 Reverse Transcriptase
PDB entry
HIV-1 RT Structure
Resolution
Range ()
R-Factor/Free R-Factor
(%)
Data Completeness
(%)
Temperature
(C)
References
85
-165
41
21.9
89.5
-173
42
1hmv
unliganded RT
6.0-3.0
25.4/29.7
1rtj
unliganded RT
25.0-2.35
1dlo
unliganded RT
8.0-2.7
23.0/33.6
99.5
-165
43
1hmi
RT/DNA/Fab
15.0-3.0
26.0
88.1
-10
38
3hvt
RT/nevirapine
8.0-2.9
26.6
95.6
-165
31,32
1vrt
RT/nevirapine
25.0-2.2
18.6
87.1
16
33
1rth
RT/1051U91
25.0-2.2
21.4
81.4
16
33
1hni
RT/-APA (R95845)
10.0-2.8
25.5/36
78.5
-15
34
1vru
RT/-APA (R90385)
25.0-2.4
18.7
86.5
16
33
1hnv
RT/8-Cl TIBO (R86183)
10.0-3.0
24.9/35.6
81
-10
35
1rev
25.0-2.6
22.4
80.7
-173
36
1tvr
10.0-3.0
25.9
72
-165
37
1rti
RT/HEPT
25.0-3.0
23.6
86.3
14
33
1hrh
RT (RNase H domain)
20.0-2.4
20.0
93.1
94
1rdh
RT (RNase H domain)
20.0-2.8
21.5
95.6
95
1har
RT (fingers and palm subdomains)
7.0-2.2
20.8/27.0
96
44
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Figure 2
Ribbon diagrams of the three types of HIV-1 RT crystal structures. (a) Structure of the HIV-1 RT/DNA/Fab ternary complex
[38]. The bound nucleic acid is shown as double-stranded helices with template strand in black and primer strand in gray. The Fab
fragment is not shown. (b) Structure of the HIV-1 RT complexed with the NNRTI TIBO R86183 [34]. For the sake of clarity, the bound
TIBO inhibitor is shown as an atomic model. (c) Structure of unliganded HIV-1 RT [41,43]. (d) A schematic diagram showing the
resemblance of the HIV-1 RT p66 subunit to a human right hand. The RNase H domain, which has no counterpart for a human hand, is
shown as an oval below the thumb. When a template-primer binds to HIV-1 RT, the fingers, palm, and thumb subdomains of p66 form a
large cleft to bind the DNA. The polymerase active site (shown as a small circle) lies at the bottom of the DNA-binding cleft. The
NNRTI binds in the highly hydrophobic NNIBP (shown as a large circle), which is located in the vicinity of the polymerase active site.
The p66 thumb subdomain in the NNRTI-bound HIV-1 RT structures is in an upright position extended beyond that observed in the
structure of RT with bound DNA. In the absence of any bound nucleic acid or NNRTI, however, the p66 thumb folds down into the
DNA-binding cleft (shown as dashed drawing).
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Figure
Continued
consequence, the p51 submit has no cleft for binding nucleic acid substrates and hence no polymerase
activity.
There is considerable evidence showing that HIV-1 RT is quite flexible and that this flexibility is
essential for DNA polymerization. Comparisons among DNA-bound, inhibitor-bound, and unliganded
HIV-1 RT structures provide a demonstration of the enzyme's flexibility. When a DNA template-primer
binds to HIV-1 RT, structural elements of the fingers, palm, and thumb subdomains of the p66 subunit
form a clamp-like structure that holds the nucleic acid (Figure 2) [38]. The template-primer substrate
interacts with amino acid residues of the fingers, palm, and thumb subdomains, especially in the regions
denoted as primer grip and template grip, believed to position the template-primer precisely relative
to the polymerase active site [38]. The primary contacts between the template-primer and the protein are
along the sugar-phosphate backbone of the DNA and thus are not sequence-specific [38]. In the absence
of nucleic acid template-primer or NNRTI, the thumb subdomain of p66 is folded down into the DNAbinding cleft and lies near the fingers subdomain (Figure 2) [40,41,43]. As a consequence, the DNAbinding cleft is closed. However, even in the absence of a bound nucleic acid, binding of an NNRTI
induces both short-range and long-range structure distortions, including a hinge-like movement near the
base of the p66 thumb that constrains the p66 thumb in a conformation that is extended beyond the
upright
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Figure 3
Overall structure of HIV-1 RT p66/p51 heterodimer [38] showing the locations
of the major target sites for anti-HIV-1 RT inhibitors. The NRTIs target the dNTP-binding
site/the polymerase active site (shown as a small striped circle) that lies at the
floor of the DNA-binding cleft. The NNRTIs bind to the NNIBP (shown as a large
dotted circle), which is near, but distinct from, the polymerase active site. The RNase H
domain is located at the C-terminal of the p66 subunit. The RNase H catalytic site
(shown as a medium circle) is an attractive target site for anti-HIV-1 drugs. The HIV-1
RT p66/p51 heterodimer interface is shown as a dashed line. Since HIV-1 RT functions
as a heterodimer, any inhibitors that could interfere with the dimerization process might
also be potential drugs for treating HIV-1 infection.
position of the thumb observed in the HIV-1 RT/DNA/Fab structure [31,3337,43].

In the unliganded structure of HIV-1 RT reported by Esnouf et al. [42], the p66 thumb subdomain is in
an upright conformation different from that observed in the other unliganded HIV-1 RT structures but
similar to that found in the DNA-bound HIV-1 RT and NNRTI-bound HIV-1 RT structures. Esnouf et
al. [42] contend that the upright conformation of the p66 thumb subdomain in their unliganded RT
structure is appropriate for unliganded HIV-1 RT and that the binding of an NNRTI does not affect the
conformation of the p66 thumb. However, we believe that the conformation of the p66 thumb in the
Esnouf et al. structure may well be a result of the method used to prepare the
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crystals, which were produced by soaking out a weakly bound NNRTI (HEPT) from pregrown crystals.
When the crystals were grown in the presence of HEPT, the p66 thumb was presumably in an upright
position similar to that seen in all of the known NNRTI-bound HIV-1 RT structures. As the weakly
bound HEPT diffused out, the molecular packing arrangement may have constrained the position of the
p66 thumb subdomain. As a consequence, this unliganded HIV-1 RT structure may represent an
intermediate between the other unliganded structures and the structure of HIV-1 RT with a bound
NNRTI. It is evident that the p66 thumb subdomain has considerable flexibility and can adopt
substantially different conformations during the binding of template-primer or inhibitors and,
presumably during DNA polymerization as well.
III. Polymerase Active Site of HIV-1 RT and the NRTIs
Polymerization of DNA by HIV-1 RT involves a sequential stepwise binding of the template-primer and
deoxynucleoside triphosphate (dNTP) substrates at the polymerase active site [45,46]. The incoming
dNTP is covalently linked via the -phosphorus to the 3'-oxygen of the primer strand, accompanied by
the release of pyrophosphate. An essential requirement for the polymerization reaction is the presence of
a 3'-OH group at the end of the primer strand. Nucleoside analogs contain a modified sugar moiety in
which the 3'-OH group is replaced by another group (e.g., hydrogen, halogen, or azido) (Figure la). To
exert their antiviral activity at the level of RT, the NRTIs must be phosphorylated successively to the 5'monophosphate, 5'-diphosphate, and 5'-triphosphate forms by a series of kinases. Once the NRTI is
converted to the triphosphate form and interacts with RT, it can inhibit polymerization in two possible
ways. One possibility is that the NRTI binds preferentially to the dNTP-binding site and competitively
inhibits the binding of natural dNTPs. Another possibility, which seems to be the predominant mode of
inhibition, is that the NRTI is incorporated into the growing chain and acts as a terminator of chain
elongation. Once an NRTI is incorporated, no additional nucleotides can be added to the DNA chain
since the primer terminal 3'-OH group (the site of phosphodiester bond formation) is absent.
Phosphorylation is a crucial step in the intracellular metabolism of the NRTI and often is a limiting
factor for the antiviral activity of the NRTI [47]. In an attempt to bypass the first phosphorylation step,
several acyclic nucleoside phosphonates have been developed in which the sugar moiety of normal
NRTIs is replaced with an acyclic phosphonate group, such as 9-(2-phosphonylmethoxyethyl)adenine
(PMEA), (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA), and (S)-9-(3-fluoro-2phosphonylmethoxyethyl) adenine (FPMPA) (Figure la) (see reviews [5,11]). These acyclic nucleoside
phosphonates are dideoxynucleoside
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monophosphate analogs which can easily be converted to the active triphosphate form by adding two
additional phosphates [48,49], and can inhibit HIV replication [50,52].
Analysis of the structure of the HIV-1 RT/DNA/Fab complex showed that the dNTP-binding site is
composed of both protein and nucleic acid. In addition to the 5'-terminus of the template nucleotide and
three carboxylate residues (Asp110, Asp185, and Asp186), the amino acid residues Asp113, Tyr115,
Phe116, Gln151, Phe160, and possibly Met184 and Lys219 form part of the putative dNTP-binding site
[12,53] (Figure 4 and Table 2). It is important to realize that the precise composition, position, and
conformation of the template-primer can influence the recognition and incorporation of incoming
nucleotides at the polymerase catalytic site. In the wild-type HIV-1 RT, the dNTP-binding
Figure 4
Stereoview of the polymerase active site of HIV-1 RT [38]. The amino acid
residues that compose the putative dNTP-binding site, including the three catalytically
essential aspartic acids, are shown with side chains. The double-stranded nucleic acid is
shown with the atomic model in the HIV-1 RT/DNA/Fab complex. The dNTP-binding
site consists of structural elements from both protein and nucleic acid. The precise
composition, position, and conformation of the template-primer can affect the recognition of
incoming dNTPs at the polymerase active site.
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Table 2 HIV-1 RT Amino Acid Residues Composing the Putative dNTP-Binding Sites and the Locations of NRTIResistance Mutations (see also [12])
dNTP-Binding Site
Nucleoside Drug-Resistance Mutation Site
Residue
Location
Mutation
Location
Possible Effects
Asp110
Met41Leu
template binding
55
Asp113
6-C loop
Ile50Thr
unclear
154
Try115
Lys65Arg
34
template binding
154
Phe116
Asp67Asn
34
template binding
155
Gln151
8-E
Thr69Asp
34
template binding
156
Phe160
Lys70Arg
34
template binding
155
Asp185
910
Leu74Val
template binding
Asp186
910
Val75Thr
template binding
163
Lys219
11
Glu89Gly
dsDNA binding
157
Tyr115Phe
dNTP binding
170
Ile135Thr
78
unclear
Gln151Met
8-E
dNTP binding
Met184Val, Ile
910
dNTP-binding/fidelity
Thr215Tyr, Phe,
Cys
11
indirect effect/dNTP
binding
Lys219Gln
11
dNTP binding
References
158,159
154,160,161
155,162
155
site can accommodate both the normal dNTP substrates and dideoxynucleoside analogs. The majority of
mutations that confer resistance to NRTIs are not located at the dNTP-binding site; however, they
appear to influence the geometry of the dNTP-binding site indirectly in a way that permits RT to
discriminate between a normal dNTP and a modified nucleoside triphosphate (see discussion in the next
section).
Document
Analysis of various HIV-1 RT structures has revealed an unusual -turn geometry for the YMDD motif
at the polymerase catalytic site of p66 [33,34,4143]. The energetically unfavorable main chain
conformation of Met184 (torsion angles ~60 and ~-120) is stabilized by a hydrogen bond of its
carbonyl oxygen to the side chain of either Gln182 [33,41,43] or Gln161 [42]. It has been suggested that
this novel -turn geometry might be required to position the aspartic acids in precisely the correct way
for catalysis [41].
IV. Mechanism of NRTI-Resistance Mutations
Development of resistance to NRTIs has been a major problem with clinical use of these drugs. Careful
analysis of mutations that confer resistance to different
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NRTIs in light of available structural data might provide information that could be used in the
development of improved NRTIs that are more effective against the commonly observed NRTI-resistant
HIV-1 variants. A viable drug-resistant RT mutant should be able to recognize and incorporate normal
nucleoside triphosphate, yet reject a nucleoside analog. The only difference between normal nucleotide
substrates and the NRTIs is the modification of the sugar moiety. This alteration may affect sugar
puckering and the conformation of the glycosyl bond. Recognition of these differences could render the
triphosphate form of the nucleoside analog a good substrate for wild-type RT but a poor substrate for a
drug-resistant variant of RT.
Structural analysis of HIV-1 RT has shown that most of the NRTI-resistance mutations are not located
close to the putative dNTP-binding site and are unlikely to have a direct impact on the binding of dNTP
analogs (Figure 5 and
Figure 5
A close-up view showing the relative locations of the commonly identified
drug-resistance mutations for NRTIs (in dark-gray) and for NNRTIs (in light-gray) with
respect to the bound DNA. Most of the NRTI-resistance mutations are not located at the
putative dNTP-binding site, but are at positions to have potential interactions with the
nucleic acid template-primer. Conversely, all the NNRTI-resistance mutations are
clustered around the NNIBP and have direct contacts with NNRTIs or have direct effect on
inhibitor binding.
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Table 2) [12,54]. For example, none of the five mutations, in HIV-1 RT Met41Leu, Asp67Asn,
Lys70Arg, Thr215Tyr, and Lys219Gln [55] (Table 2) consistently associated with resistance to AZT,
are at locations close to the dNTP-binding site. However, most (but not all, c.f. [56]) biochemical studies
have failed to show that recombinant HIV-1 RT enzymes containing these mutations are more resistant
to inhibition by AZT triphosphate than the wild-type HIV-1 RT [27,57,58]. Other mutations that confer
resistance to NRTIs have been identified at positions 50, 65, 69, 74, 75, 89, 115, 135, 151, and 184 of
HIV-1 RT (Figure 5 and Table 2). Most of these mutations do not lie close to the dNTP-binding site
(Met184Val/Ile are the exception), but instead are located at positions where they could interact with the
nucleic acid template-primer [12,59,60]. Biochemical data have shown that only when the 5'-template
extension length is greater than three nucleotides does the wild-type RT begin to incorporate
dideoxynucleotides effectively [54]. If the template extension is less than three nucleotides in length,
wild-type HIV-1 RT is resistant to dideoxynucleotides. On the other hand, HIV-1 RT variants containing
the mutations Leu74Val or Glu89Gly did not readily incorporate dideoxynucleotides either with short or
long template extensions [54]. Based on both structural and biochemical data, it was proposed that
mutations that cause HIV-1 RT to have reduced sensitivity to NRTIs exert their effects via interactions
with the nucleic acid template-primer, which consequently alter the geometry of the polymerase active
site [54]. It has been suggested that mutations that confer resistance to foscarnet might use a similar
mechanism [61]. One possible exception to this mechanism might be the mutations of Met184Val and
Met184Ile (see review [27]). Part of the highly conserved YMDD motif, Met184 is adjacent to residues
Asp185 and Asp186, which are two of the three catalytically essential aspartic acid residues at the
polymerase active site. In addition, Met184 appears to interact with the ribose moiety of the 3'-terminal
nucleotide of the primer strand [12,38,53] (Figure 4). Therefore, mutations at this position could affect
interactions with the incoming dNTP directly and/or alter the positioning of the nucleic acid. These
mechanisms are not mutually exclusive and which mechanism is responsible for resistance has not yet
been resolved [62]. There are two recent reports suggesting that the Met184Val mutant HIV-1 RT has
approximately three-fold higher fidelity than the wild-type enzyme [63,64]. Based on these data, it was
suggested that this increase in fidelity might reduce the overall rate of generation of viral variants in
patients treated with 3TC or other dideoxynucleosides [64]. However, owing to both theoretical and
technical problems with these analyses, these conclusions are controversial. Determination of crystal
structures of both wild-type and mutant HIV-1 RT complexed with individual NRTIs in the presence of
a variety of template-primers and/or dNTP substrates should provide a better understanding of the
mechanisms of dNTP selection and drug resistance.ed at Arial for catalysis [41].
IV. Mechanism of NRTI-Resistance Mutations
Development of resistance to NRTIs has been
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V. Drug Design Targeting at the Polymerase Active Site

All of the existing NRTIs contain a modified sugar moiety that lacks the 3'-OH group that is essential
for incorporation of the next nucleotide. Modification can also be made on other functional groups such
as the base and the triphosphate moieties. It may be worthwhile to try to alter the base moiety of the
NRTIs to produce compounds that will be more specific to HIV-1 RT (i.e., less cytotoxic to normal
cellular polymerases) and more effective against both wild-type or drug-resistant viral variants.
Structure-activity analysis indicates that the pyrimidine moiety of the NRTIs can be modified at the C5
position. An AZT derivative that has a 3'-azido group on the sugar moiety and a methyl group at the C5
position of the pyrimidine moiety showed potent antiviral activity [65]. Substitution of the methyl group
with a chlorine atom at position C5 of AZT results in a compound that has strong anti-HIV-1 activity
[66]. Other possible substitutions at the C5 position include other halogen atoms or an ethyl group.
Another possible drug-design strategy would be to devise compounds that can interface with the binding
of the metal ions (Mg2+ or Mn2+) at the polymerase active site. Metal ions appear to be important in
DNA polymerase catalysis. Based on the structural and biochemical data, a two-metal dependent
mechanism of polymerization has been postulated [53,67,68] that is similar to that proposed for other
DNA polymerases [6971]. In this model, the metal ions mediate interactions between the three
catalytically essential aspartic acid residues (Asp100, Asp185, and Asp186) and the -, -, and phosphates of the incoming dNTP and promote the nucleophilic attack on the -phosphate by the
oxygen atom of the 3'-OH group of the primer strand. In the structure of the fingers and palm
subdomains of the RT of Moloney murine leukemia virus (MuLV), a single Mn2+ ion was found bound
to the two aspartic acid residues at the polymerase active site [72]. In the structure of the unliganded
HIV-1 RT, an electron density peak was located at the polymerase active site with a good coordination
geometry to the O1 atoms of both Asp185 and Asp186 [43]. This electron-density peak is in a position
similar to that of the Mn2+ ion observed in the MuLV RT structure. It is possible that this position
corresponds to a Mg2+ ion-binding site [43]. It might be useful to design inhibitors that would influence
the metal-ion coordination using either computer-based calculations (such as DOCK [7375]) or based
directly on an analysis of HIV-1 RT structure. Crystal structures of HIV-1 RT complexed with
Mg2+/Mn2+ ion(s) at the polymerase catalytic site in the presence of template-primer and/or dNTP
substrates would be helpful in defining the target sites of inhibitors of this type. Further studies on the
structure activity relationship of HIV-1 RT complexes with these inhibitors, if active, might ultimately
lead to a new type of HIV-1 RT drug that would not compete with the dNTP binding but would affect
the DNA polymerization mechanism.
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It is also attractive to consider developing agents that bind to the HIV-1 RT polymerase active site but
are not nucleoside analogs. Since the amino acid residues at the dNTP-binding site are highly conserved,
viral variants resistant to such inhibitors may be significantly impaired in their polymerase activity.
However, there is a good chance that drug resistance could result from mutations in HIV-1 RT that
influence the precise positioning of template-primer [54]. Initial attempts to use this approach starting
from the crystal structure of the HIV-1 RT/DNA/Fab complex [38] (Ding, et al., in preparation) have
uncovered some interesting lead compounds (Kuntz, Kenyon, Arnold, Hughes, et al., unpublished).
VI. NNRTIs and the NNIBP
Nonnucleoside RT inhibitors (NNRTIs) constitute the other major class of HIV-1 RT inhibitors. Many
structurally distinct families of NNRTIs have been identified, including HEPT [13], TIBO [14],
nevirapine [15], BHAP [17], TBA [18,19], TSAO [76], -APA [21], pyridinones [16] and quinoxalines
(HBY) [22,23] (Figure 1b). However, development of drug resistance is a major problem when NNRTIs
are used to treat AIDS patients. An ideal drug should be able to block replication of all viable strains of
HIV-1, but should not inhibit normal cellular enzymes. In this regard, the known NNRTIs may be too
specific. While these inhibitors do not inhibit cellular polymerases, they are also inactive against HIV-2
RT (which can be viewed as an extreme variant of HIV-1 RT). In addition, drug-resistant variants of
HIV-1 RT emerge rapidly in the presence of most inhibitors. In contrast, the NRTIs inhibit a broad
spectrum of polymerases including the host cellular polymerases. Though it appears to be more difficult
for the virus to evade NRTIs than NNRTIs (in general, it takes longer for the virus to develop resistance
to NRTIs than NNRTIs), NRTI toxicity is a serious problem.
Structural and biochemical studies have shown that all NNRTIs bind in a highly hydrophobic pocket in
the p66 subunit, located approximately 10 away from the polymerase active site (Figures 2 and 3)
[31,3337]. Nevertheless, in all known structures of HIV-1 RT/NNRTI complexes, the bound NNRTIs
have not been found to have any direct interactions with residues that compose the putative dNTPbinding site. The nonnucleoside inhibitor binding pocket (NNIBP) contains primarily amino acid
residues from the 56 loop (Pro95, Leu100, Lys101, and Lys103), 6 (Val106 and Val108), the
910 hairpin (Val179, Tyr181, Tyr188, and Gly190), and the 1213 hairpin (Phe227, Trp229,
Leu234, His235, and Pro236) of the p66 palm subdomain, and 15 (Tyr318) of the p66 thumb
subdomain, as well as the 78 connecting loop (Glu138) of the p51 fingers subdomain (Figure 6 and
Table 3).
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Figure 6
Superposition of the NNRTIs in the HIV-1 RT/TIBO complex [35], HIV-1 RT/-APA complex [34], and HIV-1
RT/nevirapine complex [32]. The side chains are shown for those amino acid residues that have close contacts with bound inhibitors
and the three catalytically essential aspartic acids in the HIV-1 RT/TIBO complex. Most of the amino acid residues that form the
NNIBP are hydrophobic. Though the NNRTIs are chemically and structurally diverse, the bound NNRTIs all assume a strikingly
common butterfly-like shape.
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Table 3 HIV-1 RT Amino Acid Residues Defining the Nonnucleoside Inhibitor-Binding Pocket (NNIBP) and the
Locations of NNRTI-Resistance Mutations (see also [12])
NNIBP
Residues
Location
Mutation
Possible Effects
References
Pro95
Ala98
56
Gly
less bulky
9,164
Leu100
56
Ile
-branch
9,164-166
Lys101
56
Glu
charge change
Lys103
56
Asn, Gln
charge loss, less bulky
9,24,164
Val106
Ala
less bulky
9,58,165
Val108
Ile
bulkier
Glu138
78(p51)
Lys
charge change
166
Val179
Glu, Asp
charge gain, bulkier
164
Tyr181
Cys, Ile
aromaticity loss, less bulky
24,58,164
Tyr188
10
His, Cys, Leu
aromaticity loss, less bulky
9,167
Gly190
10
Glu
charge gain, bulkier
Phe227
12
Leu228
12
Phe
aromaticity gain, bulkier
133
Trp229
12
Glu233
13
Val
169
Leu234
13
Pro236
1314
Leu
increase flexibility, bulkier
133
Lys238
14
Thr
133
Tyr318
15
164
9,164
9,22,168
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Most of the amino acid residues that form the binding pocket are hydrophobic and five of them are
aromatic residues. The hydrophobic interactions of the side chains of these residues, especially Tyr181,
Tyr188, and Trp229, with the hydrophobic moieties of the NNRTIs appear to be important for inhibitor
binding [3234,59]. Since most of the NNRTIs also contain polar group(s), they have the potential to
form hydrogen bonds with surrounding amino acid residues either directly or via water bridges [3336].
In the structures of both liganded HIV-1 RT and the HIV-1 RT/DNA/Fab complex, there are two small
surface depressions at the base of the NNIBP that are the putative entrances to the pocket [34,43]. One
surface depression is located at the p66/p51 heterodimer interface and is composed of amino acid
residues Leu100, Lys101, Lys103, Val179, Tyr181, and Tyr188 of p66, and Glu138 of p51 [34]. This
putative entrance is narrow compared to the size of the NNRTIs. Another surface depression has been
found at the location near the base of the p66 thumb subdomain between two adjacent structural
elements: the 56 connecting loop (Lys101 and Lys103) and the 1314 hairpin (Pro236 and
Leu238) [43]. Since this site is also exposed to solvent, an NNRTI could approach the NNIBP from it.
How-
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ever, once an NNRTI is bound to RT, only the first putative entrance remains accessible; the second
disappears due to the conformational change and repositioning of the 12-13-14 sheet [43].
It is evident, from comparison of the various HIV-1 RT structures that the NNIBP has a highly flexible
structure that apparently allows the enzyme to accommodate various types of NNRTIs with different
shapes and sizes. Despite apparent differences in the structures of the bound inhibitors, comparison of
structures of several HIV-1 RT/NNRTI complexes revealed remarkable similarity in the geometry of
both the bound inhibitors and the NNIBP [33,35]. All these chemically diverse NNRTIs assume a
strikingly similar butterfly-like shape (Figure 6). The binding of NNRTIs in the NNIBP can be likened
to a butterfly sitting on the 6-10-9 sheet and facing toward the putative entrance to the pocket. The
angle between the two wings of the butterfly is approximately 112115 in the TIBO, -APA, and
nevirapine complexes [35]. This angle might be critical in inhibitor binding and could be a crucial
parameter in the design of new NNRTIs. There are many other NNRTIs that are significantly larger or
smaller in size than -APA, TIBO, or nevirapine. It is very likely that the NNIBP can adopt other
conformations. For example, BHAP appears to be too large to fit into the NNIBP in any of the reported
HIV-1 RT/NNRTI complexes. The NNIBP in the HIV-1 RT/BHAP complex would need to be
significantly larger than that observed in the structures of the known HIV-1 RT/NNRTI complexes. It is
possible that the BHAP inhibitor may not conform to a butterfly-like shape. This underscores the
importance of solving crystal structures for as many HIV-1 RT/NNRTI complexes as possible.
Additional structural and biochemical data for other HIV-1 RT/NNRTI complexes should provide the
insight needed to define the limits of the flexibility of HIV-1 RT in the NNIBP region.
VII. Process of NNRTI Binding
In crystal structures of unliganded HIV-1 RT [40,41,43] and of HIV-1 RT/DNA/Fab complex [38], the
NNIBP does not exist (although a small cavity is found in the region of the NNIBP proximal to the
polymerase active site in the unliganded HIV-1 RT structure described by Esnouf et al. [42]). In these
structures, the side chains of Tyr181 and Tyr188 in p66 point away from the polymerase active site and
toward the hydrophobic core. However, in the HIV-1 RT/NNRTI complex structures, the side chains of
Tyr181 and Tyr188 point toward the polymerase active site, and the side chain of Tyr181 is in a position
that prevents Trp229 from occupying the position it has in the unliganded or DNA bound HIV-1 RT
structures. Binding an NNRTI also moves the 12-13-14 sheet away from the hydrophobic core
[34,35,37]. These conformational
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changes create the space in the pocket required to accommodate inhibitors. In other words, significant
conformational changes occur during the process of inhibitor binding that lead to the formation of the
NNIBP [3335]. This observation also underscores the importance of determining structures of HIV-1
RT with and without bound inhibitors.
An obvious question is, what forces initiate this series of conformational changes during NNRTI
binding? One possibility is the contacts between the inhibitor and the protein. Though the NNIBP is
hydrophobic, there are three hydrophilic amino acid residues (Lys101 and Lys103 of p66, and Glu138 of
p51) at the rim of the putative entrance(s) to the pocket. The flexible and polar side chains of these
residues could assist in steering an inhibitor into the pocket and/or could block the bound inhibitor from
escaping out of the pocket. Mutagenesis studies have shown that these three residues are important in
the binding of NNRTIs. Though the importance could be explained in terms of the interactions between
these residues and the bound inhibitor in the final complexes, interactions at the initial stages of inhibitor
binding might also be crucial. The flexible and polar side chains of these residues might help in directing
the inhibitor toward the entrance to the pocket via electrostatic interactions, in part by replacing the
original hydrogen bonds between the drug and the solvent molecules. Any initial energy gains from such
polar interactions could potentially be replaced by hydrogen bonds or other types of interactions
between the inhibitor and alternative residues as the inhibitor moves deeper into the binding pocket. In
addition, significant portions of the aromatic rings of both Tyr181 and Tyr188 are exposed at the bottom
of the surface depression and offer the potential for early - interactions with the inhibitor. This type of
- interaction might also play an important role in the initial approach of inhibitors to the binding
pocket. This hypothesis may provide a kinetic explanation for the ineffectiveness of NNRTIs against
viral strains of HIV-1 that carry nonaromatic amino acids at positions 181 and 188. As the solvated
inhibitor approaches the enzyme and proceeds to enter the binding pocket, most of the water molecules
of solvation are lost. The few water molecules that remain in the NNRTI-bound complex are typically
located at the entrance to the pocket, forming water bridges between the inhibitor and one or two polar
residues around the entrance [33,35,36]. Once the inhibitor is in place, the surface residues close down
around the drug preventing it from escaping by effectively sealing the entrance to the pocket.
VIII. Mechanisms of Inhibition by NNRTIS
Based on structural, biochemical, and genetic data several hypotheses have been postulated about the
mechanism(s) of inhibition of HIV-1 RT by NNRTIs. It is
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now clear that the binding of NNRTIs provokes substantial conformational changes in both secondary
structural elements and in side chains of residues in the NNIBP. These conformational changes in the
NNIBP could directly or indirectly affect the precise geometry and/or mobility of the nearby polymerase
catalytic site, especially the highly conserved YMDD motif and/or the divalent metal ions
[31,33,34,42,68]. The binding of NNRTIs appears to lock the flexible hinge-like structure between the
palm and thumb subdomains and restrict mobility of the thumb subdomain, placing constraints on the
geometry of the DNA-binding cleft [12,31,34,43]. The primer grip (i.e., 12-13-14 sheet), which
has close interactions with the 3'-terminus of the primer strand [38], forms a part of the NNIBP and is
involved in the binding of NNRTIs. It has become apparent that binding of NNRTIs can substantially
alter the conformation of the primer grip; this could affect the precise positioning of the primer strand
relative to the polymerase active site [34,37]. Displacement of the primer grip by NNRTI binding could
lead to repositioning of the primer terminus. This could explain the observation that dNTP binding is
largely unaffected by NNRTI binding while the rate of the chemical step of DNA polymerization is
reduced [77]. Long-range distortions of the HIV-1 RT structure by NNRTI binding can potentially
account for NNRTI inhibition of polymerization [39,41,43] and alteration of RNase H cleavage
specificity [43,78]. These possible mechanisms are not mutually exclusive and the binding of inhibitors
might have multiple influences on HIV-1 RT polymerization. The exact mechanism(s) of inhibition is
still under investigation.
IX. NNRTI-Resistance Mutations
Analyses of the crystal structures of HIV-1 RT complexed with various NNRTIs have indicated that
amino acid residues whose mutations confer high levels of resistance to NNRTIs [9,11,12,26,27] are
located close to the bound inhibitors (Figure 5 and Table 2). Subunit-specific mutagenesis studies have
confirmed that mutations that confer resistance to the NNRTIs act directly through the change in the
NNIBP itself [60,79]. In these studies, recombinant HIV-1 RTs that contained amino acid substitutions
only in the p66 subunit were resistant to NNRTIs, while those containing the same amino acid
substitutions only in the p51 subunit remained susceptible to the drugs. There is one exception: the
amino acid substitution of Glu138 to Lys, which confers resistance to inhibitors only when it is present
in the p51 subunit. Amino acid residue 138 is located in the 78 connecting loop of the fingers
subdomain. In the p51 subunit this residue forms a part of the NNIBP, while its counterpart in the p66
subunit is far away from the pocket [12,60].
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The mechanism(s) of resistance may depend on the specific amino acid change. It is likely that most
NNRTI-resistance mutations exert their effects by altering interactions between protein side chains and
the inhibitors [12,34,35]. Drug-resistance mutations that result in a decrease or increase in the size of
side chains might lead to loss of favorable contacts or steric conflicts with bound inhibitors. Mutations
that alter the local electrostatic potential, i.e., gain, loss, or inversion of charge, may change the affinity
of the NNIBP for inhibitor binding. These altered interactions could interfere with the binding of
NNRTIs to the hydrophobic pocket or conceivably could even relax the geometric distortion that the
binding of an inhibitor causes in the vicinity of the polymerase active site.
X. Design of Improved NNRTIs
Different NNRTIs, even from the same class of compounds, show remarkable variations in their ability
to inhibit HIV-1 replication and can give rise to different spectra of resistance mutations [9,11,26,27].
For example, biochemical studies showed that the 8-chloro TIBO derivative R86183 is quite potent in
inhibiting an HIV-1 strain containing the Tyr181Cys mutation, which is one of the frequently occurring
HIV-1 RT mutations that gives rise to a high level of resistance to almost all NNRTIs, including other
TIBO derivatives [80]. There are several other reports of NNRTIs that are also relatively effective in
inhibiting the HIV-1 RT Tyr181Cys variant [8184]. These results suggest that although all the
inhibitors appear to bind in the NNIBP, there are differences in their specific interactions with HIV-1
RT. Structural analyses of HIV-1 RT/NNRTI complexes and computer modeling studies confirmed that
the exact conformations of the amino acid residues forming the NNIBP appear to vary in different
complexes and that there are specific interactions between individual inhibitor and surrounding residues
[33,35,36,85]. However, these differences have not been sufficiently large to allow a successful
combination therapy to be developed using two or more of the currently available NNRTIs (discussed in
more detailed in a later section) [9,11,26,27,86]. Systematic analysis of wild-type and drug-resistant
mutant HIV-1 RT structures in complexes with various NNRTIs should provide additional insights
about constraints that could be used to optimize the design of NNRTIs. This knowledge could guide
development of more effective inhibitors for AIDs treatment.
As discussed earlier, the bound NNRTIs in HIV-1 RT complexes determined so far conform to a
common butterfly-like shape (Figure 6). A close inspection of interactions between inhibitors and
protein reveals that though
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most of the amino acid residues forming the pocket adjust their side chains to make close contacts with
the inhibitor, the inhibitor is not sufficient to fill all of the space in the pocket. There is space for
additional nonpolar, polar, or charged groups. Modification of the inhibitor would result in adjustment
of the orientation of the side chains and could improve interactions between the inhibitor and
surrounding residues such as Leu100, Lys101, Lys103, Val106, or Leu234. Inhibitors designed to have
more extensive interactions with essential elements in the pocket should minimize the chances of
selecting resistant HIV-1 RT variants. From this point of view, NNRTIs that interact with the relatively
conserved residues of the pocket, such as Trp229, Leu234, and Tyr318, may reduce the risk of
encountering resistance mutations that do not have significant costs for the enzyme. In addition,
compounds could be designed to contain functional groups (for example charged or polar groups) able
to fill more of the available space of the NNIBP and also capable of specific hydrophilic interactions
with the polar or charged side chains and/or with polypeptide backbone atoms of the NNIBP (for
example the main chain amide nitrogens and carbonyl oxygens). The hydrophilic interactions between
inhibitors and protein backbone atoms should be advantageous because mutations to any amino acid
other than proline would not affect such contacts. In the structures of HIV-1 RT/NNRTI complexes, the
bound inhibitors are located very close to the polymerase active site composed of the three catalytically
essential aspartic acids Asp110, Asp185, and Asp186. It might be useful to design compounds that have
a long and branched aliphatic group or a substituted aromatic group that could not only produce
hydrophobic interactions with Tyr181, Tyr188, and Trp229, but could also be able to interact with the
three aspartic residues or interfere with the metal ion(s) binding at the polymerase active site.
XI. RNase/H-Active Site as a Potential Drug Target Site
HIV-1 RT contains RNase H, which is responsible for degradation of viral RNA and removal of RNA
primers for minus- and plus-strand DNA synthesis (see reviews [8789]). The absolute requirement for
virus-associated RNase H function [9093] offers an additional target for antiretroviral drugs. The
RNase H domain of HIV-1 RT is located at the C-terminus of the p66 subunit (Figures 2 and 3). In
contrast to the polymerase domain of HIV-1 RT, the structure of the RNase H domain is quite similar in
all known HIV-1 RT structures and conforms quite well with the structure of the isolated HIV-1 RNase
H domain [9495]. The relative stability of the structure of the RNase H domain suggests that the RNase
H active site could be a relatively well-defined target for drug
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design. Mutagenesis studies have demonstrated the interdependence of DNA polymerase and RNase H
activities. Mutations that disrupt one of the two enzymatic activities of HIV-1 RT often also impair the
second activity [9699]. Indeed, according to the crystal structure of HIV-1 RT, the polymerase active
site and the RNase H active site are separated by approximately 1718 nucleotides [38] and the RNase
H domain has many contacts with the polymerase domain, especially with the connection subdomain of
p66 and the thumb and connection subdomains of p51 [31,38,100]. Interactions between the polymerase
domain and nucleic acid can modulate RNase H activity. Because the predominant contacts of HIV-1
RT with template-primer occur in the vicinity of the polymerase active site, precise placement of the
template strand relative to the RNase H active site may be regulated by the sequence and composition of
the template-primer. Mutagenesis experiments showed that mutations located at or near the template
grip in the polymerase domain of HIV-1 RT can have a greater effect on RNase H than on polymerase
activity [99,101,102]. It was also reported that binding of the NNRTI nevirapine alters the cleavage
specificity of RNase H [78]. Structural distortions in the position and conformation of template-primer
induced by NNRTI-binding may account for alteration of the cleavage specificity of RNase H [43].
Divalent metal ions such as Mg2+ or Mn2+ are essential for the RNase H activity [103106]. The
structure of the isolated RNase H domain crystallized in the presence of MnCl2 revealed two tightly
bound Mn2+ ions in close proximity to four catalytically essential acidic residues, Asp443, Glu478,
Asp498, and Asp549, that form the active site [94]. Biochemical data have shown that mutations of
these conserved residues could either disrupt RNase H activity or lead to a highly unstable enzyme
[107109]. Based on the crystal structures, a two-metal ion-dependent catalytic mechanism for RNase H
activity has been postulated [101], which is similar to that proposed for phosphoryl transfer reactions
catalyzed by polymerases and their associated nucleases [67,6971]. In contrast, in the structure E. coli
RNase H reported by Katayanagi et al. [111] only one Mg2+ ion was observed, and that led to the
proposal of a single metal-ion catalyzed hydrolysis [112]. Interestingly, in the structure of unliganded
HIV-1 RT reported by Rodgers et al. [41] and Hsiou et al. [43] only one Mg2+ ion was found at the
RNase H active site. The mechanism of RNase H cleavage and the exact role of metal ion(s) in the
hydrolysis and formation of phosphodiester bonds are still under investigation (see review [89]).
Very few inhibitors specifically target HIV-1 RNase H activity. Illimaquinone, a natural marine product,
was shown to preferentially inhibit the HIV-1 RNase H activity [113,114]. However, this compound
appears to react with a sulfhydryl group in the polymerase domain and not with RNase H itself. It may
be possible to use the available information on structural and biochemi-
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cal properties of the polymerase and RNase H of HIV-1 RT to design compounds that would bind at the
RNase H active site or interfere with the metal ion(s) binding and inhibit the RNase H activity of HIV-1
RT. However, for optimal utility, these compounds should selectively inhibit the RNase H activity of
HIV-1 and not the RNase H activity of the host cells.
XII. Other Possible Target Sites for HIV-1 RT Inhibitors
Both polymerase and RNase H activities of HIV-1 RT require that the enzyme be in a dimeric form
[115118] (Figure 3). The exact role(s) of the p51 subunit in the enzymatic activities of HIV-1 RT are
not yet known. The three-dimensional structure of HIV-1 RT shows that the interface between p66 and
p51 primarily involves interactions between the p66 palm and the p51 fingers subdomains, between the
p66 connection and the p51 connection and fingers subdomains, and between the RNase H and the p51
thumb and connection subdomains [34,100,119] (Figure 3). A compound that would interfere with
dimerization would be a potential candidate for an anti-AIDS drug.
As discussed earlier, the flexibility of HIV-1 RT permits the enzyme to adopt different conformations.
In the absence of bound DNA, the thumb and the fingers subdomains come together and close a major
portion of the DNA-binding cleft [40,41,43]. Synthetic oligonucleotides that could interact with the
specific or conserved regions of the DNA-binding cleft could potentially block binding of templateprimer substrates. An RNA pseudoknot has been reported to bind and specifically inhibit HIV-1 RT
[120]. Chemical modification and substitution of specific groups in RNA ligands can change the
structure of the pseudoknot, which could result in considerably more effective pseudoknot inhibitors
with high binding specificity [121]. Studies employing the phosphorodithioate analogs of the primer
sequence recognized by HIV-1 RT showed that these compounds can act as inhibitors and that inhibition
is a function of both the sequence and length of these novel single-stranded nucleic acid oligomers
[122,123].
A series of natural products, i.e., trihydroxyquinolone compounds isolated from Red Sea marine
organisms, were reported to inhibit the DNA polymerase activity of HIV-1 RT [124,125]. This type of
inhibitor appears to have a mechanism of inhibition that is different from either the NRTI inhibition
mechanism or the NNRTI inhibition mechanism. The inhibition is reversible and noncompetitive with
respect to both dNTP and template-primer [125]. This result indicates that there are other potential
binding sites for inhibitors of HIV-1 RT.
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XIII. Useful Tools in Structure-Based Drug Design

Several computer modeling algorithms have been developed for structure-based drug design. Among
them, DOCK [7375] and 3D SEARCH [126] have been successfully applied in the design of HIV-1
protease inhibitors. These programs search a target protein for invaginations, grooves, and recognition
surfaces that could bind a potential receptor molecule. Compounds complementary to the putative
receptor binding site in both shape and chemical properties can be identified through searching
databases of small molecules, such as the Cambridge Crystallographic Database, the Fine Chemicals
Directory, or other commercially available databases.
An important issue in analyzing HIV-1 RT is the flexibility of the enzyme. Comparison of structures of
unliganded HIV-1 RT and NNRTI-bound HIV-1 RT complexes has shown that the NNIBP is not
present in the unliganded form [34,41,43]. This underscores the importance of searching both the
unliganded HIV-1 RT and the HIV-1 RT complexes with inhibitors and substrates in order to identify
any potential inhibitor-binding sites.
Many other approaches have been and are being developed for computeraided design of inhibitors. For
example, pharmacophore analysis can identify the spatial arrangement of groups or atoms common to all
active inhibitor molecules and then incorporate these elements into a single molecule [127,128].
Detailed analysis of the volumes occupied by different inhibitors bound to the same binding site could
also provide new suggestions for inhibitor design. For example, the volume union of all known NNRTIs
such as nevirapine, TIBO, -APA, HEPT, and 1051U91 can be calculated. This type of analysis could
be used to screen for new NNRTIs. Since the coordinates for a number of HIV-1 RT/NNRTI complex
structures are now available in the Protein Data Bank, these approaches can be applied to the design of
new or improved NNRTIs. Given the relatively high flexibility of the NNIBP region and the diversity of
NNRTI structures, the NNIBP of HIV-1 RT could be a methodologically challenging yet extremely
important target for structure-based drug design.
XIV. Enzymatic Efficiency of Drug-Resistant HIV-1 RT Variants
Analyses of viral population dynamics indicated that, although drug resistance cannot be seen as a
positive outcome of chemotherapy, clinical progress can be made through the development of drugresistant viral variants (see review [30]).alyzed hy Arial H itself. It may be possible to use the available
information on structural and biochemi-
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Biochemical data show that HIV-1 replicates extremely rapidly in infected individuals and that the viral
load is low in the early stages of the disease because the host immune system is initially successful in
limiting viral replication [28,29]. When patients are treated with either RT and/or protease inhibitors,
wild-type HIV-1 is rapidly replaced with drug-resistant variants. In fact, even in patients who have not
received treatment with any anti-RT drugs, HIV-1 variants that contain residues corresponding to both
NRTI- and NNRTI-resistance mutations in RT can be found as minor components of the viral
population [129]. Similarly, viral variants that contain residues in protease sequence corresponding to
protease inhibitor drug-resistance mutations have also been observed in patients prior to drug therapy
(see review [130]). Enzymatic components found in a wild-type virus, such as RT or protease, are
optimized for efficient viral replication [30]. In the absence of selective pressure (drug), the wild-type
virus has a fitness advantage over drug-resistant viral variants. However, in the presence of drugs, drugresistant variants have a fitness advantage over the wild-type because the drug impairs efficiency of the
target enzyme in the wild-type virus [30].
Binding of an NRTI or an NNRTI to wild-type HIV-1 RT interferes with the polymerization reaction.
However, the presence of resistant variants in the population allows the virus to escape, and the variants
to rapidly replace the wild-type virus. Nevertheless, this escape has a price. When the optimized wildtype virus is replaced by the less fit drug-resistant variants, the relative fitness of the virus decreases. In
other words, the enzymatic efficiency of a drug-resistant HIV-1 RT variant is impaired relative to the
wild-type enzyme (see review [131]). If the enzymatic efficiency of a drug-resistant viral variant is
sufficiently impaired, the replication of the variant virus would be significantly decreased. Thus, an
antiviral drug will be useful not because it would completely stop the growth of HIV-1 but because it
selects viral variants whose replication is significantly impaired. Positive clinical benefit results from the
fact that the viral load is decreased owing to reduced replication of the variant virus. As predicted by this
model, some HIV-1 RT and protease inhibitors seem to select for relatively less fit drug-resistant
variants. For example, treatment of HIV-1 infection with HBY 097, a quinoxaline inhibitor, induces
development of an HIV-1 RT variant containing the Gly190Glu mutation that appears to have
substantially decreased polymerase activity and replicates relatively slowly [22,84]. Replacement of the
hydrogen atom of Gly190 with an acidic side chain of Glu190 in the hydrophobic NNIBP apparently
interferes with the stability of the enzyme as well as the ability of the NNIBP to bind a hydrophobic
inhibitor. The relative inefficiency of HIV-1 variant containing the Gly190Glu mutation in RT can be
viewed as a positive outcome of the selection pressure provided by this particular inhibitor. However,
most of the HIV-1 variants selected by
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currently available antiviral agents are not significantly less fit than the wild-type virus and the clinical
benefits are not obvious. In this regard, new drugs should be designed that would be intended to select
HIV-1 RT variants that are significantly less fit and do not replicate efficiently.
XV. Combination Therapy Using Multiple ANTI-HIV-1 Drugs
Monotherapy using either NRTIs or NNRTIs has led to the emergence of drug-resistant viral strains of
HIV-1. Though many drug-resistance mutations confer cross-resistance to other inhibitors belonging to
the same class, there are indications that some mutations conferring resistance to certain inhibitors are
incompatible (see reviews [5,11,131]). A multidrug clinical trial with HIV-1 infected patients has shown
that AZT resistance can be reversed by mutations that confer resistance to ddI [8]. The Leu74Val
mutation appears to suppress the effects of the Thr215Tyr mutation that confers resistance to AZT
[8,27]. The Met184Val mutation, which causes resistance to 3TC or other oxathiolane-cytosine analogs,
also appears to reverse the effects of the AZT-resistance mutations [27]. Recent clinical studies have
shown that a combination of AZT and 3TC led to a considerable decrease in viral load and a substantial
increase of CD4 cells when compared with monotherapy using AZT alone, even after emergence of the
Met184Val mutation [132]. Another example is the Pro236Leu mutation that confers resistance to
BHAP. The sensitivity of this HIV-1 RT variant to TIBO, nevirapine, and pyridinone is increased ten
fold [133]. Although the NRTIs and NNRTIs target two distinct binding sites of HIV-1 RT and lead to
different sets of resistance mutations, some of the NRTI- and NNRTI-resistance mutations also appear
to be incompatible. For example, the NNRTI-resistance mutations Leu100Ile and Tyr181Cys have been
shown to suppress the effects of some AZT-resistance mutations [11,134]. This has led to the suggestion
that a combination of anti-HIV-1 drugs would be more effective in inhibiting HIV-1 replication than
using individual drugs alone. In fact, both clinical and in vitro studies have shown that combination
therapy has considerable advantages over monotherapy. At least in some cases, the effectiveness of the
therapy increases with an increase in the number of drugs in the combination [5,135]. Combination
therapy may, in addition to increasing antiviral activity, also slow emergence of drug-resistant variants
and may have the added benefit that reducing the dosage of individual drugs can reduce toxicity. It is
generally believed that synergistic drug interactions arise from the fact that certain combinations of drugresistance mutations are particularly detrimental for the enzyme (and, by extension, the virus). This has
focused attention on
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determining the mechanism(s) underlying drug resistance and, from this understanding, to devise ways
for identifying combinations of drugs which might provoke drug-resistance mutations incompatible with
viral survival.
Several protocols have been designed for combination therapy using a variety of anti-HIV-1 drugs (see
reviews [5,11]). Combinations of different drugs that interact with the same binding site of the same
viral protein but lead to mutually antagonistic or suppressive resistance mutations have been studied
extensively, especially for the combined uses of different but structurally related NRTIs (see for
example [136138]) or NNRTIs [139141]. Combinations of drugs or inhibitors that target different
sites of the same viral protein, primarily the combination of NRTIs and NNRTIs of HIV-1 RT, show
enhanced inhibition of HIV-1 RT polymerase activity and suppression of the emergence of drugresistance mutations (for example [142146]). Experiments have also been conducted with combinations
of drugs that target different viral proteins, e.g., inhibitors of virus adsorption, virus-cell fusion, and/or
uncoating proteins have been tested in combination with protease inhibitors and/or RT inhibitors.
Combinations of AZT with the glycosylation inhibitor castanospermine [147], or with the Tat inhibitor
Ro 24-7429 [148], or with the protease inhibitor Ro 31-8959 [149] have been shown to potently inhibit
HIV-1 viral replication in vitro.
Combination therapy can increase the effectiveness of inhibition and significantly impair efficiency of
viral replication. However, both NRTI- and NNRTI-resistance mutations can affect the positioning of
the nucleic acid and/or the overall structure of HIV-1 RT [23]. These two sets of resistance mutations
can communicate with each other and can result in cross resistance. Moreover, new drug-resistance
mutations that confer cross-resistance to both NRTIs and NNRTIs can be selected, which reduce the
effectiveness of some drug combinations (see reviews [5,11,26]). Biochemical studies showed that both
HIV-1 RT mutants [150] and viral variants [151] could be obtained that are resistant to the combination
of AZT, ddI, and nevirapine. In clinical trials, treatment with AZT and ddI or AZT and ddC led to a
different spectrum of NRTI-resistance mutations [152,153]. The most notable of these new mutations is
Gln151Met, which is located at a position close to the dNTP-binding site. Structural analysis of the HIV1 RT/DNA/Fab complex suggests that the side chain of Gln151 in the wild-type enzyme may interact
with the first unpaired template nucleotide. The side chain of this residue may play a role in selecting the
correct base for the incoming nucleotide [72]. Since the RT mutant containing only the Gln151Met
mutation can confer high-level resistance to a number of NRTIs, including AZT, ddI, and ddC, it is not
clear why this mutation did not emerge in monotherapy of these NRTIs. However, Gln151 is relatively
well conserved and mutations at this position may have an unfavorable impact on HIV-1 RT.
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XVI. Perspective
Substantial progress has been made in understanding the structure and function of HIV-1 RT and in the
development of anti-HIV-1 inhibitors. However, the genetic flexibility of HIV-1 will continue to make
development of a truly effective antiviral therapy for AIDS an exceptionally difficult task. We are
beginning to understand how to circumvent drug resistance. The accumulated evidence has shown that
the ability of the virus to develop drug resistance is limited and that the drug-resistant viral variants are
less efficient than the wild-type virus. If the selection pressure provided by antiviral drugs makes the
virus pay a sufficiently high price, then the viral load can be decreased and there will be a measurable
clinical benefit. Based on a better understanding of the structure-function relationships of HIV-1 RT, we
are now coming to grips with the mechanisms of polymerization, drug inhibition, and drug resistance.
This information should make it possible to develop new or improved HIV-1 RT inhibitors that have
different properties and provoke different patterns of drug-resistance mutations. Though it is likely that
there will be no single drug which would be effective against all HIV-1 variants, we have reasons to
believe that new or improved drugs or, more likely, new drug combinations, will be designed that are
broadly effective against all of the HIV-1 variants that can grow efficiently. Detailed analysis of the
conformational changes among the various HIV-1 RT structures may reveal additional sites (in addition
to the currently known NRTI- and NNRTI-binding sites) for binding new inhibitors able to interfere
with the polymerization and/or the flexibility of the enzyme required for its activity. The considerable
physical and genetic flexibility of HIV-1 RT suggests that more effective anti-RT drugs should be
designed to target the conserved portions of HIV-1 RT that the virus cannot easily afford to change.
Such conserved elements can be identified by comparing the sequences of RTs from different
retroviruses; the functions and relative importance of these conserved elements can be determined by
mutagenesis and biochemical and structural analyses. It is our hope that application of structure-based
drug design strategies may aid in the development of novel HIV-1 RT inhibitors for a more effective
treatment of HIV-1 infection.
Acknowledgments
We thank the other members of the Arnold and Hughes laboratories and our collaborators for their
helpful discussions and assistance, including Koen Andries, Gail Ferstandig Arnold, Paul Boyer, Arthur
Clark, Jr., Paul Janssen, Jrg-Peter Kleim, Luc Koymans, Tack Kuntz, Karen Lentz, Chris Michejda,
Henri Moereels, Manfred Roesner, Marilyn Kroeger Smith, Rick Smith, Jr., and
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Chris Tantillo. The work in Edward Arnold's laboratory has been supported by Janssen Research
Foundation and NIH grants (AI 27690 and AI 36144). Research in Stephen Hughes' laboratory is
sponsored in part by the National Cancer Institute, DHHS, under contract with ABL, and by NIGMS.
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3
Retroviral Integrase: Structure as a Foundation for Drug Design
Alison B. Hickman and Fred Dyda
National Institutes of Health, Bethesda, Maryland
I. Introduction
A. Retroviral Lifecycle
The human immunodeficiency virus (HIV) is one of only a few retroviruses known to infect humans. It
is estimated that approximately twenty-two million people are now infected worldwide [1]. With only a
tiny number of exceptions, infection ultimately leads to the development of the lethal condition of
acquired immunodeficiency syndrome, or AIDS. To date, only a handful of drugs have been shown to
have any effect on the course of the disease. These are, in general, relatively ineffective at significantly
prolonging life, and drug resistance develops rapidly. Equally discouraging, vaccines have not yet been
developed to prevent infection.
The retroviral lifecycle presents several steps that can be targeted as possible sites of intervention by
inhibitors. As shown in Figure 1, when a retrovirus encounters a host cell, specific recognition between
proteins on the surface of the virus and receptors on the host cell surface leads to membrane fusion. The
viral core then enters the cell cytoplasm where the process of reverse transcription begins. The
requirement of the conversion of viral RNA to double-stranded DNA is a feature unique to retroviruses.
With the recent exception of the protease inhibitor saquinavir, ritonavir, and indinavir, the drugs
approved to date by the U.S. Food and Drug Administration (FDA) for the treatment of HIV infection
have been nucleoside analogs targeted against the viral enzyme that carries out this conversion, reverse
transcriptase.
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Figure 1
Retroviral lifecycle as summarized in Reference 67. Reprinted by
permission of Springer-Verlag Publishing Co., New York, NY.
Although details of the timing of reverse transcription, nuclear localization, and integration are not yet
clear, it is generally recognized that the movement of double-stranded viral DNA across the nuclear
membrane is followed by insertion, or integration, of the viral genome into a host-cell chromosome. The
viral DNA moves as part of a larger preintegration complex, a high-molecular-weight aggregate
whose composition has not yet been completely defined.
The end result of integration is the incorporation of the viral DNA into the DNA of the host cell. Once
there, the provirus can serve as a template for the production of mRNA, allowing for the synthesis of
viral proteins. These are assembled at the cell membrane to produce new viral particles, which then bud
off to seek out new cells to infect. The integrated viral DNA is also necessarily copied whenever the
host cell undergoes cell division. The insidious nature of
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the virus arises because, once integrated, the viral DNA can no longer be distinguished from host cell
DNA and has become a permanent fixture of the host cell genome.
B. Rationale for Drug Design Against Integrase to Fight HIV and AIDS
It has been demonstrated that the chemical steps that comprise DNA integration are carried out by the
viral protein, integrase (IN). Integrase is encoded by the 3' end of the viral pol gene, which also codes
for two other viral enzymes, the protease and reverse transcriptase. These three enzymes are initially
synthesized as part of a larger polyprotein that is subsequently cleaved by the protease to the individual
proteins.
Why is integrase a good target for drug-design efforts to prevent infection by halting the viral replication
cycle? First, integration is required for replication. In the absence of integration, the virus is unable to
continue to make copies of itself. Secondly, the enzyme that carries out integration is virally encoded,
and when the viral genome is disrupted so that functional integrase is no longer made, sustained viral
replication does not occur [2]. This demonstrates that if viral integrase can be effectively inhibited, there
is no protein encoded by the host cell that can replace it and carry out viral integration. Finally, since
mammalian cells do not have enzymes capable of integrating HIV DNA, there are no vital host cell
analogs of integrase carrying out essential reactions whose function would be blocked by integrase
inhibitors.
Effective inhibition of HIV integrase would add to the number of sites at which the virus replication
cycle can be halted. One can imagine treatment protocols in which a mixture of inhibitors, each aimed at
a different viral protein, could be administered. This is known as divergent combination therapy. As
structural details are a necessary starting point for rational drug design, we present here our recent
results on the high-resolution three-dimensional structure of the catalytic core domain of HIV-1
integrase [3]. We also review the current literature discussing integrase inhibitors and present thoughts
on ways in which knowledge of the chemical reactions carried out by integrase and its structure might
direct the development of effective inhibitors.
II. Biochemical Reactions Catalyzed By HIV Integrase
A. In Vivo Integration
Details of the initial chemical reactions that occur during HIV integration are now well understood (for
reviews, see References 4,5). Once linear double-stranded DNA is available for integration, (Figure 2a)
integrase then removes
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Figure 2
In vivo reactions carried out by HIV integrase.
two nucleotides from each 3' end of the viral DNA (Figure 2b). The two nucleotides are removed as a
dinucleotide rather than in two individual steps. The specificity for this reaction is conferred by the third
and fourth nucleotides from each 3' end, a -CA sequence that is absolutely conserved. Once two
nucleotides have been removed, leaving recessed 3' hydroxyl groups, the next step is the joining of the 3'
ends to target DNA (Figure 2c, d). This process, known as double-ended integration, occurs on opposite
strands such that the joining sites on each of the target DNA strands are separated by five base pairs. The
final step in integration is the repair of the single-stranded gaps generated by the staggered insertion of
the viral 3' ends on opposite strands; this regenerates an intact double-stranded DNA molecule (Figure
2e and f). Gap repair is probably carried out by host cell DNA repair systems.
One necessary consequence of retroviral integration is the duplication of five base pairs of host cell
DNA on either side of the integrated provirus.
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Another is the loss from the ends of the viral DNA of the original two base pairs that preceded the
conserved 3'-CA.
B. In Vitro Assays to Monitor Integrase Activity
In contrast to the in vivo reaction, concerted integration in vitro of two HIV DNA ends into a target
DNA molecule separated by a 5 base-pair stagger occurs very inefficiently. However, in vitro systems
have been developed [6,7] using recombinant HIV integrase that have allowed the chemistry of the
single-ended integration event to be studied in fine detail. It is possible and routine to use short, doublestranded synthetic oligonucleotides that mimic the viral ends to monitor the removal of two nucleotides
from 3' ends (denoted 3' processing or cutting) and the subsequent insertion of one 3' processed DNA
molecule into another (known as strand transfer or joining). Typical reactions are depicted in Figure 3.
The stereochemical mechanism of 3' processing and strand transfer has been investigated using DNA
substrates that incorporate phosphorothioate link-ages [8]. For both reactions, the introduced chiral
centers are inverted in the products, implying that the reactions occur via a one-step in-line displacement
mechanism rather than via a covalent intermediate.
A third assay of integrase activity, termed disintegration, has more recently been developed [9] that
monitors the apparent reversal of strand-
Figure 3
Reactions carried out by integrase in vitro, using short oligonucleotide substrates.
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transfer (Figure 3). While disintegration probably has no physiological significance, it has been useful in
defining aspects of integrase biochemistry.
The three in vitro activities of integrase require divalent metal ions as cofactors. The only two metals
that support these activities are Mn2+ and Mg2+. Since quite high metal concentrations must be added to
assays (110 mM for optimal activity), it has been presumed that Mg2+ is the ion used in vivo.
C. Evidence for a Multimer as the Active Unit of Integrase
Several lines of evidence demonstrate that the active unit of integrase is a multimer. It is clear, as an
isolated protein in solution, that integrase forms dimers [6,1012], and it has been shown by
sedimentation equilibrium studies that Rous sarcoma virus (RSV) integrase exists in reversible
equilibrium between monomeric, dimeric, and tetrameric forms [13]. Protein-protein cross-linking
studies of HIV-1 [14] and RSV [15] integrases confirm the existence of protein dimers and tetramers in
solution, and in vivo, the yeast GAL4 two-hybrid system has demonstrated that HIV-1 integrase can
interact with itself [16].
Complementation studies using mutant proteins in vitro provide compelling evidence that the active
form of integrase must be at least a dimer [14, 17]. This can be inferred from the result that when certain
inactive forms of integrasegenerated either by truncation or point mutationare mixed, robust
activity can be reconstituted. This indicates that different monomers in a multimer are capable of
providing different essential functions in the context of an active complex.
Collectively, these studies suggest that integrase acts as a multimer. This would also seem the most
straight-forward model to explain the observation that viral integration requires two coordinated cutting
and joining reactions on the target DNA during strand transfer. However, physical studies have not yet
addressed what form of integrase actually binds to DNA and carries out the chemical reactions of
integration.
III. Properties of HIV-1 Integrase
A. Domain Structure of Retroviral Integrases
A consistent view of the domain structure of retroviral integrases has emerged by combining the results
from biochemical studies using deletion and site-specific mutants, limited proteolysis experiments, and
sequence comparisons among the family of retroviral integrases. The organization of the domains of
integrase is shown schematically in Figure 4.
The central domain of HIV-1 integrase, consisting approximately of residues 50 to 200, is largely
conserved among retroviral integrases, and forms
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Figure 4
Schematic domain structure of HIV-1 integrase as adapted from
Engelman et al. [19]. Structures of two domains, the catalytic core
extending from residues 50 to 212 [3] and the nonspecific
DNA-binding domain from residues 220 to 270 [28,29], have
recently been determined by x-ray crystallography and NMR
spectroscopy, respectively.
the protease-resistant core of the protein [18,19]. Within this domain are three invariant residues that
comprise the D,D-35-E motif (see alignment in Figure 5). These are residues Asp64, Asp116, and
Glu152. Even conservative substitution of any of these residues leads to loss of all three in vitro
activities of integrase in parallel [1921]. The D,D-35-E motif is also observed in retrotransposons and
some prokaryotic transposases. A truncated form of HIV-1 integrase consisting of residues 50 to 212 is
capable of disintegration [22], implying that the catalytic site is contained within this domain. These
observations and the absolute requirement for metals for in vitro activity have led to the proposal that
the three acidic residues constitute a divalent metal-binding site capable of binding one or two Mg2+ or
Mn2+ ions to form a catalytically active enzyme. As will be seen in later sections, the three-dimensional
structure of the core domain of HIV-1 integrase is consistent with this hypothesis. The catalytic
mechanism may be, therefore, similar to the one proposed by Beese and Steitz for the 3'5' exonuclease
of E. coli DNA polymerase I [23]. It is proposed that for phosphate bond cleavage, one metal ion helps
form the attacking hydroxide ion while the other stabilizes a pentacovalent intermediate around the
phosphorus.
The C-terminus of HIV-1 integrase, consisting approximately of residues 210 to 288, includes the
dominant nonspecific DNA binding domain [24, 25], which has been more finely mapped to residues
220270 [26]. The C-terminus is the least conserved region of retroviral integrases; only one residue,
Trp235, is absolutely invariant. However, it has been reported that removal of only five amino acids
from the C-terminus of HIV-1 integrase is enough to severely reduce its 3' processing and strand transfer
activities [27]. One notable feature of the C-terminus is its high proportion of positively charged
residues. As discussed in Section IV.E, the structure of part of this region has recently been determined
using NMR spectroscopic methods [28,29].
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Figure 5
Alignment of amino acid sequences of retroviruses. See Engelman et al. [19] for details.
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The role of the N-terminus of integrase, residues 1 to 50, is still unclear. Within this region are four
strictly conserved amino acids: two His and two Cys residues. In HIV-1 integrase, the spacing is His-X3His-X23-Cys-X2-Cys. This cluster of His and Cys residues is reminiscent of a zinc-binding motif, and it
has been demonstrated that the full-length protein binds Zn2+ [22,30], and that the separately expressed
domain consisting only of residues 1 to 55 also binds Zn2+ stoichiometrically [31]. However, it has not
been shown that either the structural integrity or the enzymatic activities of integrase require Zn2+. While
truncation of residues from the N-terminus of HIV integrase results in loss of 3' processing and strand
transfer activities [22,25], in the case of RSV integrase, the N-terminal region can be replaced by
unrelated sequences, and the enzyme is still capable of all three in vitro activities [32].
B. Biophysical Properties of Full-Length Recombinant HIV-1 Integrase
It has been known for some time that recombinant HIV-1 integrase is a particularly poorly behaved
protein in solution. Its solubility in most usual buffers is limited to approximately 1 mg/mL, and even
then only in the presence of high concentrations of NaCl. At ~1 mg/mL, HIV-1 integrase slowly
precipitates out of solution, revealing one of its characteristic features, a tendency towards aggregation.
These properties of the protein are not unreasonable, since in its viral environment integrase is probably
never required to be a soluble protein. To maintain the integrity of preintegration complexes, it may
even be advantageous for the protein to have the properties of being rather insoluble and sticking to
itself, nucleic acid, and perhaps other proteins.
C. Properties of Truncated Versions of HIV-1 Integrase
It has been our approach to protein structure determination by x-ray crystallography that it is imperative
to begin with well-characterized and well-behaved protein. In particular, it is important that the protein
be reasonably soluble and monodisperse in solution. Unfortunately, as discussed above, recombinant
HIV-1 integrase satisfies neither of these conditions. One approach we and others have taken to
circumvent these problems has been to examine truncated versions of HIV-1 integrase to determine if
removal of amino acids from either terminus or both affects solubility and aggregation properties.
Although we observed that two proteins we constructed, IN213288 and IN50288, were more soluble than
the full-length HIV-1 integrase, IN1288 [33, and unpublished observations], our first target protein for
crystallization efforts was the core domain of HIV-1 integrase consisting of residues 50 to 212, IN50212.
We reasoned that this protein domain was likely to be compact and
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well folded since it is relatively resistant to proteolysis. As it is also active for disintegration, we
concluded that it contained the enzyme active site.
We exploited the convenience of histidine-tag (HT) technology to develop methods to purify large
quantities of IN50212 [12]. A 20-amino-acid histidine-containing tag was added to the N-terminus of
HIV-1 IN50212 [22] to allow rapid purification on nickel affinity columns. It was subsequently removed
by thrombin cleavage. Biophysical studies showed that although buffer conditions could be identified
where the protein was soluble to ~ 4 mg/mL, under these conditions the protein was highly aggregated
(unpublished observations). Although the aggregation problem could be largely avoided by the addition
of high concentrations of the zwitterionic detergent CHAPS, conditions could not be identified under
which protein crystals formed in the presence of CHAPS.
D. Systematic Mutation of Hydrophobic Residues to Improve Protein Solubility
As it became clear that IN50212 was crystallographically challenged, a condition readily understood in
terms of its aggregation problems and low solubility, a more radical approach was undertaken to try and
improve its biophysical properties. Hydrophobic residues in the catalytic core were targeted for sitespecific mutation according to the following criteria: where two or more hydrophobic residues were
encountered close together in the primary amino acid sequence, they were each changed to an alanine
residue. When a hydrophobic residue stood alone, it was mutated to lysine. In this way, 29 different
mutant proteins of IN50212 were rapidly generated using the overlapping polymerase chain reaction
(PCR) and screened for improved solubility properties [34]. Three mutated proteins were identified that
were more soluble at lower NaCl concentration than the unmutated core (V165K, F185K, and the
double mutation of W131A/W132A). However, one of these in which Phe185 was mutated to Lys had
dramatically improved solubility and was ultimately crystallized and its three-dimensional structure
determined [3]. The remarkable biophysical properties of this single point mutant of IN50212 have
recently been described [34].
IV. Structure Of The Catalytic Core Domain Of HIV-1 Integrase
A. Description of the Structure
The Overall Protein Fold
The three-dimensional structure of the catalytic core domain of HIV-1 integrase is centered on a mixed
five stranded sheet flanked by several helices forming
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Figure 6
Molscript stereo figure of the three-dimensional structure of the catalytic core of
HIV-1 integrase. The two catalytically essential aspartic acid residues (D64 and D116)
visible in the x-ray structure are highlighted.
an - meander sandwich (see Figure 6) [35]. In the crystal structure, interpretable electron density
starts at Cys56, leading to a short loop. The first strand starts at Gly59 and runs until Val68. The two
central residues of a type I' reverse turn, Glu69 and Gly70, change the polypeptide chain direction to
form the second strand between residues Lys71 and His78, which runs antiparallel with the first
strand. A type I' turn follows, with Val79 and Ala80 changing the chain direction again to form the
third strand between Ser81 and Ile89, which runs antiparallel with the second strand. A short loop
between Pro90 and Glu92 leads to the first helix (helix A) between Thr93 and Trp108. This helix
packs against the bottom face of the sheet formed by the first three antiparallel strands by several
hydrophobic interactions. A short loop (Pro109 and Val110) leads to the fourth strand between Lys111
and His114. This strand is parallel with the first. A short loop starting at Thr115 leads to helix B, a one
turn helix between Gly118 and Thr122, followed by helix C between Ser123 and Ala133. This helix
runs parallel to and packs against helix A. The residues Gly134 and Ile135 form a short loop prior to the
fifth and last strand of the structure between Lys136 and Ala138. This short strand is parallel with the
first and the fourth. There is no interpretable electron density due to disorder between Gly140 and
Met154. At Met154, the fourth helix (helix D) starts and runs until Ala169 on the top face of the sheet
formed by the first three strands. The residue Glu170 leads into the next helix (helix E) running
between His171 and Lys186, the first residue of a short basic sequence (Lys186, Arg187, and Lys188).
Together with
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Gly193 and Tyr194, Lys188 and Gly189 form two short antiparallel strands separated by a turn of
three residues (Gly190, Ile191, and Gly192) not involved in main chain hydrogen bonds. The first
residue of the last helix, (helix F), which runs until Asp212, is Ser194.
Three Conserved Acidic Residues at the Enzyme Active Site
There are four amino acids in the core domain sequence that are absolutely conserved among retroviral
integrases: Asp64, Asp116, Glu152, and Lys159. The three acidic residues form the conserved D,D-35E motif and have been shown to be essential for catalysis (see Section III.A). The role of Lys159 in
retroviral integrases is not obvious; its replacement with Val does not abolish catalytic activity, although
there is a decrease in strand transfer activity [20].
The first essential acidic residue, Asp64, is located in the middle of the first strand, while Asp116 is in
a loop region right after the fourth strand. These two residues define the active-site area and they are
right next to each other three-dimensionally with their -carbons separated by only 6.7 . The closest
approach is 3.4 between O1 of Asp64 and C of Asp116. These residues are on the surface of the
molecule, not part of any obvious substrate-binding cleft. The third catalytically essential acidic residue,
Glu152, is in the disordered and hence crystallographically invisible region between Gly140 and
Met154. Its location therefore must be inferred from other parts of the structure and from available threedimensional structures of related proteins. The location of Met154, the residue only two positions
upstream from Glu152, is known because of interpretable electron density. The distance between the carbons of Glu152 and Met154 cannot be larger than about 7.3 , which constrains Glu152 to the
neighborhood of the two other essential carboxylates, allowing it to contribute to the formation of a
divalent metal-binding site.
More recently, a crystal structure of the avian sarcoma virus (ASV) integrase core domain was solved
[36]. Within this domain, ASV integrase has 24% sequence identity to the HIV-1 integrase core and, as
expected, its three-dimensional structure is remarkably similar. The ASV integrase core in its native
form has much better solution properties than the HIV-1 integrase core, and did not require any point
mutations to render it crystallizable. Due to this fact and perhaps also due to its different crystal packing
interactions, the crystal lattice of the ASV integrase core domain is somewhat more ordered than that of
HIV-1. The two three-dimensional structures can be aligned quite well, using 74 -carbons, with an rms
deviation of only 1.4 in these -carbon positions. The most remarkable difference between the two
structures is that in the ASV structure the electron density is interpretable in all parts of the molecule.
This is not to say, however, that serious disorder is not present. For example, in one particular loop, the
temperature factors are above 70 2 for the carbons, indicating larger than 1 mean displacement
value for these atoms. The corresponding
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region in the HIV-1 structure is the uninterpretable stretch from residues 140 to 153, showing that
beyond the three-dimensional similarity, the molecules also share a similar disorder pattern despite their
different crystal packing interactions. It is clear that in the apoenzyme (metal-free) form of the HIV-1
integrase core, disorder is present in parts of the active site. However, in the holo form, structural
stability must be necessary to form a metal-binding site.
Position of the Third Essential Carboxylate
Does the structure of ASV integrase give us a hint about the likely conformation of the polypeptide
chain around Glu152 in the holoenzyme form of the HIV-1 integrase core? The answer is probably yes,
considering the overall similarity of the structures. The first residue after the disordered part in the HIV1 integrase core is Met154, which is also the first residue in helix C. The corresponding helix in the
ASV core is longer, running between Gln153 and Gly175. The residue analogous to Glu152 is Glu157
in the ASV integrase core structure, located a half-turn upstream from Ala159, which corresponds to
Met154 in the HIV-1 integrase structure. It is plausible to assume, therefore, that the polypeptide chain
in the holoenzyme form of HIV-1 integrase would also be in a helical conformation around Glu152, and
its location would be very close to the one that can be inferred from the location of Glu157 in the ASV
integrase core. Secondary structure prediction also supports this assumption, assigning -helical
structure around Glu152. Why does this helical turn show significant disorder in the HIV-1 integrase
structure? The answer might be found in the amino acid sequence: Pro145 is a highly conserved residue
among retroviral integrases, the only exception being ASV integrase where it is substituted with a Ser.
Since the main chain nitrogen of a proline is not capable of participating in hydrogen bonding, it is very
rarely found in helices. It is likely that if the polypeptide chain around Glu152 were helical in the
holoenzyme form of the HIV-1 integrase core, then this helix would start after Pro145. There is no such
restriction in the ASV integrase core, and it is possible that this is why helix C is longer in ASV than in
HIV. This may also explain the disorder around Glu152 in the HIV-1 integrase core, since it is closer to
the end of the helix and more susceptible to disordering effects. A longer helix and therefore a more
ordered active site in the apo form may be a unique feature of the ASV integrase core.
B. Similarity to Other Polynucleotidyl Transferases
Overall Protein Folds
The catalytic core domain of HIV-1 integrase has a topologically identical fold with the RNase H
domain of HIV-1 reverse transcriptase [37], the RuvC Holli-
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Figure 7
Structures of the catalytic core of HIV-1 integrase, HIV-1 RNase
H, RuvC, and the core domain of MuA transposase demonstrating
similarities in folding topology. The catalytically essential residues
are highlighted.
Document
day junction resolving enzyme [38], and also the core domain of the phage MuA transposase [39] (see
Figure 7). In the case of HIV-1 integrase, RNase H, and RuvC, definite three-dimensional similarity
extends only to the ends of the last strand; from this point, the structures diverge. In HIV-1 RNase H,
there is only one more helix corresponding to helix D in the HIV-1 integrase core but in a 40
different orientation. In RuvC there are three more helices, with the last one running parallel to helix D
of HIV-1 integrase, but in the opposite direction and also 4.6 closer to the sheet. In contrast, the
homology between HIV-1 inte-
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grase and the MuA transposase extends until the carboxyl termini of their respective catalytic domains
with three very similarly positioned and oriented helices.
Limited three-dimensional alignment between the four molecules can be accomplished by identifying
structurally homologous stretches along the polypeptide chains if this search is restricted to between the
first well-ordered amino terminal residue and the end of the last strand. For the core domain of HIV-1
integrase, this corresponds to the region between Ile60 and Gln137. Using the corresponding region in
the MuA transposase, the two structures can be aligned with an rms deviation of 1.7 over 69 -carbon
positions. The main differences between these structures are two insertions in the transposase core: an
11-residue -stranded extension replacing the turn between the first and the second strand in the HIV-1
integrase core, and a 15-residue extension before helix B with no secondary structure. Both of these
extensions interact with the downstream nonspecific DNA-binding domain of the transposase. For HIV1 RNase H, the alignment results in a rms deviation of 2.0 over 48 alignable -carbon positions. The
position of helix A is significantly different in RNase H, as it shifts more than 5 toward the sheet.
There is also an additional 2.5 turn helix following the fourth strand and a 5-residue loop after this
helix. For RuvC the alignment yields an rms deviation of 2.0 over 50 alignable -carbon positions. In
this case, the differences are mostly the result of longer secondary structure elements in RuvC. Of all the
molecules compared, the HIV-1 integrase core is the smallest, with the most compact design in the
region where these alignments were performed. For comparison, let us mention again that the
homologous ASV integrase core can be aligned with an rms deviation of 1.4 over 74 -carbon
positions in this region.
Both topological similarity and three-dimensional homology with the MuA transposase was expected
based on the similarity of the reactions the enzymes catalyze, but the relationship with RNase H and
RuvC was a surprise. This discovery led to the proposal of a new polynucleotidyl transferase
superfamily. All the members of the superfamily are divalent metal ion-dependent endonucleases, and
they all leave 3'-OH and 5'-phosphate groups at the site of cleavage. All the members of the superfamily
display their catalytically essential acidic residues at the same general location. There are three such
residues in HIV-1 integrase, RNase H, and the MuA transposase, while there are four in RuvC. Two of
these residues are always located on the same three-dimensional structural elements, while the location
of the third varies. The Asp64 residue HIV-1 integrase corresponds to Asp443 in HIV-1 RNaseH, Asp7
in RuvC, and Asp269 in the MuA transposase. Based on the three-dimensionally aligned structures, the
-carbon positions of these residues cluster quite well around that of HIV-1 integrase, with an rms
deviation of 0.84 . All these residues are located in the middle of first strand. The side chain torsion
angle, Chi 1, is
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-62 for HIV-1 integrase, a frequently observed rotamer. For the other three molecules this Chi 1 value
varies between -142 and -156, a common range for rotamer angles. The preference for the first
rotamer of the HIV side chain is probably due to the 2.73 -long hydrogen bond between O2 of
Asp64 and N2 of Gln62. There is no such interaction in the other molecules. The different rotamer
causes a 2.46 rms separation of the C positions around the HIV-1 C, compared to only 1.63
around the C of Asp443 in HIV-1 RNase H, indicting the carboxylate of Asp64 in HIV-1 integrase as
the outlier.
Position of the Second Carboxylate Residue
In all the analogous structures, the second essential carboxylate resides just after the end of the fourth
strand. The main-chain atoms are not involved in strand-forming direct hydrogen bonds, therefore the
chain diverts from running parallel with the first strand, forming a small cleft. The equivalent residues
are Asp116 in HIV-1, Asp498 in HIV-1 RNase H, and Glu66 in RuvC. The clustering is weaker than for
Asp64; the rms deviation is 1.77 in -carbon position around the HIV-1 integrase residue.
Interestingly, by including the structurally otherwise highly homologous ASV integrase core, the rms
deviation increases to 2.15 due to the 3 distance between the C of Asp116 of HIV-1 integrase and
that of the corresponding residue, Asp121 of ASV integrase. The rms separation between the ASV
position and the rest of the cluster (now excluding HIV-1 integrase) is 2.2 , which is rather high,
identifying the ASV residue as the outlier. For the Chi 1 torsion angles, all three preferred rotamers are
present: Chi 1 is -86 for HIV-1 integrase, 73 for HIV-1 RNase H, and -173 for the MuA transposase.
The RuvC Chi 1 value is not included in this comparison because it has a Glu in this position. The
different Chi 1 values combined with the variation in -carbon positions leads to a somewhat more
scattered C (or C for Glu66 in RuvC) position with an rms deviation of 2.71 around C of Asp116
of HIV-1 integrase. By including the ASV molecule, the scatter increases to 3.82 due to the 6
distance between C of Asp116 in HIV-1 integrase and C of Asp121 of ASV integrase.
The Third Essential Carboxylate
The location of the third essential catalytic carboxylate varies between different members of the
superfamily. For HIV-1 integrase, Glu152 is in a disordered region with no interpretable electron
density. Based on the location of the equivalent residue in the ASV integrase, its position is assumed to
be on helix D, as discussed above. For RNase H, Glu478 is located on helix A, with its side chain
pointing toward the other two carboxylates to complete the divalentmetal-binding site. Such metal
binding has been observed crystallographically
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[37]. For RuvC, four residues have been shown to be essential for catalysis [40]. The third of these,
Asp138, is at the amino end of the last helix of the structure. In the three-dimensionally aligned
structures, this helix is parallel with the helix D in HIV-1 integrase, although it is running in the opposite
direction. The position of helix D in RuvC is also significantly different, mainly due to a 13 shift
along its axis toward the active site, placing Asp138 close to the other two carboxylates. The fourth
essential residue, Asp141, is on the first turn of the same helix, very close in the aligned structures to the
essential Glu157 of ASV integrase, their carbons separated by only 1.6 . For the MuA transposase,
its third essential carboxylate, Glu392, is in a loop region, just one residue upstream from the amino end
of a helix, the topological equivalent of helix D. Unexpectedly, this residue turns away from the region
defined by the two other carboxylates to a position where it clearly cannot contribute to the formation of
a metal-binding site. It is likely, therefore, that the conformation of the polypeptide chain around Glu392
in the transposase core observed in the crystal structure belongs to an inactive form. In this case, a
conformational change upon transposase tetramer assembly or perhaps upon substrate binding is
required for activity.
Significance of the Disordered Region
From the point of view of HIV-1 integrase, it is interesting to note that the apparently flexible part of the
MuA transposase structure is topologically equivalent with the disordered and uninterpretable part of the
integrase. Similarly, in the crystal structure of the isolated RNase H domain of HIV-1 reverse
transcriptase, a five-residue loop in a topologically equivalent location is disordered and therefore
uninterpretable. In the ASV integrase core, the corresponding loop is visible but with rather high
mobility. It seems that some kind of disorder or flexibility in this region is a common feature of the
superfamily. Crystal structures of enzyme-substrate or enzyme-inhibitor complexes will tell us the
functional significance of this flexibility as well as the exact configuration of the active site.
C. The Dimer Interface
HIV-1 integrase is active as a multimer, and the catalytic core domain alone forms dimers in solution,
even at low protein concentration (see Section II.C). In the crystal structure, a roughly spherical dimer of
about 45 diameter was observed, formed by a crystallographic two-fold axis. The dimer has a large
solvent-excluded surface of 1300 2 per monomer. This area is close to what is expected for dimers in
this molecular weight range [41]. Therefore, we are convinced that in the crystal structure the authentic
dimer is present. This was subsequently confirmed by the structure of the ASV integrase core. Although
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crystallized under different conditions, forming crystals that are in a different space group with different
crystal-packing interactions, the dimer observed for the ASV core is essentially identical with that of
HIV-1 integrase, although the solvent-excluded surface is smaller (only 740 2). This difference is
largely due to the absence of helix F in the ASV structure.
The core domain dimer of HIV-1 integrase is held together by several hydrophobic and polar
interactions. Hydrophobic interactions dominate the interface between helix E from one monomer and
helices A and B from the other. There is a buried salt bridge between Glu87 of the third strand and
Lys103 on helix A. There are also some water-mediated polar interactions between these two secondary
structure elements. There are direct hydrogen bonds between residues on helix A and residues on helix E
across the interface including one between Lys185 (the substitution responsible for the improved
solubility and therefore crystallizability) and the main-chain carbonyl on Ala105. In the ASV core,
His198 is in this position, forming a very similar hydrogen bond with the carbonyl oxygen of Ala110.
There are about 10 water molecules buried in the interface, all involved in hydrogen bonds. The part of
the solvent-accessible surface of the monomer which becomes buried upon dimer formation displays a
high degree of shape compatibility with itself: by rotating it 180 around the crystallographic two-fold
axis, the resulting surface will fit the original one without forming large pockets. It is possible that the
core domain of HIV-1 integrase has evolved to optimize this compatibility in order to increase its
stability. It would be interesting to see the effect on protein activity of site-directed mutations aimed at
disrupting this interface and hence the dimer (or possibly the higher order multimers in the context of
the full-length protein).
D. Implications of Crystallographic Dimer for the Chemistry of Catalysis
The nearly spherical nature of the dimer formed by two monomers of the integrase catalytic core places
active sites on respective monomers on opposite sides of the dimer: approximately 35 separates the
carboxylate oxygens of Asp64 of each monomer. While we are convinced that the observed dimer is not
an artifact or consequence of crystallization, it would seem difficult to reconcile this distance with the
observation that, during in vivo strand transfer, cuts on the target DNA occur with a separation of five
base pairs, corresponding to 1520 in B-form DNA. How can a single dimer accomplish this? One
possibility is that the cuts do not occur simultaneously. One end of the viral DNA could be joined by a
reaction at one active site, followed by carefully controlled movement of DNA and protein relative to
one another such that the second active site is now positioned five base pairs away from the initial site of
strand transfer. It has been proposed, in a variation on this theme, that the first strand-transfer
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reaction is followed by DNA relaxation (unwinding, rotation, etc.), resulting in the site on the target
DNA for the second transfer reaction site now being located close to the active site of the second
monomer [42]. An alternate possibility is that a multimer larger than a dimer is responsible for the
coordinated cutting and strand-transfer reactions. For example, two contacting dimers can be modeled
such that two active sites of the resulting tetramer are located 1520 apart. It is also possible that even
higher order multimers are involved.
There is, as yet, no convincing evidence in support of any one model. The observation that RSV
integrase cuts target DNA with a six-base-pair stagger rather than the five observed for HIV correlates
intriguingly with the apparently longer distance (~ 38 vs. 35 ) between active sites in the RSV
dimer. However, understanding the coordinated cutting and joining reaction awaits three-dimensional
information on the arrangement of monomers within an integrase multimer binding to DNA.
E. Three-Dimensional Structures of Other Domains of HIV-1 Integrase
Three-dimensional structural information has not yet been obtained for a full-length integrase protein. In
its absence, attempts have been made to determine the structure of the smaller domains consisting of the
separately expressed N- and C-termini that flank the core whose structure is now known.
The Amino Terminus of Integrase
While the N-terminus of HIV-1 integrase, consisting of residues 1 to 55, has been separately expressed,
purified, and biophysically characterized [31], structural data has not yet been obtained. This protein
domain binds metal ions such as Zn2+, Co2+, and Cd2+ stoichiometrically, and is monomeric at low
protein concentrations. Dramatic changes in helix content (from 14% to 32%) are observed in the
circular dichroism (CD) spectrum upon addition of metal. Analysis of CD spectral features led
researchers to conclude that it is highly probably that integrase contains a zinc finger that folds in much
the same way as the TFIIIA-like DNA binding proteins, with two His residues located on an helix and
two cysteines part of a sheet [31]. However, confirmation of such a model awaits structure
determination by x-ray crystallography or NMR spectroscopy.
The Carboxy Terminus of Integrase
When the C-terminal domain is expressed as a separate polypeptide, IN213288 can be purified from the
initial soluble fraction from cell lysates [33]. This small protein fragment, therefore, was an attractive
target for structure determination.
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Figure 8
Molscript stereo figure of the structure of the nonspecific DNA-binding domain
of HIV-1 integrase, IN220270, determined by heteronuclear NMR spectroscopy [28].
The structure of this domain is of particular interest as it represents the dominant nonspecific DNAbinding region of integrase. Gel filtration and sedimentation equilibrium results indicated that purified
IN213288 partitioned between dimers and highly aggregated material (unpublished observations).
However, a smaller domain consisting of residues 220 to 270 maintains the DNA-binding properties of
the longer C-terminal domain and is better behaved in solution. Recently, two groups have reported the
structure of this smaller fragment, IN220270, determined using multidimensional heteronuclear NMR
spectroscopic methods [28,29].
As shown in Figure 8, the overall structure of IN220270, is that of a sandwich formed by two threestranded sheets. As anticipated by biophysical studies, the polypeptide is a dimer in solution. The
interface between monomers is formed by the antiparallel interaction of three strands from each
subunit and is stabilized predominantly by hydrophobic interactions. There is a long loop between
strands 1 and 2 which, in the dimer, defines the sides of a cleft that is of the appropriate dimensions
(about 24 24 12 ) to accommodate double-stranded DNA. The folding topology is very similar to
that of SH3 domains that are found in several proteins involved in signal transduction, despite the lack
of significant sequence homology. This is rather unusual since SH3 domains are generally involved in
protein binding rather than interactions with DNA.
V. Prospects for Inhibitors
A. Overview of Inhibitor Studies to Date
The investigation of HIV integrase inhibitors has been largely restricted to testing available compounds
that inhibit other enzymes with similar substrates or
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Table 1 Representative Inhibitors of HIV-1 Integrase and IC50 values of compounds that inhibit 3' processing and
strand transfer activities of HIV-1 integrase
Compound
IC50 of 3' processing

(M)
IC50 of strand
transfer (M)
Active against
IN50212?
Reference
I aurintricarboxylic acid
monomer
1050
n.d.
n.d.
44
II cosalane
n.d.
25
n.d.
45
III DHNQ
5.7
2.5
yes
47
IV primaquine
15
3.6
n.d.
46
V CAPE
220
19
yes*
46
VI quercetin
24
14
n.d.
47
VII quercetagetin
0.8
0.1
yes
47
VIII AG1717
0.4
0.16
yes
50
IX -conidendrol
0.5
0.5
n.d.
51
X suramin
0.25
0.11
n.d.
53
XI curcumin
95
40
yes
55
XII (neocuproine)2-Cu+
yes
56
XIII AZT-monoPi
100150
100150
yes
57
XIV GT 17-mer
0.092
0.046
n.d.
59
XV HCKFWW
yes
43
The third column indicates if these compounds also inhibit disintegration by IN50212. n.d. = not determined.* = only if
pre-incubated.
Document
proposed mechanisms. For example, as will be seen, many topoisomerase II inhibitors also inhibit HIV
integrase. While screening of chemical databases is likely underway at several pharmaceutical
companies, results have either not been made available or are discouraging (see below). One foray into
integrase inhibitor designrather than discoveryhas recently been described using a peptide
combinatorial library approach [43]. We summarize below published reports to date (March 1996) in
which compounds have been identified that inhibit integrase in in vitro assays with IC50 values of 100
M or less (IC50 is the concentration at which the measured activity is inhibited by 50%). In vitro
inhibition data is compiled in Table 1; structures of selected compounds are shown in Figure 9.
An Effective Pharmacophore: Multiple Hydroxyl Groups on Aromatic Rings
The first report of a class of compounds that inhibits HIV integrase appeared in 1992 [44].
Aurintricarboxylic acid (I) and its derivatives, known to inhibit other enzymes that process nucleic acids,
were determined to inhibit 3' processing with moderate IC50 values of 1050 M. As shown in Figure 9,
a recurring structural theme was established early on in which integrase inhibitors often possess
aromatic rings with multiple hydroxy substituents that are either located
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Figure 9
Chemical structures of reported inhibitors of HIV-1 integrase. (I) aurintricarboxylic
acid monomer; (II) cosalane; (III) dihydronaphthoquinone or DHNQ; (IV)
primaquine; (V) caffeic acid phenethyl ester or CAPE; (VI) quercetin; (VII)
quercetagetin; (VIII) AG1717; (IX) -conidendrol; (X) suramin; (XI) curcumin.
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on the same ring or can potentially be positioned close together in three-dimensional space if rings stack
on top of each other. More recently it was shown that cosalane (II), a steroid-substituted derivative of
(I), was no better in inhibiting integrase in vitro than the parent compound, but showed promise in HIV
cytopathicity cell-culture assays [45]. Although cosalane and a number of related analogues inhibit both
HIV protease and integrase in vitro, the primary site of action is believed to be inhibition of gp120
binding to CD4 receptors.
In 1993, the effects of selected topoisomerase II inhibitors, antimalarial agents, DNA binders,
naphthoquinones, and various other agents on integrase activity in vitro were investigated [46].
Although certain effective topoisomerase inhibitors are also good HIV integrase inhibitors, this is not a
generalizable correlation. Since many topoisomerase inhibitors also bind DNA, it is difficult to assess
whether the observed in vitro effects result from specific interaction with integrase or from the
sequestering or distorting of the DNA substrate. However, several compounds were identified that are
not known to be DNA binders but that inhibited integrase with reasonable IC50 values. These included
dihydroxynaphthoquinone (III), primaquine (IV), caffeic acid phenethyl ester (CAPE, V), and quercetin
(VI).
Motivated by the structural similarities between compounds IIIVI, a more intensive structure-function
study of flavones was undertaken in which approximately 50 related compounds were screened for
inhibition of in vitro integrase activity [47]. Flavones are planar compounds containing three aromatic
rings substituted with various polar groups such as hydroxy substituents. General trends were observed
relating structure to inhibitory effectiveness; for example, inhibition required at least three hydroxy
groups, the most favorable arrangement being when they were located ortho to one another. The most
effective compound, quercetagetin (VII), is a potent topoisomerase II inhibitor and a known DNA
intercalator. It was noted by the authors that many flavones are not integrase-specific; rather, they inhibit
a broad range of enzymes including DNA polymerases, ATPases, and NAPDH-monooxygenases. They
are also, in general, capable of DNA intercalation. It has not been established that their inhibitory effects
are due to direct interactions with integrase.
A subsequent detailed structure-activity relationship study of CAPE (V) revealed that while the ortho
hydroxys were important for in vitro integrase inhibition, both the caffeic acid and phenethyl moieties
could be substantially modified [48]. Ortho hydroxyl groups in the context of other classes of
compounds also confer anti-integrase potency. For example, several semisynthetic compounds derived
from arctigenin, a topoisomerase I inhibitor that itself is not active against integrase, have been shown to
inhibit HIV integrase [49]. More compelling evidence of the importance of ortho hydroxyl groups was
provided by the tyrphostins, a group of synthetic compounds that are tyrosine kinase inhibitors. Several
of these also inhibit integrase in the submicromolar range
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(for example, compound VIII, aka AG1717). In cell-based screening assays, AG1717 demonstrated
some antiviral activity [50].
Another study that demonstrated a role for ortho hydroxyl groups in in vitro integrase inhibition
identified -conidendrol (IX) via random screening as an inhibitor with an IC50 value of less than 1 M
[51]. Although -conidendrol did not inhibit several other nucleic-acid processing enzymes, indicating
some specificity for integrase, it was not active in cell-based antiviral assays at concentrations as high as
100 M.
Other Classes of Integrase Inhibitors
Several compounds and their derivatives that do not contain adjacent hydroxy groups on a phenyl ring
have recently been identified as HIV integrase inhibitors. These include suramin (X), curcumin (XI),
phenanthroline-Cu+ complexes (XII), and 3'-azido-3'-deoxythymidylate (AZT) monophosphate (XIII).
Suramin (X) is a known inhibitor of DNA and RNA polymerases, retroviral reverse transcriptases, and
topoisomerase II. It has also been shown to prevent the infection of T lymphocytes by HIV in vitro [52].
Its six sulfonic acid groups confer a strong negative charge, and it was reasoned that there might be an
inhibitory electrostatic interaction with the positive residues of the HIV C-terminus domain. Suramin
was shown to be an effective inhibitor of 3' processing and strand transfer, with IC50 values of 0.25 M
and 0.11 M, respectively [53]. It was not demonstrated, however, that the mechanism of inhibition does
involve binding to the C-terminus of integrase, although this could be readily addressed using Cterminal truncated mutants active for disintegration.
Curcumin (XI), the coloring dye in the spice turmeric, is structurally related to CAPE (V). It has also
been shown to inhibit HIV replication by inhibiting p24 antigen production and tat-mediated transcription
[54]. As shown in Table 1, it also has moderate integrase inhibitory properties [55]. Although its twoOH groups are neither adjacent to each other nor on the same phenyl ring, its conformations can be
modeled to bring the hydroxy groups into close proximity by stacking the two phenyl rings on each
other.
Several tetrahedral cuprous phenanthroline complexes, known inhibitors of transcription, were tested
against integrase and shown to be reasonably effective inhibitors [56]: IC50 values in the range of 110
M were determined (for example, the neocuproine-Cu+ complex, XII). Analyses of the mode of
inhibition demonstrated that these compounds act noncompetitively, and that inhibition does not
correlate with inhibition of DNA binding. Thus, it has been proposed that these metal chelates may act
at a site distant from the active site, or perhaps in the context of an enzyme-DNA complex.
3'-Azido-3'-deoxythymidine, or AZT, is a nucleoside analog approved for use to treat AIDS. Its
metabolites, the mono-, di- and triphosphate forms, accu-
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mulate during treatment; in particular, AZT-monoPi (XIII) accumulates in cells to millimolar levels.
These metabolites were investigated as possible integrase inhibitors and it was shown that all three
phosphate derivatives inhibit with IC50 values of 100150 M, although AZT itself is not inhibitory [57].
These results suggest that despite the weak inhibition by these particular compounds, nucleoside analogs
may serve as lead compounds for the development of integrase inhibitors.
It was recently observed that oligonucleotides that form guanosine quartet structures inhibit HIV
replication [58]. In light of the AZT results that suggested that there may be a nucleotide binding site on
integraseand because integrase binds DNAthese oligonucleotides (for example, 5'GTGGTGGGTGGGTGGGT-3', XIV) were investigated as possible inhibitors [59]. These compounds
have the lowest inhibition constants reported to date (see Table 1), and suggest an exciting new avenue
for integrase inhibitor development.
In a completely different approach to integrase inhibitors, a synthetic peptide combinatorial library was
used to select a hexapeptide capable of inhibiting integrase proteins [43]. The first two amino acids were
selected using a library of 400 dipeptides, and the remaining amino acids selected one-by-one in an
iterative process. The optimal hexapeptide, HCKFWW (XV), inhibits HIV-1 integrase with an IC50 of 2
M. The peptide does not compete for DNA binding to viral DNA, nor does it represent a sequence
present in integrase itself. Although it is not expected that a peptide consisting of L-amino acids would
be a suitable drug in itself, the use of D-amino acids or peptidomimetic backbones may be fruitful
directions to pursue.
Summary
Many of the compounds identified to date that inhibit HIV integrase in vitro have common structural
features as illustrated in Figure 9. Most notably, these include hydroxy-substitued phenyl rings.
However, these compounds may inhibit in vitro activity for reasons unrelated to enzyme binding. For
example, it is difficult to know what to make of inhibition studies where the compounds added to the
assays are known to bind DNA. Do the compounds affect in vitro activity because they bind and
sequester the substrate? It is possible that they distort the DNA or intercalate between base pairs,
preventing appropriate binding to the enzyme. While this is itself is a valid basis for the design of
inhibitors against HIV infection, particularly if it targets DNA specific to the virus such as the LTR
sequences [60], it is not an approach that builds on knowledge of the three-dimensional structure of the
protein. To this end, valuable information could be obtained from studies in which direct binding of
compounds to integrase is measured. This is also true for those inhibitors that do not contain the hydroxysubstituted phenyl pharmacophore. Binding studies could be carried
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out, for example, using radiolabeled inhibitors or possibly by monitoring any UV-spectral shifts in the
case of aromatic compounds.
B. What We Need to Know before We can Start Structure-Based Drug Design
The review of known integrase inhibitors presented in the previous section demonstrates the paucity of
effective inhibitors reported in the literature. A limited number of structural types have been
investigated, focused heavily on compounds with aromatic ring systems and hydroxy substituents. Most
inhibitors reported to date are only moderately effective in in vitro assays, with IC50 values residing in
the low M range (see Table 1).
To build on this knowledge base, and to use known molecules as lead compounds for the development
of more effective inhibitors, it would be extremely valuable to obtain a high-resolution crystal structure
of an integrase-inhibitor complex. We and others are vigorously pursuing this goal. It is not clear if the
lack of success to date is because the inhibitors identified so far manifest their effects in in vitro assays
predominantly at the level of the DNA, or if some aspect of the structure of the HIV-1 integrase core
itselffor example, its mobility in certain regoinsprevents the formation of a tight complex. It is also
possible that binding is weaker to the HIV-1 core domain than to the full-length protein because of
missing enzyme-inhibitor contacts. Co-crystallization attempts would benefit from in vitro studies to
determine relative binding constants as a guide in selecting the most tightly bound inhibitors. It would
also be useful to obtain information on the effect of variables, such as Mn2+ or Mg2+, on binding
constants.
It may be futile at this stage to attempt to model the binding of known inhibitors to the catalytic core
domain of HIV-1 integrase in the absence of more complete information. It cannot a priori be assumed
that the site of action of all these inhibitors is the enzyme active site identified by the constellation of
conserved acidic residues. For example, certain very effective nonnucleoside inhibitors of HIV reverse
transcriptase bind not to the enzyme active site, but rather to a small pocket adjacent to it. There are no
obvious structural features of the integrase coresuch as the deep trough surrounding active site
residues in the case of HIV protease [61,62]that can be readily identified as a potential inhibitor
binding site. Furthermore, since part of the region defining the active site of HIV-1 integrase is
disordered in our crystal structure, this prevents a surface rendering of the region around the conserved
acidic residues. It also seems unlikely, given the known differences in active-site geometries between
the HIV and ASV integrase (see Section IV.B), that the structure of the related ASV integrase core
domain by itself would be particularly useful in this regard. As the active form of the enzyme
presumably binds divalent metal ions, it will be important to determine how or if the structure of HIV
integrase changes
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when metals are bound. Finally, there is no evidence to rule out the possibility that the two termini
contribute to part of the active site, occlude some of it, or restrict access to it in as yet undetermined
ways. For this and other obvious reasons, three-dimensional structures of larger versions of HIV-1
integrase, such as IN1212, IN50288, and the full-length protein, IN1288, will be required.
We also lack a clear picture of how the enzyme substrates, the viral DNA ends and the target DNA, bind
to integrase. The DNA must at some point approach the region defined by the three conserved acidic
residues so that bond cleavage and joining can occur. However, the dominant DNA binding domain is
defined by residues in the C-terminus. It would be extremely valuable to determine the relative
orientation of these domains in the context of a larger version of integrase. Even more revealing would
be the structure of the full-length protein with bound DNA. Once we possess this information, it should
be possible to rapidly progress with structure-based drug design.
C. Possible Approaches to the Design of Effective Integrase Inhibitors
There are a variety of approaches to the design of integrase inhibitors that are obvious and do not depend
on knowing its three-dimensional structure. However, the rational implementation and refinement of
these approaches will require high-resolutional structural data, much of which, as indicated above, is not
yet available. It still may be useful to discuss here different classes of inhibitors that can be envisioned.
Preventing DNA Binding
One approach to the inhibition of integrase would be to prevent binding of the DNA substrate.
Unfortunately, we do not yet know how or where DNA binds. There are likely to be several sites on the
enzyme that contact DNA, including the C-terminus and the region around the active site. In the absence
of the structure of an integrase-DNA complex, structures of related enzymes (RNase H, the MuA
transposase, and RuvC) with their DNA substrates would be useful guides in suggesting ways in which
DNA could interact with integrase. However, there is no guarantee that modes of DNA binding are
conserved among members of this polynucleotidyl transferase family. The overall structure of the Cterminus fragment suggests that it should be possible to develop compounds that bind specifically in the
cleft formed by dimers of IN220270 (see Section IV.E) which may prevent DNA binding.
Inhibition at the Active Site
It may be possible to inhibit integrase by preventing the binding of the required metal cofactor(s) to the
active site acidic residues that are presumed to provide
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coordinating ligands to the metal(s). This could be accomplished by sterically blocking access to the
active site or by specifically binding the acidic residues themselves. Such a mechanism has been
suggested to explain the inhibition of integrase by curcumin [55], which could bind to the active site
aspartates or glutamates via its hydroxy groups. Compounds could also be devised that chelate the
metal(s) once bound, preventing access of the active site to the substrate or distorting the active site
geometry preventing phosphate bond cleavage. An intriguing approach to disrupting metal binding has
recently been reported for the Zn2+-binding HIV nucleocapsid (NC) protein [63]. In this case,
compounds were developed that specifically eject Zn2+ from the zinc-finger region of NC, interfering
with viral replication. Such an approach might be applied to Mn2+ or Mg2+ binding at the active site,
although the Zn2+-binding domain at the N terminus of integrase also suggests itself as a target.
It is curious that approaches have not been devised for mechanism-based inhibitors, particularly since
the stereochemical mechanism of integration has been understood for some time now [8].
Interfering with Multimerization
The structures of the domains of HIV-1 integrase determined to date both reveal dimers [3,28,29,36]. It
may be possible to develop compounds that bind specifically to the dimer interfaces, preventing
interactions between monomers that may be necessary for activity. The success of this approach
presumes that during the retroviral lifecycle there is a point where the monomer surfaces are accessible.
It is not clear that integrase in the context of preintegration complexes is in equilibrium between the
monomeric and higher order forms. This approach need not be restricted to preventing dimer formation
if higher order interactions (e.g. formation of a tetramer) are also mechanistically relevant. To this end,
the structure of an integrase tetramer, such as that formed by the full-length protein, would be useful in
identifying dimer-dimer interface(s).
Other Ways to Confound Integrase
There are other parts of the retroviral life cycle involving integrase that could be targeted for inhibition.
For example, integrase can bind other proteins such as human Ini1 [64], and most likely interacts with
the viral proteins that are part of the preintegration complex [65]. Although it is not known if these
interactions are essential for viral replication, preventing protein-protein binding would provide another
site at which to attack integrase. It is possible that interactions between integrase and other proteins in
the preintegration complex are crucial for maintaining the integrity of this complex and its ability to
migrate to the cell nucleus. Interfering with these protein-protein or protein-nucleic acid interactions
may be another approach to halting viral replication. For example, prein-
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cubation of phosphotyrosine with integrase has been shown to inhibit interaction with the MA protein
[65]. Therefore, phosphotyrosine analogs could be a unique approach to antiviral development.
D. Concluding Statement
It is known from drug design studies with HIV reverse transcriptase and protease that the virus is able to
escape from the pressures of inhibitors by mutation of the drug targets [66]. Although integrase is, a
priori, a reasonable target for drug-design efforts, it must be anticipated that integrase will also be able
to rapidly mutate and thereby avoid inhibition.
By analogy with recent approaches to reverse transcriptase inhibitors, it may be possible to design a set
of integrase inhibitors that act at slightly different binding sites and from which the virus cannot
simultaneously escape. That is, the combination of mutations required to avoid inhibition may be severe
enough to prevent integrase from carrying out its required chemistry. (In the absence of direct structural
information on the sites of inhibitor binding to integrase, the generation of escape mutants in vitro and
their subsequent sequencing may be an indirect way to identify inhibitor binding sites.) It will also be
important to determine if the virus will be able to simultaneously mutate reverse transcriptase, integrase,
and the protease in response to a combination of inhibitors targeted against all three pol gene products.
The answers to these questions will require the development of suitable inhibitors and the beginning of
in vitro testing. To this endwhile large-scale screening and the development of combinatorial
chemistry methods should continuethe structure of the catalytic core domain of HIV-1 integrase is a
starting point for the rational design of integrase inhibitors. There is much more structural information
that must be obtained for the full-length protein and its complexes with metals, inhibitors, and
substrates. We and others are aggressively pursuing results on these fronts.
E. Recent Developments
Several studies published since March 1996 have expanded the list of in vitro integrase inhibitors
effective at IC50 values below 100 M. These include two dicaffeoylquinic acids obtained from
medicinal plants and a synthetic analog, L-chicoric acid [68], the HIV protease inhibitors NSC 117027
and NSC 158393 [69], certain anthraquinone derivatives [70], coumermycin, and pyridoxal phosphate
[71]. In addition to exhibiting in vitro inhibition, the dicaffeoylquinic acids effectively inhibited HIV-1
replication in T-lymphoblastoid cell lines [68].
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Follow-up studies were also reported for two previously identified classes of integrase inhibitors.
Several nucleotides that were more effective inhibitors than the originally tested AZT nucleotides were
identified [71]. For example, the L-enantiomers of 5-fluoro-2',3'-dideoxycytidine monophosphate and
triphosphate inhibit 3' processing and strand transfer with IC50 values of ~40 M. A structure-function
study on GT-containing oligonucleotides showed that both the number of quartets formed and the loop
sequences between the quartets are important for activity, and that inhibitors of this type may function
by interacting with the N-terminus of integrase [72].
A particularly important contribution was the demonstration that preintegration complexes isolated from
HIV-infected lymphoid cells can be used in assays to screen for inhibition of integration [70].
Intriguingly, many compounds previously identified as in vitro inhibitors of 3' processing and strand
transfer had no effect on integration carried out by either crude or partially purified preintegration
complexes. Thus, such an assay may be a valuable method of screening out false positives identified
using in vitro oligonucleotide assays, or corroborating the evidence that a particular compound is indeed
active against integrase.
Acknowledgments
Our work described here was carried out in the laboratories of R. Craigie and D. R. Davies. We express
our gratitude to our co-workers who, over the years, participated in the effort to determine the structure
of HIV integrase. In particular, we acknowledge the contributions of F. D.Bushman, M. Carmichael, A.
Engelman, S. Hosseini, T. Jenkins, K. Mizuuchi, I. Palmer, P. Rice, P. Sun, and P. Wingfield. We would
also like to thank D. R. Davies and T. Jenkins for their comments on the manuscript and A. Mazumder
for alerting us to the most recent work on integrase inhibitors and for his contributions to Section V.A.
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4
Bradykinin Receptor Antagonists
Donald J. Kyle
Scios Nova Inc., Sunnyvale, California
I. Introduction
The term kinins is generally made in reference to either the nonapeptide bradykinin (Arg1-Pro2-Pro3Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) or the decapeptide kallidin (Lys1-Arg2-Pro3-Pro4-Gly5-Phe6-Ser7-Pro8Phe9-Arg10). In rats another kinin, Ile1-Ser2-Arg3-Pro4-Pro5-Gly6-Phe7-Ser8-Pro9-Phe10-Arg11 (T-kinin) is
produced under certain circumstances and binds to the same receptors as bradykinin [1,2]. A schematic
of the human kinin-kallikrein system is shown in Figure 1.
The release of kinins from precursor proteins (known as kininogens) is mediated by enzymes called
kininogenases [35]. The predominant enzymes responsible are kallikreins, but others, which include
trypsin, plasmin, and some snake venoms, also release kinins. Kininogens are primarily synthesized in
the liver and represent an abundant source of the precursors that are required for kinin generation. These
proteins are produced from alternative splicing of a single gene product and there are two forms: high
molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) [6]. Unlike
HMWK, which exists in the circulation as a complex with plasma pre-kallikrein, LMWK circulates
freely.
During immunological reaction, charged surfaceswhich may be derived from bacterial
lipopolysaccharide, oligosaccharides, connective tissue proteoglycans, or damaged basement
membranesfacilitate the conversion of factor XII to factor XIIa. Once factor XIIa is present, prekallikrein can be cleaved to its active form, known as plasma kallikrein. This enzyme acts upon its
preferred substrate, HMWK, to release bradykinin. Plasma kallikrein is further able to convert inactive
factor XII to active XIIa, thereby participating in a positive
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Figure 1
Diagram of the human kinin-kallikrein system including the native ligands for B1 and B2
receptor subtypes.
feedback loop. The cleavage of bradykinin from HMWK is highly localized since pre-kallikrein and
substrate (HMWK) circulate as a complex.
Another kinin, Lys-bradykinin (also known as kallidin), is produced via the action of the tissuekallikrein enzyme on LMWK. This enzyme is found in many tissues, either in the form of a precursor
requiring activation or as an active enzyme. In contrast to plasma kallikrein, which preferentially acts
upon HMWK, tissue kallikrein can release kallidin from either HMWK or LMWK. Through the action
of aminopeptidases, kallidin can subsequently be converted directly into bradykinin. This enzyme is
present in both the plasma and on the surface of epithelial cells.
Both bradykinin and kallidin can be degraded by a variety of plasma and cell surface enzymes
(kininases) [7]. The most widely recognized of these enzymes are kininase I, kininase II (angiotensin
converting enzyme, ACE), and carboxypeptidase N. In plasma, kininase I cleaves the C-terminal
arginine from both bradykinin and kallidin to form [des-Arg9] kinins. These [des-Arg9] kinins are known
to act as agonists of B1 receptors that are present in some species and have been implicated in the
pathophysiology associated with prolonged inflammation [810].
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Nearly all cells express kinin receptors that mediate the activities of both bradykinin and kallidin. The
activation of these G-protein coupled receptors causes relaxation of venular smooth muscle and
hypotension, increased vascular permeability, contraction of smooth muscle of the gut and airway
leading to increased airway resistance, stimulation of sensory neurons, alteration of ion secretion of
epithelial cells, production of nitric oxide, release of cytokines from leukocytes, and the production of
eicosanoids from various cell types [11,12]. Because of this broad spectrum of activity, kinins have been
implicated as an important mediator in many pathophysiologies including pain, sepsis, asthma,
rheumatoid arthritis, pancreatitis, and a wide variety of other inflammatory diseases. Moreover, a recent
report demonstrated that bradykinin B2 receptors on the surface of human fibroblasts were upregulated
three-fold beyond normal in patients with Alzheimer's disease, implicating bradykinin as a participant in
the peripheral inflammatory processes associated with that disease [13].
In contrast to the adverse physiologies associated with bradykinin release, there is a growing body of
literature that implicates bradykinin as a protective agent during periods of cardiac or renal stress
[1416]. In this regard there is substantial evidence that the cardioprotective effects afforded by ACEinhibitor treatment are a result of metabolically preserving bradykinin and are therefore mediated by
bradykinin B2 (and possibly B1) receptors [1718]. These results point to a possible therapeutic role for
a kinin receptor agonist.
Overall, the kinins are an important part of a well-organized physiological system. The various aspects
and interdependencies of the kinin system have been, and continue to be, the focus of intensive research
efforts in many laboratories. Many pharmaceutical companies have identified this system as an ideal site
for therapeutic intervention in many inflammatory diseases. Hence, there have been many diverse
approaches taken toward the discovery of antagonists (peptide and nonpeptide) of B2 and B1 receptors.
This review focuses on the structure-based design strategies pursued in our laboratories during the past
several years.
II. Ligand-Based Investigations
A. The Solution Conformation of Bradykinin
In the late 1980s when we began the pursuit of bradykinin receptor antagonists, information of relevance
to medicinal chemists was scarce. For example, not one nonpeptide antagonist of this receptor was
known, nor were any series upon which to base a structure-activity relationship. Moreover, all
publications described bradykinin as being highly flexible in an aqueous environment, such that no
structural mimetics could be rationalized. Of course the receptors had not been cloned at that time so
nothing was known about the primary sequence of the receptor or the three-dimensional structure.
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Initially our approach was to complete a detailed examination of bradykinin using two-dimensional
NMR methods in combination with empirical energy calculations [19]. Our strategy was derived on the
basis of spectral data, biological results from conformationally restricted analogs, as well as the
relationship between ordering in bradykinin and the dielectric environment of the solvent. Our guiding
hypothesis was that, although in aqueous solution bradykinin is conformationally random, the
biologically active form of the peptide is likely ordered and stabilized within the lipid bilayer of the cell
membrane prior to binding with its receptor. Alternatively, the receptor binding environment might also
be hydrophobic and thereby lead to similar conformational biases in the ligand. We presumed that an
appropriate solvent environment should be able to stimulate, at least in terms of hydrophobicity and
dielectric constant, the nature of a cell membrane, and a 90:10 d8-dioxane-H2O mixture was selected for
NMR experiments. It was anticipated that under these nonsolvating conditions the conformational
diversity of bradykinin might be severely restricted. The ultimate analysis of the two-dimensional NMR
data collected at 500 MHz supported a single major conformational species. There were five HN-CH
connectivities, one for each amide. This was confirmed in the 13C NMR spectrum where only nine
carbonyl resonances, one for each amino acid, were present.
In order to provide a starting point for subsequent molecular dynamics simulations the assumption was
made, based on multiple observed long-range amide-to-amide nuclear overhauser effects (NOEs), that it
was indeed a single major conformational species. Although bradykinin contains three proline residues,
the absence of any strong CHi-CHj+, cross peaks in the nuclear overhauser enhancement
spectroscopy (NOESY) spectrum was taken as proof that all peptide bonds were trans. In total, 35
interproton distances were extracted from the NOESY spectrum and, whenever possible, stereospecific
assignments for pro-R and pro-S hydrogens were made explicitly. A temperature-dependent study of the
chemical shifts of the amide protons resulted in a near-linear dependence suggesting no major
conformational changes were coinciding with the temperature change and thereby allowing a
comparison of slopes (/t). The lowest values obtained for these slopes corresponded to Phe8 and
Arg9 suggesting solvent sequestering for these amides.
Given the high Chou and Fasman probability of -turns in the sequences Pro2-Pro3-Gly4-Phe5 and Ser6Pro7-Phe8-Arg9 (3.79 10-4 and 1.99 10-4, respectively), the computational strategy employed was to
begin from two initial structures: (a) an extended strand, and (b) a structure containing these two
predicted turns. Utilizing custom routines written using the program CHARMm, version 21 [21], the
interproton distances were incorporated into the potential-energy expression in the form of an additional
potential-energy term. During the 3-ps heating step of the molecular dynamics, the temperature was
raised from 0K to 300K in steps of 20K every 0.2 ps. Since the target distances
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were poorly satisfied in the starting structures, the potential-energy term corresponding to the imposed
NOE data (ENOE) was applied gradually by increasing the scale factor in a nonlinear fashion such that it
was 0.0 after 0.2 ps, 0.1 after 1.2 ps, and 1.0 after the full 3.0 ps. Following 15 ps of equilibration, 7 ps
of incremental production dynamics was completed. During this stage the NOE scale factor was raised
from 1.0 to 4.5. By slowly raising the force constants for the NOE restraints as the target distances
became better satisfied, no dramatic increase in temperature was observed. Finally the NOE scale factor
was set to 5.0, 10 ps of production dynamics were completed, and an average structure was extracted
from the last 5 ps of the coordinate trajectory.
Analysis of the two average structures obtained from the two unique starting points demonstrated
convergence to a similar conformational species. In each, the sum of the NOE restraint energy was less
than 4.7 kcal/mol and the RMS deviation from the target distances was below 0.25 . Similar results
were obtained for each simulation when they were repeated without the electrostatic term being included
in the total potential-energy function. This important data lends credence to the hypothesis that the final
structures are derived from the NOE restraints and not by poorly represented electrostatic interactions.
The average dynamic structures are characterized as having all trans peptide bonds and hydrophobic side
chain groups oriented outward into solution, perhaps ready to interact with the receptor. There is a
possible 13 hydrogen-bonded turn bridging Phe5, although it is not explicitly defined by the NOE
data set. If present, then the preferred overall geometry would be U shaped and, if absent, an S shaped
geometry is possible based on coincident conformational analyses. According to the dihedral angle
values for Pro7 and Phe8, a type-II turn extending from Ser6 to Arg9 also exists. A variety of reports
have subsequently appeared that are in agreement with the conformation we described in this work.
A similar C-terminal turn structure was observed in an analogous NMR study of a first-generation kinin
antagonist, NPC 567 (DArg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-DPhe7-Phe8-Arg9), although the type of
turn was not the same. Our initial speculation was that this slight structural difference might partially
account for the functional differences of bradykinin and NPC 567. These solution conformations, one of
an agonist and the other of an antagonist, were subsequently used to focus the design and synthesis of
conformationally constrained peptide analogues of NPC 567.
B. Conformationally Constrained Bradykinin Antagonist Peptides
The ligand-based approach of conformationally constrained peptides has been widely used. The process
involves the incorporation of conformational constraints into known peptides, either agonist or
antagonist, which enforce a
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predictable geometry. A series of peptides containing these types of constraints can be useful for
extrapolating the steric and electronic environment of a given binding site. This structural information
can be derived regardless of whether or not the constrained peptide binds to the target receptor. Since
peptides can be prepared rapidly, it is typical to establish a structure-activity relationship using them and
then at some later time transpose that information onto a nonpeptide lead molecule in an attempt to
improve its potency.
As part of an expansion upon the hypothesis that a C-terminal turn was a structural prerequisite to highaffinity antagonist binding, a novel series of constrained decapeptides was prepared [2224]. These
peptides are of the sequence DArg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-DHype7-Y8-Arg9, where Y is either
tetrahydroisoquinoline-3-carboxylic acid (Tic), or octahydroindole-2-carboxylic acid (Oic). DHype
denotes an organic ether of D-4-hydroxyproline in either the cis or trans geometric form. The C-terminal
portion of a representative member of this class of peptides was shownfirst by empirical calculation
[22], then by NMR at 600 MHzto adopt a turn nearly unambiguously (figure 2) [25,26]. Moreover,
it was shown by calculation that the turn was adopted regardless of the nature of the ether group (alkyl,
aryl, etc.) or its geometry (cis or trans). Hence, a diverse series of these peptides was initially used as a
tool to probe the steric and electrostatic topology of an antagonist
Figure 2
Lowest 5 kcal
of the calculated overall potential energy surface for a model
peptide of Ser-DHype(trans propyl)-Oic-Arg. The contour interval is 0.5 Kcalmol-1
and the highest (outermost) and lowest contour energy values are labeled.
Superimposed on the contour plots are values for i+1 and i+2 from each of the
thirty structures generated from the NMR data corresponding to the tetrapeptide
Ser-DHype(trans propyl)-Oic-Arg.
mol-1
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binding site on the bradykinin B2 receptor in the guinea pig ileum. The cis ethers, in all cases, bound to
the receptor with significantly lower affinity than did the trans. A more complete listing of the peptides
used in the study is shown in Table 1. These results support the hypothesis that the domain of the
receptor that binds these antagonist ligands is partly made up of a hydrophobic cavity about one side of
the C-terminal turn. However, adjacent to the other side of the
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Figure 3
Receptor binding curves for the binding of NPC 17410 and NPC 17643 to B2 receptors
from the guinea pig ileum and cloned rat and human B2 receptors. Legends are noted on
the figure.
turn, there appears to be some type of steric interference (or lack of a pocket) that might otherwise
accommodate the ethers of the cis configuration.
More recently, bradykinin B2 receptors have been cloned from both rat and human sources [27,28]. In
receptor-binding experiments using these new receptors, selected members of the DHype-containing
decapeptides were used to probe these receptors [24], a representative sample of the data is shown in
Figure 3. Specifically, NPC 17643 (a trans propyl ether of D-4-hydroxyproline at position 7) and NPC
17410 (a cis propyl ether of D-4-hydroxyproline at position 7) were used. Although the trans ethercontaining decapeptide behaved similarly in binding assays directed toward the bradykinin B2 receptors
in guinea pig, rat, and human, the cis ether-containing decapeptide, NPC 17410, displayed an interesting
pharmacology. In particular, NPC 17410 bound with similar affinity to both the guinea pig and rat
bradykinin B2 receptors, but had an appreciably higher affinity for the human B2 receptor. This result
strongly suggests that there are slight structural differences in the antagonist binding sites of the rat and
human B2 receptors. With clones available for the rat and human bradykinin B2 receptors, this
prompted a systematic search using NPC 17410 binding to rat/human bradykinin B2 receptor chimeras
and point mutations in an attempt to discover residues on the receptor that comprise this antagonist site.
The details and results of the subsequent application of these novel receptor-probing ligands is fully
described later in this chapter.
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The des-Arg9 forms of these peptides have also been shown to have high affinity for the recently cloned
human B1 receptor [24]. An extension of the work described herein would be to use a more complete
series of des-Arg9 DHype-containing nonapeptides to probe the binding site of this new receptor where
other interesting pharmacological differences are likely to exist since the B1 receptor is only 33%
homologous to the human B2 [29].
In summary, the DHype-containing decapeptides have been useful in many regards. First, they
incorporate a novel -turn mimetic that was alternatively functionalized and used to probe the unknown
topology of the guinea pig, rat, and human bradykinin B2 receptors. In this role, one of these tools
showed differential pharmacology between rat and human forms of the receptor. This tool was used in a
synergistic fashion with subsequent molecular biological and computational procedures in the
elucidation of an antagonist binding site. Second, these peptides, together with another potent
decapeptide antagonist with similar conformational constraints [30,31], provide the first strong
experimental evidence that high-affinity decapeptide bradykinin receptor antagonists adopt a C-terminal
turn in the receptor-bound conformation. Third, certain members of this series of decapeptides contain
alkyl ethers of D-4-hydroxyproline at position seven. In this regard, they are the very first examples of
decapeptide bradykinin receptor antagonists that do not contain a D-aromatic amino acid at the seventh
position as had been previously deemed to be essential. Commercially, this renders the series patentably
distinct from all other known bradykinin receptor antagonists. Finally, several members of the series
(i.e., NPC 17731, NPC 17761, NPC 17974) are among the most potent antagonists for this receptor yet
reported. Hence, there may be applications for these compounds as human therapeutics.
Several second generation decapeptide antagonists have been reported, but the prototype from the
class, which was first to be reported, is HOE 140 (DArg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-DTic7-Oic8Arg9) [30,31]. This decapeptide has also been shown to preferentially adopt a C-terminal turn,
consistent with the previous discussion [26,32,33]. The side chain of DTic at position seven is, however,
flexible. While side-chain rotational movement is not allowed, the saturated six-membered ring easily
undergoes an endo/exo ring-flipping motion. Hence, the turn predominates about the backbone
dihedral angles, but the side chain of DTic could be either endo or exo ring flipped in the receptor-bound
conformation. In the absence of an appropriate x-ray crystallographic structure, there is no definitive
means of establishing which possibility is correct. This type of ring-flipping conformational change
serves to orient the bulky hydrophobic side chain of the DTic residue to either one side of the turn, or
the other. The data collected from the decapeptides containing either cis or trans ethers of D-4hydroxyproline at the analogous position in the
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sequence (discussed above) support a hypothesis that the DTic is exo ring flipped in the receptor-bound
state.
There are two factors that must be considered when applying structure-activity-relationship (SAR)
information from a series of peptides toward the design of nonpeptide mimetics and putative library
scaffolds. One is in regard to the backbone conformation that primarily serves as a structural scaffold
upon which the various functionalities (side chains) are attached. The other factor is the side chains
themselves whose spatial positions are primarily dictated by the backbone structure. Usually, the threedimensional arrangement of these differing chemical groups are responsible for affinity and triggering of
the receptor. Knowledge of the relative importance of the individual side chains and amide bonds for
receptor affinity is therefore a critical aspect of small molecule design from a peptidic structure-activity
relationship.
Conformationally constrained derivatives of HOE 140 have been prepared in continuing efforts to
elucidate the ideal backbone conformation peptide antagonists must adopt for bradykinin B2 receptor
interaction. One such series made use of C- or N-methyl substituted amino acids, incorporated at
position(s) Gly4, Phe5, or both, in the peptide D-Arg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-D-Tic7-Oic8-Arg9
(NPC 18545) [34]. An N-methyl substitution in the backbone of an L-amino acid is known to disfavor
helical, or twisted, backbone conformations while favoring an extended backbone. The contrasting Cmethyl modification tends to favor a helical (twisted), rather than extended, conformation [35,36]. These
conformational preferences apply only to the backbone , angles (where i and i correspond to
backbone dihedral angles for residue i, defined by the four adjacent amino acid backbone atoms Ci-1-NiCi-Ci band Ni-Ci-Ci-Ni+1, respectively) of the amino-acid residues bearing the modification. Receptor
binding assays were performed in membrane preparations of the guinea pig ileum, a source of B2
receptors, wherein these constrained peptides were evaluated for their abilities to compete with
bradykinin binding.
With the exception of the C-methyl-Phe5-containing peptide (NPC 18540), each conformational
constraint caused a significant, at least 1000-fold, loss in binding affinity with respect to the
unconstrained parent peptide, NPC 18545. There are several factors that could contribute to the poor
receptor affinities measured for these peptides. In addition to the possible induction of an adverse
conformation via the N-methyl substitution, this modification also eliminates an amide proton that might
be an important hydrogen-bond donor during ligand-receptor interaction. Furthermore, the N-methyl
substitution enhances the likelihood of trans-cis amide bond isomerization, which could also disrupt an
optimal ligand-receptor interaction by altering the spatial display of the local side chains. The C-methylPhe5 substitution of NPC 18540 is well
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tolerated by the receptor as evidenced by only a seven-fold loss in receptor affinity (Ki=0.54 nM) with
respect to the parent of the series, NPC 18545. This implies that the , backbone dihedral angles about
Phe5 are on the order of -60, -60 in the biologically active conformation. This combination of dihedral
angles represents a helical twist or kink in the midsection of the peptide.
Since the original submission of the manuscript describing these constrained linear and cyclic peptides,
bradykinin B2 receptors have been cloned from other species including human [27,28]. Toward the goal
of designing a nonpeptide antagonist as a human therapeutic agent, it would be interesting to evaluate
these analogues against these newly reported receptor homologues. This would likely be fruitful and
valuable given that there is evidence, including that presented in this chapter, showing that guinea pig
and rat B2 receptors differ structurally from the human B2 receptor at the antagonist binding site. Hence,
a structure-activity relationship established against the guinea pig or rat receptors could be misleading in
the context of potential human therapeutics.
A systematic study of the relative importances of amides and side chains in a prototypical second
generation antagonist, NPC 18545 (DArg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-DTic7-Oic8-Arg9) has
recently been described [37,38]. The D-Arg0 and Ser6-DTic7-Oic8-Arg9 segments were left intact in all
peptides on the assumption that N-terminal positive charge(s) and a hydrophobic C-terminal turn are
minimally required for binding. In a systematic fashion, the amino acids in the core of the peptide (Arg1Pro2-Hyp3-Gly4-Phe5) were substituted with glycine, an amino acid bearing no chirality or side chain.
Binding assays, either in membranes from the guinea pig ileum or in membranes from a stable cell line
expressing the human B2 receptor, were performed on each peptide and the results compared with the
parent, NPC 18545, which has a Ki against [3H]-bradykinin of 0.08 nM. The elimination of all chirality
and sidechain moieties in the segment Arg1-Pro2-Hyp3-Gly4-Phe5 via replacement by Gly1-Gly2-Gly3Gly4-Gly5 (NPC 18152), led to a peptide that no longer binds the receptor. This demonstrated that one or
more of the side chains in this segment are critical during ligand-receptor interaction. Incorporation of
either the Arg1 or Phe5 side chains led to improved potency (285 nM and 483 nM, respectively).
Constructing a peptide with side chains of both Arg1 and Phe5 in place (NPC 18149) yielded a peptide
with good affinity, Ki of 13.7 nM. Overall, this study shows that to maintain potency in the low
picomolar range, peptides in this series require Arg1, Phe5, and either Pro2 or Hyp3 (but not both). The Nterminal charged moieties and the hydrophobic C-terminal turn are also required. Potencies in the low
nanomolar range are attainable without including the side chains or chirality associated with Pro2 and
Pro3. These data have
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subsequently been successfully applied toward the design and synthesis of several nopeptide scaffolds
and mimetics. Ultimately these mimetics were assembled in a combinatorial fashion as discussed in
Section IV.
In a related study, wherein NPC 18149 (DArg0-Arg1-Gly2-Gly3-Gly4-Phe5-Ser6-DTic7-Oic8-Arg9; Ki =
13.7 nM; Guinea pig ileum) was taken as the lead peptide, the relative contributions to binding affinity
from each amide bond in the segment Arg1-Gly2-Gly3-Gly4-Phe5 were examined. Aminovaleric acid was
used in a systematic fashion as a surrogate for any pair of adjacent Gly-Gly residues in the peptide.
Aminovaleric acid is atomically identical to Gly-Gly with the exception that the amide bond linking the
two glycines is replaced by two methylenes. The synthesis of Gly4-Phe5 required a special Gly-Phe
mimic that has since been reported [39]. Since this substitution introduces flexibility into the peptide, it
is a means of probing the structural role a given amide bond plays during receptor interaction. Potential
hydrogen-bond donor and acceptor groups in the amide bond are removed via this substitution, which
yields additional insights into potential electrostatic interactions that may also be important during
ligand-receptor interactions.
The conclusions drawn from the data are that in terms of structural or electrostatic interactions with this
antagonist site on the receptor, the amide bond linking residues two and three may not be as critical as
those linking residues three to four and four to five.
Each of these investigations was aimed toward an understanding of either the backbone conformation of
this prototypical decapeptide or the relative importance of the functional groups in the side chains that
make significant contributions to receptor affinity. From the former, nonpeptide frameworks and
scaffolds can be imagined. From the latter, insights into which functionality is required for high-affinity
binding is derived. The remaining challenge is to reassemble these fragments onto synthetically feasible
nonpeptide frameworks as potential new lead compounds. Our approach toward addressing this
challenging problem is described later in this chapter.
III. Receptor Structure-Based Investigations
A. Elucidation of an Agonist Binding Site on the B2 Receptor
In addition to the deductions one might make about a receptor binding site on the basis of receptor
binding data from conformationally constrained ligands as previously described, models of bradykinin
and bradykinin antagonists bound to their respective sites on the receptor as complimentary aspects of
the overall strategy are also valuable. Unfortunately, due to the nature of the bradykinin
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receptor, it has not yet been obtained in crystalline form, nor is it likely to be in the near future.
The bradykinin receptor is a member of a family of receptors for which an intracellular interaction with
a G-protein is a critical part of the signal transduction pathway following agonist binding. Structurally,
these G-protein-coupled receptors extend from beyond the extracellular boundary of the cell membrane
into the cytoplasm. The tertiary structure is such that the protein crosses the bilayer of the cell membrane
seven times, thus forming three intracellular loops, three extracellular loops, and giving rise to
cytoplasmic C-terminal and extra-cellular N-terminal strands. It is generally presumed that the
transmembrane domains of these receptors exist as a bundle of helical strands. This assumption is
derived primarily from the known structure of the trans-membrane portions of a structurally related
protein, bacteriorhodopsin [40].
G-Protein-coupled receptors do not lend themselves to analysis by either NMR or x-ray crystallography
due to their structural dependence on an intact cell membrane. In our laboratories we pursued this
valuable structural information by utilizing a combination of structural homology modeling, molecular
dynamics, systematic conformational searching methods, and mutagenesis experiments. The
combination of these techniques led to a proposed model of bradykinin bound to the agonist site on its
receptor [41].
A hydrophobicity (Kyte-Doolittle) calculation [42] on the amino acid sequence of the rat bradykinin
receptor yielded seven segments, each of which were 21 to 25 contiguous residues with predominantly
hydrophobic side chains. These were presumed to be the seven transmembrane portions of the receptor.
Cartesian coordinates of the backbone atoms within each of these seven segments were built by
structural homology from the cryomicroscopic structure of the analogous segments of
bacteriorhodopsin. Subsequently, side chains were added to these seven segments as appropriate for the
rat bradykinin receptor, and the resulting geometry was optimized via constrained energy minimization
to alleviate bad contacts. Extracellular and intracellular loops were extracted from the Protein Data Bank
library, following a geometric search based upon a vector defined by terminal alpha carbons in adjacent
helices. The model was subsequently subjected to a series of constrained and unconstrained energy
minimizations as well as molecular dynamics simulations. The resulting structure of the receptor was
used in a novel two-step docking procedure.
Following our hypothesis that bradykinin adopts a C-terminal turn upon complexation with the
receptor, the , backbone dihedral angles in the tetrapeptide corresponding to the C-terminus of
bradykinin (Ser-Pro-Phe-Agr) were constrained in a harmonic fashion (force constant = 15 Kcal -1 mol1) to values that define a type II' -turn [43]. This tetrapeptide probe was then systematically translated
about the interior of a theoretical box inscribing the rat
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receptor model. The translations were such that the tetrapeptide probe molecule was incrementally
repositioned within the receptor by following a 3 3 3 grid pattern. At each new position, both
the probe and receptor were reset to their initial conformations, then the geometry of the complex was
optimized using 200 steps of steepest descent followed by 500 steps of Adopted-Basis Newton-Raphson
energy minimization. Subsequently, the sum of the steric and electrostatic contributions to the overall
potential energy (interaction energy)as measured only between the tetrapeptide probe molecule and
the atoms of the receptorwere calculated. Slices through the receptor illustrating the energy of
interaction as grayscale contour lines (darker gray = lower interaction energy) for that portion of
receptor that was sampled by the tetrapeptide probe molecule is shown as an edge-on, frontal view in
Figure 4. In this Figure it is qualitatively clear where the transmembrane domains are located (white), as
well as where the most favorable sites of probe interaction are located (black).
Figure 4
Complete group of contour plots showing energy of interaction between probe
and receptor. Each contour plot corresponds to a different horizontal slice as part
of the first stage in the conformational search. Darker gray indicates most favorable
interaction and the light shades represent least favorable interactions.
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This initial stage of the docking process was used to reduce the computational difficulties that would be
inherent in tumbling a complete bradykinin molecule (which has great flexibility) about the receptor
in a similar fashion. However, following this initial stage, insight into those regions of the receptor
capable of accommodating the C-terminal portion of the bradykinin molecule was obtained. On the basis
of energetics, and as qualitatively shown in Figure 4, those particular regions are clustered in the central
part of the receptor near to the extracellular domain. Using this information as a steering device to limit
the size of the problem, an exhaustive conformational search was performed using the entire nineresidue sequence of bradykinin as a probe molecule, again enforcing a C-terminal turn via dihedral
angle constraints. Specifically, 24 unique geometric orientations (eight on each of three axes) of the
bradykinin molecule were sampled at each of 100 grid points identified during the initial stage as likely
zones to bind the tetrapeptide probe molecule. Bradykinin-receptor complexes within the lowest 150
kcal mol-1 interaction energy with respect to the lowest found (17 complexes out of 2400) were grouped
into sets of related conformational families, of which there were five. Computationally, each of the five
complexes were presumed to be equally likely. All of these simulations were accomplished using
custom routines written using the program CHARMm [21].
To guide the selection of which of the five bradykinin-receptor complexes to consider a lead model,
supporting experimental evidence was sought from site-directed mutagenesis experiments. This support
was taken primarily from work describing bradykinin binding assays performed of mutant rat B2
receptors [44,45]. The underlying strategy of the mutation studies was based on the hypothesis that,
since bradykinin has positive charges at either end of its sequence (Arg1 and Arg9), separated by a group
of rather hydrophobic amino acids (Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8), it was likely that some acidic
residues in the receptor participated during ligand binding. Several mutant receptors were made such
that each contained either a point mutation or a small cluster of point mutations, wherein native residues,
having negatively charged side chains (Asp, Glu), were replaced by alanine(s). Table 2 lists the initial
cluster mutations (rat) that were prepared as well as the follow-up single point mutations (rat).
Figure 5 shows a stereoview of the selected ligand-receptor complex chosen on the basis of best
agreement with the results of these mutagenesis studies. None of the other four putative complexes were
in agreement with this experimental data and were not considered further. Of particular significance in
this work was that the trans-membrane residue Glu49, when mutated to alanine, showed no adverse
effect on bradykinin receptor affinity with respect to rat wild type. A similar result was reported for the
Glu196 rarrow.gif Ala196 mutation. These residues are remotely situated with respect to the proposed site
of bradykinin
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binding and are colored light gray in Figure 5. In contrast, the [Asp175, Glu178,179] rarrow.gif
Ala175,178,179 cluster mutation showed a 12-fold loss in bradykinin binding affinity, and the [Glu282,
Asp286] rarrow.gif Ala282,286 cluster mutation lost 17-fold with respect to the wild type receptor. The
Asp268 rarrow.gif Ala268 and Asp286 rarrow.gif Ala286 point mutations caused 19-and 28-fold
respective losses in affinity for bradykinin. Close inspection of the bradykinin Arg1 side chain location
and surrounding receptor interactions led to the suspicion that Asp286 and Asp268
Figure 5
Proposed model of bradykinin bound to the rat B2 receptor at the agonist binding site.
Only the upper portion of the receptor is shown as gray helical ribbons. Bradykinin
backbone and side chain atoms are shown as thick white licorice. Positions of point
mutations having no significant adverse effects on bradykinin binding are shown as
light gray spheres. Positions of mutations affecting bradykinin binding are shown as
dark gray spheres.
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might be jointly interacting either with the guanidino group in the side chain of Arg1 or the N-terminal
amino group in bradykinin. Therefore a receptor containing a double mutation (Asp268,286 rarrow.gif
Ala268,286) would be expected to show a much more dramatic loss in affinity for bradykinin than would
receptors containing the individual point mutations. The appropriate double mutation experiment
confirmed this by causing a 500-fold loss in affinity for bradykinin, as predicted (Table 2). The
mutagenized residues of this double mutant B2 receptor are colored dark gray in Figure 5. This type of
an ionic interaction is also precedented by the body of literature that exists supporting the requirement of
an N-terminal arginine residue and a free N-terminal amino group in both bradykinin peptide agonists
and antagonists for high affinity binding. All of these mutant receptors were demonstrated to be
functional receptors on the basis of bradykinin-induced membrane depolarization in a Xenopus oocyte
expression system [44,45].
The selected agonist site model is characterized by an overall twisted S-shape ligand, similar to the
conformation of bradykinin determined previously in a hydrophobic environment by NMR [19,46].
Overall, the model suggested that the N-terminal amino and guanidine groups of Arg1 interact directly
with negatively charged amino acids in extracellular loop three, and the C-terminal end is in a -turn
conformation buried just below the extracellular boundary of the trans-membrane domain of the
receptor. Noteworthy is the presence of a hydrophobic cavity in our receptor model located adjacent to
Pro7 of the bradykinin ligand. This cavity is made up, in part, by the residues Phe261, Leu104, Val108, and
Ile112. Given the historical significance of position seven in peptide bradykinin-like ligands, these
residues represent interesting targets for further mutagenesis experiments. One such result, the mutation
of Phe261 to Ala261, has already been described, and the results were supportive of this proposed model
[47]. Antibodies to the extracellular loops two and three have also been shown to compete with
bradykinin binding, lending further experimental support for an extracellular domain on this agonist
binding site.
More recently, chemical crosslinking combined with site-directed mutagenesis was used to analyze the
bradykinin binding site in the human B2 bradykinin receptor [48]. Previous studies using the bovine B2
receptor showed that heterobifiunctional reagents reactive to amines and free sulfhydryls crosslink the
bound bradykinin N-terminus to a sulfhydryl(s) in the receptor [49]. To identify this sulfhydryl(s), two
conserved candidate residues in the human B2 receptorCys20 in the N-terminal domain and Cys277 in
extracellular loop 3were mutated to serine residues. Single and double mutants were expressed in Cos
7 cells. All mutants bound [3H]bradykinin with typical B2 receptor specificity. The heterobifunctional
reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester crosslinked bradykinin to wild-type and
mutants with maximum efficiencies of 35% (wild type), 40% (Ser20), 20% (Ser277), and 0%
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(Ser20, Ser277). This clearly demonstrated that Cys20 and Cys277 are the only sulfhydryls available for
crosslinking receptor-bound bradykinin. These results provided direct biochemical evidence that the Nterminus of bradykinin, when bound to the B2 receptor, is adjacent to extracellular loop 3 and the Nterminal domain in the receptor.
Further consideration of the model led to the hypothesis that agonist peptides may minimally require an
intact C-terminal -turn structure with appropriate side chains in place and N-terminal amino and
guanidine groups for primary electrostatic interaction(s) with Asp286 and Asp268 in extracellular loop 3.
As a test of this hypothesis, the prototypical second generation antagonist, NPC 18545, (DArg0-Arg1Pro2-Hyp3-Gly4-Phe5-Ser6-DTic7-Oic8-Arg9) was modified such that residues 25 were replaced by a
simple twelve carbon chain spacer (12-aminotridecanoic acid). The resulting compound, NPC 18325,
contains only the appropriately charged moieties at the N-terminus, separated by a simple organic spacer
moiety from a known turn forming tetrapeptide [25,26]. This pseudopeptide was tested in the human
bradykinin B2 receptor binding assay and found to have a Ki of 44 nM against [3H]bradykinin binding
[50]. Functionally, this pseudopeptide was an agonist as measured by its ability to stimulate IP
production in a stable CHO cell line expressing the human B2 receptor and in WI-38 cells. Since it was
designed on the basis of an agonist site on the receptor, this result was not completely surprising despite
the incorporation of the DTic-Oic pair at the C-terminus that previously had been shown to be critical in
high affinity antagonists.
Subsequently the length of the linear carbon chain was varied to further explore the hypothesis that the
agonist site on the receptor had two domains, a hydrophilic site in the extracellular loop area and a
hydrophobic domain in the transmembrane area [50]. Presumably, the two terminal portions of NPC
18325 can only simultaneously interact with each putative domain of the receptor binding site when the
carbon chain is 1213 methylenes long. But if the carbon chain length is shortened too far, this ligand
might be unable to simultaneously interact with both domains, resulting in an affinity loss. This series of
pseudopeptides and their respective human bradykinin B2 receptor affinities are presented in Table 3.
The data are consistent with the hypotyhesis since the receptor affinity decreases as the carbon chain
length is shortened. An alternative explanation of the data is that a certain hydrophobicity profile is
required of the compounds in this series for good receptor affinity. These results indicated that there
may be additional hydrophobic or flexibility prerequisites to binding in this series of pseudopeptides.
One noteworthy observation was that NPC 18325 showed divergent behavior when evaluated against
different species homologues of the bradykinin B2 receptor. Notably, in the guinea pig ileal membrane
preparation assay, the affinity for the receptor was approximately 10-fold less than what had been
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observed for the human B2 [37]. Furthermore, in contrast to the functional activity of NPC 18325 at the
human B2 receptor, the compound is a functional antagonist as measured against bradykinin-induced
contraction of the isolated guinea pig ileum (pA2 = 5.5). These findings are in agreement with the
concept that as a ligand is made smaller (i.e., fewer contact points possible with the receptor), the subtle
structural differences in the binding sites on species variants of the same receptor become amplified.
This observation further supports a cautionary posture toward developing nonpeptide antagonists for use
in human diseases on the basis of results obtained in some animals including the guinea pig. Taking this
new molecule as a lead structure, together with the receptor model and structure-activity relationship
associated with related peptides including cyclic antagonists, the pursuit of several related
pseudopeptides was undertaken.
B. Elucidation of an Antagonist Site on the B2 Receptor
There have been a variety of single alanine point mutations experimentally introduced into both rat and
human bradykinin B2 receptors. Several of these have been shown to decrease the affinity of bradykinin
to the receptor and have been implicated structurally near the agonist binding site. In contrast, at the
time of this manuscript, there have been no mutations reported that adversely affect the ability of any
peptide antagonists to bind to the receptor. Furthermore, antibodies raised against the certain
extracellular domains of the kinin receptor compete with bradykinin for binding to the receptor but have
no inhibitory
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action on the binding of antagonist peptides. In addition, it has been shown that bradykinin can be
covalently crosslinked to the B2 receptor while antagonists cannot. These observations have fostered the
belief that the agonist and antagonist binding sites of the receptor are not the same. At best, they may be
partially overlapping, although there is no direct evidence for this. The ultimate identification of the
amino acid residues that make up the antagonist site would be another important step toward the goal of
structure-based design of novel nonpeptide antagonists.
As described in a previous section of this chapter, characterizations of the bradykinin B2 receptors from
rat and human using NPC 17410 (Figure 3) revealed different pharmacologies. Specifically, it showed a
higher affinity for the human B2 receptor than it did for the rat B2 (human IC50 = 0.95 nM, rat IC50 =
48.0). This ligand tool provided a means for evaluating a series of bradykinin rat/human B2 receptor
chimeras [5153]. Several different chimeras were prepared in a systematic fashion and the affinity of
NPC 17410 was determined for each. The chimeras are depicted schematically in Figure 6 together with
the IC50 values determined for NPC 17410. Chimeras I through III sample the N-and C-terminal sections
of the receptor for any contributions to an antagonist binding site. The remaining chimeras sample the
core transmembrane domains of the receptor. Each chimera was shown to induce a membrane
depolarization similar to wild type receptor in response to bradykinin when expressed in Xenopus
oocytes. For each NPC 17410 assay, [3H]-NPC 17731 was used as the radioligand.
From this systematic approach, specific groups of contiguous residues within the receptor were
identified as possible contributors to an antagonist binding site. The NPC 17410 binding to chimeras III,
IV, and VIII showed rat-like pharmacology (low NPC 17410 affinity). The NPC 17410 binding to
chimeras I, II, VI, and VII showed human-like NPC 17410 pharmacology (high receptor affinity).
Binding to chimeras V and VIII, however, was similar to rat-like NPC 17410 pharmacology, but the
affinity of the compound was slightly shifted back toward human-like results. Comparisons of rat and
human receptor sequences in the regions sampled by the chimeras reveals that only two clusters of
residues differ between rat and human B2 receptors. Specifically, TM2 has the same sequence in rat and
human receptors so it is unlikely that the differential pharmacology associated with NPC 17410 binding
can be attributed to residues there. However, TM3 has a cluster of 3 residues that differ (rat rarrow.gif
human: Thr110 rarrow.gif Ala108, Met111 rarrow.gif Ile109, Tyr113 rarrow.gif Ser111) and TM6 has a
cluster of 5 residues that differ (rat rarrow.gif human: Phe259 rarrow.gif Leu257, Leu256 rarrow.gif
Ile254, Val255 rarrow.gif Ile253, Gly252 rarrow.gif
Leu250, Ala249 rarrow.gif Val247) in rat and human receptors. These differences represent important
targets for follow-up point (and cluster) mutation experiments. Our current thinking is that the largest
effects on NPC 17410 pharmacology, if any, might be derived from the TM3 mutants since
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Figure 6
Rat and human B2 receptor chimera constructs and affinity data
for binding NPC 17410. Also shown is the affinity NPC 17410 to
rat and human wild type receptors.
between rat and human, these are quite diverse. However, it is also possible that the cluster of residues
identified in TM6, while not radically dissimilar, may as a group create different hydrophobic
environments between these species homologues. The most significant individual difference within the
TM6 zone is the Phe259 (rat) rarrow.gif Leu257 (human) swap and might therefore be most significant
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Figure 7
Schematic of the primary amino sequence of the human B2 receptor. Shown
in black are residues experimentally identified as contributing to an agonist binding site.
The dark gray residues are suspect positions for contributing to an antagonist site. The
residues colored light gray have been mutagenized only in the rat B2 receptor, but they
are conserved in the human. The Thr263 rarrow.gif Ala mutation interferes with agonist binding
only, while Gln260 partially interferes with agonist and first generation antagonist
binding.
within the context of these TM6 residues. Currently, we have prepared these mutant receptors, but at this
time binding to NPC 17410 remains unfinished.
A summary of the amino acids in the human B2 receptor implicated in comprising either agonist or
antagonist sites are highlighted in Figure 7. Marked in dark black are the residues of extracellular loop 3,
TM 6, and the extracellular N-terminal segment that have been shown to participate in agonist binding.
Marked in dark gray in TM 6 and TM 3 are residues likely to partially comprise an antagonist binding
site based primarily on the chimeric receptor studies described previously, although there is no explicit
experimental evidence as yet. Shown in light gray are two residues that are conserved between rat and
human B2 receptors. Mutagenesis experiments have been done on this pair in the rat B2
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receptor with interesting results [54]. Mutations in Thr263 only affect agonist binding, not antagonist.
Mutations in Gln260 affect binding of bradykinin and first generation antagonist peptides. As depicted in
the figure, it is possible that the agonist and antagonist binding sites have domains on opposite sides of
the helix that makes up TM 6, with Gln260 being situated partly in both.
IV. Design and Combinatorial Synthesis of Nonpeptidic Antagonists
As was previously described, a significant body of information was generated that provides insights into
the key structural features of bradykinin receptor binding sites and the residues that participate in ligand
binding. In addition, from the ligand-based studies, knowledge about relevant structure-activity
relationships was acquired. Our modular synthetic strategy was based primarily upon the recognition
that high-affinity ligands appear to be comprised of three domains. These domains are (1) a positively
charged N-terminal segment, (2) a midsection containing a bend or twist with some hydrophobic
substituent attached and, (3) a C-terminal segment of appropriate hydrophobicity and structurally
simulating a type II' turn. Models of potent cyclic and linear peptide bradykinin receptor antagonists
(described previously) were used in a comparative fashion to select nonpeptide ring systems from a
database of chemical structures fine chemicals database. For each, some degree of chemical diversity
was achieved by altering one of several parameters including, o, m, or p substitution of an aromatic ring
or nature of alkyl substituent(s) as well as point(s) of synthetic attachment [55,56].
Each nonpeptide fragment was designed within the framework of several criterion. First, a given
scaffold must closely match the known SAR and be compatible with the putative ligand binding site
structure. Second, each scaffold must be a relatively simple synthetic target, having readily available
starting material, no chiral centers and having a total synthesis of not more than 45 steps. Finally, each
template must have a C-terminal carboxylate and an N-terminal amino group with no interfering
functionality such that it could be readily used in a solid phase synthetic strategy. Given that each
nonpeptide we identified was a viable surrogate for either the second or third domain of high-affinity
ligands (as described above) our goal was to rapidly explore the receptor affinities of all possible
combinations of these nonpeptide templates at position X and Y of the sequence DArg-Arg-X-Y-Arg,
hence a combinatorial synthetic approach was taken.
In this study, there were four linear aminoalkanoic acids [50], four different cinnamic acids, three
different carbolines, three different phenanthridinones, and five different spirocyclics. The variability in
the phenanthridinione series
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was that the central ring could be opened or cleaved and the amino group could be meta or para
substituted on the latter. In the carboline series, the cyclic amino group was either at the or position
of the cyclohexenyl ring and the methylene chain bearing the C-terminal carboxylate could be of
variable length. The spirocyclic series was varied by alkyl, cycloalkyl, and aryl substitution on the fivemembered ring amine nitrogen. The cinnamic acids had two carbon chains that could be of varying
length, one of which had the further possibility of containing a double bond(s).
Rather than perform individual syntheses of all possible combinations of these nonpeptide units,
members of each ring type or scaffold family were pooled in equimolar amounts prior to incorporation
into the sequence DArg-Arg-X-Y-Arg. Since each individual member of a given pool was constructed
on a similar carbocyclic scaffold, the chemical environment of the N-terminal amino group and Cterminal carboxylate groups were expected to follow similar kinetic and thermodynamic controls during
the attachment of the nonpeptide residue to the growing peptide chain. The use of these smaller, directed
libraries made it readily practical to obtain HPLC and mass spectral data for each and therefore confirm
the composition of the library.
Ultimately, 10 libraries of novel nonpeptidic structures were synthesized following typical solid-phase
methodologies. Each library contained from nine to thirty-six different compounds in approximately
equimolar amounts. Unpurified libraries were tested in a receptor binding assay utilizing membrane
preparations from a stable CHO cell line expressing the human B2 receptor. Each library was tested at
concentrations between 10 nM and 1M. The ability of each library to inhibit[3H]-bradykinin binding
was assessed and the results are presented in Figure 8a. Although this type of screening is highly
qualitative, certain libraries appear in Figure 8a that show higher affinity to the receptor than other
libraries. Library one (of the series DArg-Arg-PH-CN-Arg) was ultimately selected for further
deconvolution. This library was further broken down (decoded) in order to determine which
compound(s) were responsible for the apparent activity. It is important to note that breaking these
libraries down to elucidate the structure of the hit(s) was feasible due to the inherently small size of each
library.
Library one contained 12 different structures (recall that there were originally three different
phenanthridinones and four different cinnamic acids). The first deconvolution step of the approach is
shown in Figure 9. Here, only the CN position was randomized, and the PH moieties were specific. This
led to the preparation of three new libraries of 4 compounds each. Receptor binding was again
performed as before and only one of these three new sublibraries showed affinity for the receptor at 10
M (Figure 8b). The final step in the process required to elucidate the active component(s) was to
synthesize and purify each of the 4 members of this library as shown in Figure 9. Receptor binding on
these
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Figure 8
(a) Binding assay results for 10 original nonpeptidic libraries
of the sequence DArg-Arg-X-Y-Arg, where X and Y are defined
as PH = phenanthridinone, CB = carboline, SP = spirocycle,
SC = straight chain, CN = cinnamic acid. Each library was tested at
1 nM and 10 nM. Results were compared to cold bradykinin
binding, which was tested at two lower concentrations, 0.1 nM
and 1 nM. Panels (b) and (c) correspond to the receptor binding
results obtained using the two breakdown steps from original
library number 1, as shown in Figure 9.
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Figure 9
Composition of ten original nonpeptidic libraries of the sequence DArg-Arg-X-Y-Arg.
X and Y were selected from the set of scaffolds shown in Table 1. Also shown
are the subsequent breakdown libraries from original library number 1. Two-letter codes
used in the figure correspond to the different nonpeptide moieties described in Table 1.
Specifically, PH = phenanthridinone, CB = carboline, SP = spirocycle, SC = Straight
chain, CN = cinnamic acid.
four novel nonpeptidic structures showed that only one of the four had affinity to the receptor (Figure
8c). This new compound, I, was subsequently shown to be an antagonist in a cellular assay measuring
bradykinin-stimulated IP turnover [18]. Overall, there were 285 possible structures to survey due to the
number of structure-based scaffolds that were prepared. This was rapidly accomplished via 19 synthetic
couplings, 19 assays, and 4 purifications.
Not surprisingly, compound I showed divergent potency when assayed in different species homologues
of the bradykinin B2 receptor. In particular, in a model of bradykinin-induced hypotension in rats and
rabbits, it showed no activity. Likewise, it did not block bradykinin-induced contraction of the isolated
guinea pig ileum. Since compound I is considerably smaller than previously reported decapeptide
antagonists, subtle structural differences (which are
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known to exist in species homologs of the bradykinin B2 receptor) are likely amplified. A more
comprehensive pharmacological analysis of compound I is currently underway.
A. Lead Optimization
We have previously reported that the C-terminal guanidinyl moiety of Arg [9] in prototypical peptide
bradykinin antagonists is likely to behave more as an aromatic functional group rather than a hydrogenbond donor/acceptor. This speculation was based on proposed models of the agonist and antagonist
binding sites of this receptor that have been elucidated using molecular biological and computational
procedures. On this premise, the newly discovered lead compound, I, was altered such that the Cterminal arginine was replaced by 3',5'-dimethylpyrimidylornithine in an attempt to increase potency.
This known mimetic of arginine contains an aromatic 3',5'-dimethylpyrimidyl ring in the side chain
rather than the guanidino group on naturally occurring arginine.
The results of the receptor binding assay performed using this compound, IA, are shown in Table 4
where it is clear that affinity to the human B2 receptor is improved with respect to compound I. This
data is supportive of the notion that the C-terminal residue(s) in this new series of bradykinin antagonist
compounds interact with a hydrophobic environment, perhaps within the transmembrane domain of the
receptor as previously suggested.
The discovery of I and IA is significant in many regards. First, they are highly nonpeptidic lead
compounds that could be further modified to improve potency and/or reduce molecular weight. Such
improvements might lead to novel therapeutic agents for the treatment of inflammatory diseases. Thus
far in the kinin antagonist literature there is significant evidence showing that, for compounds containing
a C-terminal arginine residue, removal of that arginine generally yields compounds that are antagonists
of the B1 subtype of the bradykinin receptor. Following a similar strategy with compound I could lead to
the discovery of a novel series of nonpeptidic B1 receptor antagonists, although this remains to be
demonstrated.
V. Conclusions
There has been a significant effort invested toward the discovery of novel bradykinin receptor
antagonists during the past decade. In that time, several generations of peptide antagonists have been
developed and a few are in human clinical trials. The pursuit of nonpeptide antagonists of the human
bradykinin B2 receptor continues and incorporates a wide range of strategic approaches. The approach
described herein is an early and very good example of a combinatorial synthesis of nonpeptide building
blocks that mimic peptide structure,
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ultimately tested in a nontagged, solution-phase form. Perhaps more significant is that the success
described here demonstrates a possible synergy between structure-based design and combinatorial
methodology. This approach has many merits, but the most significant is the application of structurally
directed libraries toward target binding-site structures which, for one reason or another, may not be fully
characterized. This method serves to aim the combinatorial syntheses in a logical direction, rather than
attempt to prepare libraries of vast diversify (and numbers).
Finally, since the libraries of compounds that were prepared contained few members, it was possible to
analytically characterize each of the pools to assess integrity of their composition. Overall, there are
many important advantages in the paradigm we have adopted that make the strategy generally viable in
the context of structure-based lead compound discovery.
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56. Chakravarty S, Mavunkel BJ, Andy R, Kyle DJ. 14th American Peptide Symposium, Columbus,
Ohio, June, 1995.

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5
Design of Purine Nucleoside Phosphorylase Inhibitors
Y. Sudhakara Babu, John A. Montgomery, and Charles E. Bugg
BioCryst Pharmaceuticals, Inc., Birmingham, Alabama
W. Michael Carson, Sthanam V. L. Narayana, and William J. Cook
The University of Alabama at Birmingham, Birmingham, Alabama
Steven E. Ealick
Cornell University, Ithaca, New York
Wayne C. Guida and Mark D. Erion*
Ciba-Geigy Corporation, Summit, New Jersey
John A. Secrist, III
Southern Research Institute, Birmingham, Alabama
I. Introduction
A. Enzymology
Purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) catalyzes the reversible phosphorylysis of
ribonucleosides and 2'-deoxyribonucleosides of guanine, hypoxanthine, and related nucleoside analogs
[1]. It normally acts in the phosphorolytic direction in intact cells, although the isolated enzyme
catalyzes the nucleoside synthesis under equilibrium conditions. Figure 1 shows the chemical reaction.
The enzyme has been isolated from both eukaryotic and prokaryotic organisms [2] and functions in the
purine salvage pathway [1,3]. Purine nucleoside phosphorylase isolated from human erythrocytes is
specific for the 6-oxypurines and many of their analogs [4] while PNPs from other organisms vary in
their specificity [5]. The human enzyme is a trimer with identical subunits and a total molecular mass of
about 97,000 daltons [6,7]. Each subunit contains 289 amino acid residues.
*Current
affiliation: Gensia, Inc., San Diego, California.
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Figure 1
The reaction catalyzed by PNP.
B. Pharmacology
Interest in PNP as a drug target arises from its ability to rapidly metabolize purine nucleosides and from
its role in the T-cell branch of the immune system. Unfortunately, PNP can also cleave certain
anticancer and antiviral agents that are synthetic mimics of natural purine nucleosides, thus interfering
with therapy. One such substance is ddI (2'3'-dideoxyinosine), which the Food and Drug Administration
approved as a treatment for AIDS in 1991. Another is the potential anticancer agent 2'-deoxy-6thioguanosine [8]. Our goal was to develop a compound that when administered with the nucleoside
analogs would inhibit PNP while the anticancer and antiviral agents accomplished their therapeutic
missions. The combination of purine nucleoside analogs and a PNP inhibitor might prove to be a more
effective treatment.
The PNP inhibitors alone have potential therapeutic value based on the importance of PNP to the
immune system. Patients lacking PNP activity exhibit severe T-cell immunodeficiency while
maintaining normal or exaggerated B-cell function [9]. We, like other researchers, quickly recognized
that PNP inhibitors might selectively suppress the T-cell proliferation associated with an array of
autoimmune disorders such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus, multiple
sclerosis, and insulin-dependent (juvenile-onset) diabetes [10]. This profile also suggests that PNP
inhibitors might be useful in the treatment of T-cell proliferative diseasessuch as T-cell leukemia or Tcell lymphomaand in the prevention of organ transplant rejection.
C. Drug Design Strategy
Recent advances in biotechnology, macromolecular crystallography, computer graphics, and related
fields have led to a new approach in drug discovery called structure-based drug design. Structure-based
drug design requires a detailed structural knowledge of the target (enzyme or receptor) and the
interaction of small molecules with it.
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Figure 2
Structure-based drug design strategy.
A tight fit is necessary for potency and specificity. A drug that binds to its target and inactivates it for a
long time can be administered in lower doses than one that rapidly separates from its target. A substance
designed to mesh perfectly with a particular binding site of one target is unlikely to interact well with
any other molecule, minimizing unwanted interactions and side effects.
Having chosen PNP as the target, we followed a systematic strategy for designing inhibitory
compounds. Figure 2 outlines the overall strategy of this approach. To serve as a drug, an inhibitor has
to readily cross cell membranes to the interior of cells, where PNP is located.
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We determined the structure of human PNP by x-ray crystallography and used these results in
combination with computer-assisted molecular modeling to design inhibitor candidates. We examined
how well the shape and chemical
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structure of a candidate would complement the active site of PNP. We used computational chemistry to
estimate the strength of the attractive and repulsive forces between a candidate and the enzyme.
We synthesized only those candidates suggested by chemical intuition and computer simulation to have
high affinity for the target. Then we measured the inhibition of PNP and compared the proposed with the
actual fit. Because modeling programs and expert opinion are imperfect, certain compounds did not
meet expectations. After exploring the reasons for the successes and the failures, we returned to
interactive computer graphics to propose modifications that might increase the effectiveness of drug
candidates.
The resulting compounds were evaluated by determination of their IC50 values (the inhibitor
concentration causing 50% inhibition of PNP) and by x-ray diffraction analysis using difference Fourier
maps. This iterative strategymodeling, synthesis, and structural analysisled us to a number of highly
potent compounds that tested well in whole cells and in animals.
D. Previously Known Inhibitors
At the beginning of our studies several PNP inhibitors had been reported with Ki values in the 10-6 to 107 range, including 8-aminoguanine [11], 9-benzyl-8-aminoguanine [12], and 5'-iodo-9-deazainosine [13].
Acyclovir diphosphate had been shown to have a Ki near 10-8 if assayed at 1 mM phosphate rather than
the more frequently used value of 50 mM phosphate [14]. During our studies, the synthesis of 8-amino9(2-thienylmethyl)guanine was reported with a Ki of 6.7 10-8 M [15]. Figure 3 illustrates some of these
structures.
Despite the potential benefits of PNP inhibitors and the large number of PNP inhibitors that had been
synthesized, no compound had reached clinical trials. None of these compounds were potent enough to
be useful for therapy and also capable of crossing the cell membrane intact. Although potencies for the
best compounds had affinities 10100 fold higher than the natural substrate (Km = 20 M), it is expected
that T-cell immunotoxicity will only occur with very tight binding inhibitors (Ki < 10 nM) due to the
high level of in vivo PNP activity and competition with substrate.
II. Crystallography
At the present time, x-ray crystallography is the preferred technique for obtaining the required atomic
resolution structural data. In the late 1970s when this project was first conceived, determining the
structure of a protein was far from routine. The x-ray structural determination occupied a team of
crystallographers led by Steven E. Ealick, then at the University of Alabama at Birmingham through
most of the 1980s.
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Figure 3
Previously known inhibitors.
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The stumbling block did not lie with obtaining pure PNP or converting the protein into crystals. Robert
E. Parks, Jr. and Johanna D. Stoeckler of Brown University had already isolated the enzyme from
human cells. They supplied quantities of protein to William J. Cook, who succeeded in preparing the
well-ordered crystals required for x-ray studies [16]. We established that PNP crystals function normally
as a catalyst. Thus crystalline PNP is essentially identical to PNP in the body. If it were profoundly
different, one would have no justification for basing drug design on the crystal structure.
In the early years we had to depend on a relatively low-intensity x-ray source. High-resolution data was
obtained through collaboration with John R. Helliwell and his group at the Daresbury Laboratory
Synchrotron Radiation Source in England. Today greatly improved equipment and more synchrotron
facilities are available for protein crystallography.
The three-dimensional structure was determined by multiple isomorphous replacement techniques using
synchrotron radiation [17]. The native and guanine-PNP complex structures have been refined to 2.8
resolution [18,19].
A. Structure of the Enzyme
Crystals of human PNP are grown from ammonium sulfate solution and stored in artificial mother liquor
solution made of 60% ammonium sulfate in 0.05 M citrate buffer at pH 5.4. The space group is R32 with
hexagonal cell parameters a=142.9(1) and c=165.2(1) . The PNP crystals contain about 76% solvent
and diffract to around 2.8 resolution.
The x-ray data established that PNP crystals contain a high percentage of water. This feature proved
very useful; proposed drugs could easily be soaked into the active site without disrupting the crystal
packing. Figure 4A shows the
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Figure 4
Structure of PNP as stereo drawings. (A) Crystal packing. The low-resolution
surfaces of six trimers are shown. The top level is related to the bottom level by a 2-fold
axis along X. The distance between the 3-fold axes is 143 . A drug molecule is shown
in the solvent channel near the entrance to an active site. (B) Ribbon drawing. The
lowermost trimer of Figure 4A is shown. This is the native structure; the guanine and
phosphate are shown to mark the active site. (C) The swinging gate. The trimer of Figure
4B is rotated about 30 counterclockwise in the plane, followed by a roughly 90 rotation
about X to view the entrance to the active site. A model of the transition state is shown
as a line drawing. Conformational changes of the protein on binding guanine are shown.
Arrows are drawn from the C positions in the native structure to their positions in the
complex. (D) Active site of PNP. The orientation is approximately that of Figure 4C, but
enlarged and clipped to focus on the substrate. Key side-chain residues are labeled.
Residue 159'F, in the center of figure toward the viewer, is the only residue from the
adjacent subunit. The guanosine and phosphate are shown with thicker bonds. Oxygen
and sulfur atoms are shown as white spheres, nitrogen and phosphorus as black spheres.
(E) Purine binding pocket. The style is the same as Figure 4D, but the figure is rotated
slightly and enlarged. The key group interacting with bound guanine are highlighted. (F)
The best inhibitor. The style is the same as Figure 4D, but the figure has been enlarged
and rotated to place the phosphate binding site far from the viewer in the upper left.
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Figure 4
(Continued) Only one nitrogen atom from Arg 84 is visible, whichalong
with Ser 220 and His 86interact with the acetyl group branching from the benzylic
carbon. The chlorinated phenyl group is in the center of the figure, interacting with the
aromatic groups in the sugar binding site. The guanine group interactions are the same as
seen in Figure 4E. Figure prepared with ribbons (http://www.cmc.uab.edu/ribbons).
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large solvent channels and the position of the active site. The x-ray analysis confirmed the trimeric
nature of the enzyme, as the subunits are related by the crystallographic three-fold axis.
A ribbon diagram of the trimer is shown in Figure 4B. Each monomer contains an eight-stranded sheet
and a five-stranded sheet that join to form a distorted barrel. Seven helices surround this sheet
structure.
The active site is an irregular indentation on the surface of the enzyme, located from the position of a
tightly bound sulfate ion and various substrate analogs. These investigations revealed the identity of the
exact amino acids constituting the active site region; such detail was a prerequisite to drug design.
Information of greater import emerged from analyses of the complexes formed when synthetic
nucleosides, including previously discovered inhibitors, were diffused into the active site.
B. The Active Site
The structural determinations also yielded a surprise. The shape of the enzyme changes when a purine is
bound. The famous lock-and-key analogy [20] has a fallacy; the shape of the lock is not static, but
flexible. Awareness of these conformational changes critically aided our modeling efforts, allowing
prediction of which parts of PNP could change shape to interact with a proposed inhibitor.
A swinging gate consisting of residues 241260 controls access to the active site (Figure 4C). These
residues in the native structure had poorly defined electron density with high thermal motion. The gate
opens in the native enzyme to accommodate the substrate or inhibitor. The maximum movement caused
by substrate or inhibitor binding occurs at His 257, which is displaced outwards by several angstroms.
After binding, the electron density becomes well defined. The gate is anchored near the central sheet
at one end and near the C-terminal helix at the other end. The gate movement is complex and appears to
involve a helical transformation near residues 257261.
Consequently, initial inhibitor modeling attempts using the native PNP structure were far less successful
than subsequent analyses in which coordinates for the guanine-PNP complex were used. Because of the
magnitude of the changes that occur during substrate binding, it is unlikely that modeling studies based
on the native structure alone would have accurately predicted the structure of PNP/inhibitor complexes.
The active site is located near the subunit-subunit boundary within the trimer and involves seven
polypeptide segments from one subunit and a short loop from the adjacent subunit (Figure 4D). The
purine binding site employs residues Glu 201, Lys 244, and Asn 243 to form hydrogen bonds with N1,
O6, and N7 of purine. The remainder of the purine binding pocket is largely
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hydrophobic, composed of residues Ala 116, Phe 200, and Val 217. The phosphate binding site uses
residues Ser 33, Arg 84, His 86, and Ser 220 with the phosphate positioned for nucleophilic attack at C1'
of the nucleoside. The sugar binding site is mostly hydrophobic consisting of residues Tyr 88, Phe 200,
His 257 from one subunit and Phe 159 of the adjacent subunit. This hydrophobic pocket orients the
sugar to facilitate nucleophilic attack by phosphate and subsequent inversion of C1'.
C. Initial Inhibitor Complexes
In order to understand the interaction of inhibitors with the active site residues, the previously known
inhibitors were obtained and crystallographic analyses were carried out. The most important findings
were (1) 8-amino substituents enhance binding of guanines by forming hydrogen bonds with Thr 242
and possibly the carbonyl oxygen atom of Ala 116; (2) substitution by hydrophobic groups at the 9position of a purine enhances binding through interaction with the hydrophobic region of the ribose
binding site; and (3) acyclovir diphosphate is a multisubstrate inhibitor with the acyclic spacer between
the purine N9 and the phosphate of near optimal length to accommodate these two binding sites. Based
on these results, a number of starting compounds were proposed that incorporated these and other
features predicted to enhance inhibitor binding.
III. Molecular Modeling
Structural information in combination with graphical methods for displaying accessible volume,
electrostatic potential, and hydrophobicity of the active site of the target macromolecule greatly
facilitates the drug design process. Accurate prediction of binding affinities and protein conformational
changes are currently not routinely possible, although significant advances are being made.
Proposed compounds were screened by modeling the enzyme-inhibitor complex using interactive
computer graphics. Macromodel [21] and AMBER [22] based molecular energetics were used along
with Monte Carlo/energy minimization techniques [23] to sample the conformational space available to
potential inhibitors docked into the PNP active site. Methods based on the work of Goodford [24]
employing custom software were also used. Qualitative evaluation of the enzyme-inhibitor complexes
by molecular graphics and semiquantitative evaluation of the interaction energies between the inhibitors
and the enzyme aided in the prioritization of compounds for chemical synthesis.
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IV. Drug Design Progression

We focused initially on filling the purine binding region of the active site. That done, we planned to fill
the sugar binding region and, finally, the phosphate binding site. We expected that each successive step,
moving the compound closer toward fully occupying the active site, would enhance the affinity of the
drug candidate for the enzyme.
A. The Purine Site
From our crystallographic examinations, we knew that three amino acids in the purine binding pocket of
PNP formed hydrogen bonds with purines and their mimics. Such linkages are among the strongest
reversible chemical bonds that exist. In proposing inhibitor candidates, we concentrated on compounds
that would at least form hydrogen bonds with the same three amino acids. Figure 4e shows a close-up of
the purine site.
We favored exchanging a carbon atom for the nitrogen atom that normally occupies position nine, since
there was no interaction of this nitrogen with the active site and earlier studies showed such a change
promotes binding to PNP. Guanine modified in this way is called 9-deazaguanine. The first structures
selected for synthesis were 9-deazaguanines substituted by an arylmethyl group at the 9 position. These
compounds were prepared by adaption of a literature procedure [25]. We further expected that attaching
an amino group to the carbon atom in position eight on 9-deazaguanine would enhance affinity, since 8aminoguanine was the first significant inhibitor of PNP.
Both 8-aminoguanine analogs and 9-deazaguanine analogs are good inhibitors of PNP. However,
introduction of an 8-amino group into the 9-deazaguanine derivatives resulted in decreased potency. To
understand this poor binding, we undertook crystallographic analysis of PNP complexes with four
compounds having the 9-thienyl substituent attached to guanine (G), 8-aminoguanine (8AG), 9deazaguanine (DG), and 8-amino-9-deazaguanine (8ADG). The results of this analysis are summarized
in Figure 5. These data show one mode of binding for compounds that accept a hydrogen bond from Asn
243 at N7 (G and 8AG) and another for compounds that donate hydrogen to Asn 243 from N7 (DG and
8ADG). The 8AG analogs make use of the Thr 242 side chain to form an additional hydrogen bond,
which improves binding affinity. In the 9-deazaguanine series, where N7 has an attached hydrogen
atom, Asn 243 undergoes a shift that is clearly seen in difference Fourier maps. This shift is caused by
the formation of the N7-HOD(243) hydrogen bond. A concomitant shift by Thr 242 prevents it from
hydrogen bonding to the 8-amino group of 8ADG. Furthermore, the shift moves the methyl group of Thr
242 towards the 8-amino
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Figure 5
Comparison of 8-amino and 9-deaza substitutions on guanine. Details are extensively
discussed in the text. Cross-hatching schematically indicates the enzyme. Ball and
stick diagrams show the inhibitor and key side-chain residues. Nitrogens are dark
gray spheres, oxygens are light gray. Arrows indicate hydrogen bonding, with the
arrow size showing relative strength.
amino group, generating a hydrophobic environment for the group and decreasing binding affinity.
The carbon-for-nitrogen switch in the 9-deaza variant favors association with PNP by substituting a
strong hydrogen bond for the relatively weak one occurring between Asn 243 and guanine. Formation of
a simple 8-aminoguanine variant leads to tight binding by giving rise to an extra hydrogen bond between
the purine derivative and Thr 242.
The combination of the two improvementsthe carbon-for-nitrogen substitution and the addition of
the amino group to position eightwas counterproductive because the carbon in position nine prevented
the amino group at
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position eight from forming the extra bond with Thr 242. In fact, it set up an unfavorable, repulsive
clash between the threonine and the added amino group.
In the absence of detailed structural information, it would have been extremely difficult to explain why
affixing the amino group to the carbon in position eight proved unhelpful. But crystallography quickly
provided the explanation. 9-Deazaguanine itself would be a better choice for the purine component of an
inhibitor. This experience underscores the wonderful economy of the structure-based approach. Without
crystallographic data, we might have pursued a logical but unproductive avenue of research much longer
than we did.
B. Ribose Site
The next task was to fill the sugar binding site. The sugar in a nucleoside does not attach to PNP
primarily by forming hydrogen bonds, but through hydrophobic attractions. The sugar binding pocket of
PNP consists of three hydrophobic amino acids: Phe 200 and Tyr 88 from the same monomer that binds
guanine and Phe 159 from the adjacent monomer. Several known inhibitors carried a benzene group
attached to position 9 of the purine in place of the sugar in the nucleoside. An initial series of
compounds was synthesized to exploit the hydrophobic region in the ribose binding site.
A number of 9-substituted 9-deazapurine analogs were prepared with various aromatic, heteroaromatic,
and cycloaliphatic substituents. The first 9-deazaguanine derivatives synthesized, such as 9-benzyl-9deazaguanine, were three to six times more potent than the most potent known inhibitor, 8-amino-9-(2thienylmethyl)guanine. The optimum spacer between the purine base and the aromatic substituent
proved to be a single methylene group. Crystallographic data showed that generally the planes of the
aromatic rings tend to orient in a reproducible conformation. The aromatic groups optimize their
interaction with Phe 159 and Phe 200, which results in the classic herringbone arrangement reported
in a variety of aromatic systems [26].
Inhibitors with cycloaliphatic substituents at N9 of deazaguanine were also as potent as the aromatic
analogs. The cycloaliphatic substituents occupied the same general volume as the aromatic groups. As
with the aromatic series, the optimum spacer between the 9-deazaguanine and the hydrophobic
substituent is one carbon atom. X-ray analysis of the PNP complexes of 9-cyclohexyl-9-deazaguanine, a
relatively poor inhibitor, and the complex of 9-cyclohexylmethyl-9-deazaguanine, a potent inhibitor,
showed the two cyclohexyl groups occupy approximately the same space in the active site with the
purine base pulled out of its optimal position in the former.
The chemistry is more straightforward with the aromatic series. From modeling studies, we saw that the
sugar binding pocket could be filled more
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completely by adding any of several chemical groupings to the benzene ring. The best fit came from
adding a chlorine atom to position 3 of the benzene ring.
C. Phosphate Site
The final step added a group that would interact with the phosphate binding site either directly or via
electrostatic interactions. We could not use phosphate itself, because phosphate-containing compounds
are not metabolically stable and have difficulty passing through cell membranes intact. Acyclovir
diphosphate, which is not membrane permeable and is subject to extracellular metabolism, is a good
example.
Our results suggested that an ideal PNP inhibitor in the 9-deazapurine series would contain an aromatic
group and a substituent with affinity for the phosphate site interlinked by spacers with optimum lengths.
Crystallographic and modeling studies suggested a two-to-four-atom spacer. Initial modeling studies
encouraged us to prepare several structures, but they failed to improve the binding affinity of our twopart structure.
Crystallographic analysis of a number of PNP inhibitor complexes revealed significant displacement of
the inhibitors. These displacements appear to be the result of close contacts between the inhibitor and
the ion in the phosphate binding site. Sulfate ions occupy the phosphate site in PNP crystals as they are
grown from ammonium sulfate solution. These inhibitors were more potent when the binding was
measured in 1 mM phosphate solution rather than in 50 mM phosphate. Kinetic studies showed that
these inhibitors were competitive not only with inosine but also with phosphate, in keeping with the
above observation.
These results, summarized in Table 1, show that the IC50 (50 mM) is equal to or larger than the IC50 (1
mM), in some cases by as much as 100-fold. The ratio and the dimension of the 9-substituent show some
correlation. Compounds such as 8-aminoguanosine and 8-amino-9-(2-thienylmethyl)guanine show no
difference. Since the concentration of phosphate in intact cells is
1 mM, we routinely used this assay
condition for all PNP inhibitors.
Starting with a model of the 9-benzyl-9-deazaguanine/PNP complex, we concluded that two of the
positions on the 9-benzyl group, namely the 2-position of the phenyl ring and one of the benzylic sites,
appeared to be oriented so that a group attached to either one could interact favorably with the phosphate
binding site. The first compound made in this series, 9-[2-(3-phosphonopropoxy)benzyl]guanine, turned
out to be a poor PNP inhibitor. Subsequent crystallographic analysis revealed that the plane of the
aromatic ring had rotated approximately 90 from its optimum position in the hydrophobic pocket. This
reorientation of the ring was presumably necessary to accommodate the four atom spacer between the
phenyl ring and the phosphonate group. A compound
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Table 1 Inhibition Data for Selected PNP Inhibitors by Increasing IC50 Value
IC50, M
R2a
R2
(S)-3-Chlorophenyl
3-Chlorophenyl
CH2CO2H
CH2CN
50 mM
phosphateb
Ratiod
1 mM
phosphatec
0.031
0.0059
5.3
1.8
0.010
180
2-Tetrehydrothienyl
0.22
0.011
20
3,4-Dichlorophenyl
0.25
0.012
21
3-Thienyl
0.08
0.020
3-Trifluoromethylcyclohexyl
0.74
0.020
37
Cyclopentyl
1.8
0.029
62
Cycloheptyl
0.86
0.030
29
Pyridin-3-yl
0.20
0.030
2-(Phosphonoethyl)phenyle
0.45
0.035
13
Cyclohexyl
2.0
0.043
47
2-Furanyl
0.31
0.085
3.6
CH2CO2H
0.90
0.16
5.6
42
1.0
(R)-3-Chlorophenyl
2-Phosphonopropoxyphenyle
aCompounds
with R2 not equal to H are racemic mixtures unless the R or S isomer is designated.
bCalf
spleen PNP assayed in 50 mM phosphate buffer.
cCalf
spleen PNP assayed in 1 mM phosphate buffer.
dIC
at 50 mM phosphate divided by IC50 at 1 mM phosphate.
50
7.3
42
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eGuanine
base.
Source: Ref. 27.
with a two-carbon spacer was a much better PNP inhibitor; however, it was clear from x-ray analysis
that the aromatic ring was unable to form the ideal herringbone packing interaction.
Alternatively, compounds were modeled in which the spacer to the phosphate binding site branched
from the benzylic carbon, thus placing no restrictions on the tilt of the aromatic ring. Examination of the
9-benzyl-9-deazaguanine/PNP complex indicated that of the two benzylic positions, one (pro-R) pointed
into a sterically crowded area within the active site, whereas the other (pro-S) pointed into a relatively
empty space adjacent to the phosphate binding site. This analysis led to the synthesis of racemic 9-[1-(3chlorophenyl)-2-carboxyethyl]-9-deazaguanine. This compound adds an acetate group (CH2COO-) to
the methylene carbon atom that joined 9-deazaguanine to the chlorinated benzene ring. This compound
was resolved into its (S) and (R) enantiomers.
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As predicated, the (S) acid was a 30-fold more potent inhibitor of PNP than the (R) form. X-ray
crystallographic analysis of the complexes revealed that the (S) acid was oriented properly for optimal
interactions with all three subsites (Figure 4F), whereas the (R) acid was not. This series of compounds
contains the most potent membrane-permeable inhibitors of PNP yet reported [27].
V. Summary
Recently, scientists at BioCryst have successfully completed a project to design and synthesize potent
inhibitors of the enzyme Purine Nucleoside Phosphorylase (PNP) using the three-dimensional structure
of the active site. Crystallographic and modeling methods have been combined with organic synthesis to
produce inhibitors. Our experience in creating a set of potential drugsone of which (BCX-34) is now
in human trials for treating psoriasis and a form of T-cell lymphomaillustrates the process and the
power of structure-based design.
This structure-based inhibitor design approach led to a number of inhibitors more than 100 times more
potent than any membrane-permeable inhibitor available at the beginning of this project. During the two
and half years of this project, about 60 active compounds were synthesized. This is a remarkably small
number compared with the extensive synthesis programs generally involved in drug discovery by trial
and error techniques. The large number of active compounds and the enhancement of inhibitor potency
stand as proof that crystallographic and modeling techniques are now capable of playing a critical role in
the rapid discovery of novel therapeutic agents. The entire protocol, from choosing the target to creating
a drug suitable for clinical trials, can probably be accomplished today in two or three years.
A. Obstacles Encountered and Lessons Learned
Crystallographic analysis was based primarily on the results of difference Fourier maps in which the
interactions between residues in the active site and the inhibitor could be characterized. During these
studies, about 35 inhibitor complexes were evaluated by x-ray crystallographic techniques. It is
noteworthy that the resolution of the PNP model extends to only 2.8 and that all of the difference
Fourier maps were calculated at 3.2 resolution, much lower than often considered essential for drug
design. Crystallographic analysis was facilitated by the large solvent content that allowed for free
diffusion of inhibitors into enzymatically active crystals.
Initial inhibitor modeling attempts using the native PNP structure were far less successful than
subsequent analyses in which coordinates for the guanine-
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PNP complex were used, mainly because of the magnitude of the changes that occur during substrate
binding. We found that computer modeling required significant tuning in order to provide useful results.
Crystallographic results were useful in testing and modifying modeling parameters. The most useful
modeling results were achieved after incorporation of the conformational searching techniques described
earlier and when the coordinates for the PNP-guanine complex model were used. Visual inspection and
chemical intuition were very important.
B. Perspectives of Treating Targeted Disease
One of the inhibitors designed during the drug discovery process, 9-(3-pyridylmethy)-9-deazaguanine
(BCX-34), was selected for initial clinical development. Current clinical trials utilize both topical and
oral formulations of the drug.
Researchers at the University of Alabama at Birmingham and Washington University School of
Medicine have recently completed small Phase II clinical trials of two indicated applications, cutaneous
T-cell lymphoma (CTCL) and psoriasis, using a topical formulation of BCX-34. Although patients
showed improvement in both trials, the duration of each was too short (six weeks) to adequately assess
the efficacy of the drug. Subsequently 80% of the patients from the CTCL trial (24 patients) entered an
open label trial for treatment of their disease for up to twelve months. At the end of the first six months
of treatment, seven of the patients were in complete remission (verified by biopsy), two patients showed
a clinical complete response, and nine patients had shown definite improvement. The other six patients
had shown no change or progression of disease. No serious, drug-related adverse events were reported
during the study.
The process of structure-based drug design helped to ensure that the inhibitor would be highly selective
for the PNP enzyme, and thus far no other targets for the drug have been identified. The mechanism of
action of BCX-34 appears to be entirely related to its effect on the proliferation of human T-cells. This
high degree of specificity probably also contributes to the high safety profile of the drug. Although longterm studies in more patients will be necessary to substantiate these results, it appears likely that BCX34 will have a significant clinical effect on at least some T-cell mediated diseases.
Based on the results from these three trials, BioCryst has initiated a multicenter Phase III trial for the
treatment of CTCL, as well as a large, multicenter Phase II trial for psoriasis. In addition to the two
clinical trials using the topical formulation, a Phase I clinical trial in CTCL and T-cell
lymphoma/leukemia has begun using an oral formulation of BCX-34. In the future, a number of other Tcell mediated diseases or processes are possible targets for BCX-34, including rheumatoid arthritis,
multiple sclerosis, inflammatory bowel disease, and organ transplant rejection.
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References
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7:483514.
2. Stoeckler JD. In: Glazer, RE, ed. Developments in Cancer Chemotherapy. Florida: CRC, Baco Raton,
1984; 3560.
3. Friedkin M, Kalckar H. In: Boyer PD, Lardy H, Myrback K, eds. The Enzymes. 2nd Ed. New York:
Academic, 1961; 5:23755.
4. Agarwal KC, Agarwal RP, Stoeckler JD, Parks RE Jr. Purine nucleoside phosphorylase.
Microheterogeneity and comparison of kinetic behavior of the enzyme from several tissues and species.
Biochemistry 1975; 14:7984.
5. Bzowska A, Kulikowska E, Shugar D. Properties of purine nucleoside phosphorylase (PNP) of
mammalian and bacterial origin. Z Naturforschung C Biosci 1990; 45:5970.
6. Stoeckler JD, Agarwal RP, Agarwal KC, Schmid K, Parks RE Jr. Purine nucleoside phosphorylase
from human erythrocytes: physiocochemical properties of the crystalline enzyme. Biochemistry 1978;
17:27883.
7. Williams SR, Goddard JM, Martin DW Jr. Human purine nucleoside phosphorylase cDNA sequence
and genomic clone characterization. Nucleic Acids Res 1984; 12:577987.
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6
Structural Implications in the Design of Matrix-Metalloproteinase
Inhibitors
John C. Spurlino
3-Dimensional Pharmaceuticals, Inc., Exton, Pennsylvania
I. Matrix-Metalloproteinases
The matrix metalloproteinases (MMPs) are a family of ubiquitous enzymes that are involved in
extracellular matrix degradation and remodeling. They are critical for the processes of morphogenesis
and wound healing, but are also implicated in many human diseases including arthritis, metastasis, and
cancer tumor growth [1, 2]. This family includes matrilysin, fibroblast collagenase (HFC), neutrophil
collagenase (HNC), stromelysin 1 (HFS), stromelysin-2, stromelysin-3, gelatinases A and B, collagenase3, and the membrane type MMP. In addition to the destructive involvement in diseases, MMPs play a
critical role in the remodeling of the extracellular matrix [3].
The MMP enzyme family is part of the superfamily of metzincins. The metzincin superfamily is
distinguished by a conserved zinc binding motif for the catalytic zinc and a Met-turn region [4]. The
MMPs are unique in that they also contain a second structural zinc, however this zinc may be absent in
the intact full-length enzyme [5]. The presence of one to four structural calcium ions has been detected
in the MMPs that have been characterized to date. The importance of the zinc ions and at least one of the
structural calcium ions to enzymatic activity has been proven [6].
The MMPs are secreted as inactive proenzymes, which are activated by proteolytic cleavage. Once
activated they are subject to control by tissue inhibitors of metalloproteinases (TIMPs). It is the
imbalance between the active enzymes and the TIMPs that leads to destructive tissue degradation that
potent directed pharmaceuticals can overcome. These enzymes have been the target of
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drug design since the late 1970s [7]. Batimastat, [{4-N-hydroxyamino}-2R-isobutyl-3S-{thienylthiomethyl} succinyl]-L-phenyl-alanine-N-methylamide, a potent nonspecific MMP inhibitor from
British Bio-Tech, is now in Phase III trials. The information gained from current studies indicate that
there is some efficacy in the treatment of disease states by MMP inhibitors [810].
There is still much debate about whether a broad spectrum or directed MMP inhibitor is the best course
of treatment for a variety of diseases, partly because the exact role of individual MMPs is still unclear.
Both collagenase-3 and HFC are suspected to cause osteoarthritis [11]. It is currently believed that
gelatinase and collagenase-3 have a role in breast cancer [12]. Gelatinase A and B have been implicated
in hemorrhagic brain injury [13]. Gelatinase A and B, HFC, and stromelysin may all be involved in
gastric cancer [14]. Matrilysin may be implicated in colon cancer [15]. Increased gelatinase A and B
activity has also been seen in response to beta-amyloid production [16]. The role that individual MMPs
play in causing these diseases, however, remains unclear. This uncertainty underscores the need to
develop selective inhibitors of individual MMPs to ferret out the roles each play in the development of
specific disease states.
The MMPs consist of one or more structural domains (Figure 1). The first domain, the propeptide
domain, confers a self-inhibitory action on the full-length MMP. The second domain contains the active
site residues and is referred to as the catalytic domain. The catalytic domain is characterized by the
conserved zinc-binding sequence (HEXGHXXGXXHS), which also contains the glutamate residue that
is essential for activity [17]. The MMPs are activated by cleavage of the prodomain. All MMPs contain
these first two domains. Matrilysin, the simplest of the MMPs, is an example of a two-domain enzyme,
where the active enzyme consists solely of the catalytic domain.
The remainder of the MMPs also contain a hemopexin-like domain connected to the catalytic domain by
a proline-rich linker. This domain is involved in the interaction of the collagenases and stromelysins
with collagen and, in the case of the collagenases, is essential for activity against collagen [18]. The
cleavage of the proline-rich linker region in HFC and HNC is another route to control collagenase
activity. The hemopexin domain of the gelatinases is not necessary for collagen binding, but may be
involved in receptor recognition [19].
The gelatinases also contain a fibronectin-like insert in the catalytic domain that is involved in binding
collagen [20]. The fibronectin domain has also been shown to be essential for elastolytic activity [21]. A
structural picture of these additional domains is essential for an understanding of the mode of action for
these larger MMPs, but is not necessary for a structure-based drug design strategy. Differences in the
catalytic domains of the MMPs can be used to drive a targeted drug discovery effort.
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Figure 1
A ribbon model of the full-length collagenase structure (1fbl.pdb). The
prodomain would precede and include the portion labeled in the figure. The
catalytic domain is shown, with a highlighted region where the fibronectin-like
domain of the gelatinases is inserted.
II. 3-Dimensional Structure of MMPs

Catalytic domain structures for fibroblast collagenase [2225], neutrophil collagenase [26,27],
matrilysin [28], and stomelysin [29, 30] have all been determined and deposited in the Protein Data
Bank [31]. The catalytic domains of MMPs, as seen in the archetypal collagenase structure (shown as a
ribbon drawing [32] in Figure 1), consist of an upper 5-stranded sheet flanked by two helices on one
side of the active site cleft and a long loop that contains the Met-turn flanked by a single helix on the
other side of the cleft.
The active-site groove as seen in the solvent-accessible surface [33] is an obvious structural feature
(Figure 2). The top wall of the cleft (as seen in Figure 2) is formed by the top strand of the sheet and
the loop that contains the calcium binding site. The lower wall of the cleft is formed from the residues
on either side of the Met-turn. These residues can be considered as an interrupted strand which, together
with the substrate, complete the twisted sheet of the catalytic domain. The bottom of the cleft is
formed by the second helix, which
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Figure 2
The accessible surface of HFC with a modeled substrate from human collagen
showing the binding sites.
contains the HExxH motif, the catalytic zinc, and the S1' pocket. Substrates bind in an extended
conformation that approximates an antiparallel strand. The cleft, however, is not large enough to
accommodate a triple helix collagen molecule.
A structure for the full-length active porcine synovial collagenase [34] has been determined. The
structure of the catalytic domain of this full-length enzyme is equivalent to the structures of the isolated
catalytic domains of HFC, HNC, and matrilysin. The flexible linker domain between the catalytic and
hemopexin domains is disordered and the orientation of the hemopexin domain in the structure offers no
real clue as to the mode of action for the full-length collagenases. Furthermore, the matrilysin structure
of the full-length active enzyme has almost identical secondary structural features (a Ca overlap of
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Figure 3
The sequence alignment of MMPs with the catalytic domain region highlighted.
The residues that line the subtrate pockets are marked: S3 (3), S2 (2), S1 (1), S1
(*), S2' (@), and S3' (#). The highlighted catalytic domain alignment was dominated by
the structural alignment of the determined structures of HFC, HNC, and matrilysin.
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0.43 ) as the catalytic-domain structure of HFC. This demonstrates that the absence or presence of the
hemopexin domain does not affect the overall structure of the catalytic domain.
The sequence homology of the catalytic domains of the collagenases is 62%. This can be extended to the
other members of the MMP family as seen in Figure 3. An understanding of the structural features of the
target enzyme is essential for structure-based drug design. In this example we will be looking at
inhibiting MMPs by binding to the active site. The numbering system used throughout this chapter will
be in regard to the HFC sequence used in 1hfc.pdb.
III. Surface Features
The surface features of matrilysin, fibroblast collagenase, and neutrophil collagenase are all similar
(Figure 4). The active-site groove can be plainly seen on the surface: two main pockets punctuated by
the active-site zinc. Modeling
Figure 4
The accessible surfaces of HFC (a), HNC (b), and matrilysin
(c) are shown with a bound P'-side hydroxamate inhibitor.
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studies show that there is not sufficient room in the S1'-S3' cleft to accommodate the native coiled triple
collagen bundle.
The active site consists of a series of subsites on either side of the catalytic zinc. These subsites are
numbered starting at the catalytic zinc and preceding from N to C as S1', S2', etc., corresponding to the
residues (P1', P2', etc.) of the substrate that is bound (Figure 2). Likewise the subsites are numbered S1,
S2, etc. outward from the other side of the catalytic zinc.
While the interaction of the substrate (and the inhibitor) with the catalytic zinc is the most important
interaction, the remainder of the substrate (inhibitor) also forms hydrogen bonds with residues from the
top strand of the sheet and the loop region posterior to the Met-turn. These interactions with the
substrate in the binding pockets of the MMPs are the prime targets for engineering specific MMP
inhibitors. An in-depth understanding of the differences of the properties of these pockets in the different
MMPs and the interactions of specific residues within these pockets is essential for structure-based
design of inhibitors.
IV. Main-Chain Substrate Interactions
Most of the hydrogen bonds between the substrate and the MMP occur with the top strand of the sheet.
The P3 residue does not make any direct hydrogen bonds with the MMP. The P2 residue makes two
hydrogen bonds with residue 184, which is a conserved alanine residue in all the aligned MMP
sequences. Residue 183 is a conserved histidine, which is bound to the structural zinc, further stabilizing
the conformation of the top strand. The carbonyl oxygen of P1 is liganded to the catalytic zinc. The lefthand side of the substrate is thus held in place by only two hydrogen bonds with the enzyme and one
interaction with the catalytic zinc. Although the P3 residue does not make any hydrogenbond
contributions to substrate binding, it is essential for catalytic activity [43].
The right-hand side of the substrate is held much tighter. The P1' residue's carbonyl oxygen makes a
hydrogen bond with the amide nitrogen of residue 181, which is a conserved leucine residue. The amide
nitrogen of the P1' residue is hydrogen bonded to the conserved alanine-182 carbonyl oxygen. The P2'
substrate residue is held in place by hydrogen bonds to proline 238 and tyrosine 240, two more
conserved residues. The amide nitrogen of P3' makes a hydrogen bond with the carbonyl oxygen of
residue 179, the only nonconserved residue, making a main-chain interaction with the substrate.
The use of conserved residues to maintain the main-chain interactions along the substrate backbone
makes differentiation of the MMPs through these interactions difficult. Instead, differences in the
regions where the side chains of the substrate interact can be used to drive the discovery of specific
MMP inhibitors. The hydrogen-bonding pattern also indicates that right-hand side (P'-
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side) inhibitors will bind with greater affinity. Indeed, most of the structures of MMPs were determined
with right-hand side inhibitors, and most of the pharmaceuticals currently in development are also righthand side binders. A closer look at the binding pockets themselves also demonstrates the reasons for the
preference of researchers for the right-hand side.
V. Substrate-Binding Pockets
The nonprimed or left-hand side of the cleft consists of a large shallow depression. The S1 pocket
consists of a shallow ridge that complements the glycine residue of the collagen strands. Most of the
interactions with the glycine residue are brought about due to its interaction with the catalytic zinc.
Asparagine 180 approaches the P1 residue in HFC. Crystallographic evidence indicates an interaction of
the thiophene ring of batimastat via electrostatic interactions of the p orbitals and the catalytic zinc and
the possibility of a water-mediated hydrogen bond to the carbonyl O of residue 184 [35]. Larger
substituents can be accommodated in regions adjacent to the P1 pocket, possibly in the large pocket
above the S1 site (see Figure 2). Increased potency was noted for several compounds with cyclic imido
P1 substituents that could bind here [2].
The S2 pocket is a large shallow depression offering no real binding cavities. One side of the pocket is
made up from the conserved histidine at position 228 and the main chain from residue 227. The bottom
of the pocket is formed by histidine 222. Both of these histidines are liganded to the catalytic zinc. The
other side consists mostly of the residue 186 side chain with some hydrogen-bonding contacts possible
from the tip of the glutamine side chain in the case of HFC and HNC.
The S3 pocket offers a shallow cavity to bind the conserved proline. The proline residue of the substrate
would lie between the side chains of His 183, Phe 185, and Ser172 [36]. Residues 183 and 185 are
conserved among the MMPs with the minor exception of a tyrosine replacing phenylalanine 164 in
stromelysin. Residue 172 shows some variability among the MMPs existing as a serine in HNC and
HFC and a tyrosine in the remainder of the aligned MMPs.
The primed or right-hand side of the active site exists as a narrow canyon with a large well at the
beginning. The S1' pocket is a narrow, deep cavity providing an ideal binding site for inhibitor design.
The S1' pocket is the most significant feature of the surface, extending as a tunnel completely through
the enzyme in the case of neutrophil collagenase and stromelysin. This feature makes the S1' pocket an
ideal candidate for use in designing an inhibitor with specificity for HNC, HFC, or HFS.
The volumes of the S1' pockets vary greatly. Matrilysin has the smallest S1' pocket at 111 3. The
fibroblast collagenase pocket is not much larger at
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Figure 5
(a) A cut away view of the S1' pocket of HFC showing the termination of
the pocket by Arg214. Matrilysin also has a truncated pocket. (b) A cut away
view of the S1' pocket of HNC showing the clear path through to the other
side of the enzyme. The gelatinases are also likely to have large extended S1'
pockets.
123 3. The pocket of the neutrophil collagenase that travels through the enzyme has a volume of 305
3. Figure 5 demonstrates the differences in the relative sizes of the S1' pockets of HFC and HNC.
Stromelysin has a pocket that should be of similar size as that of neutrophil collagenase [21]. The
gelatinases and collagenase-3, based on sequence alignment, should also posses long tunnel-like S1'
pockets.
The residues that line the S1' pocket are mostly hydrophobic residues (see Figure 3) and show a
remarkable overall similarity. The specificity of a number of inhibitors of MMPs can be linked to
differences in the S1' pockets. The S1' pocket of HFC is terminated by arginine 214, while matrilysin
has a tyrosine residue that accomplishes the same thing. The remainder of the aligned MMPs have
leucine residues at that position. In addition the conformation of the leucine residue is swung back,
forming the tunnel. There are three additional residues that are significant in their differences within the
S1' pocket: residues 239 and a two-residue insert, relative to HFC, after residue 242. These residues
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form the lower end of the tunnel. The major effect of the different residues found here is on the diameter
of the exit hole in the S1' tunnel. However, there are some residues that could be targeted for hydrogenbond formation.
The S2' binding cavity is a narrow cleft that can easily accommodate a peptide backbone, but with no
room for a side chain. The interaction of the P2' side chain is made with the exterior surface of the
enzyme. The S2' site is exposed to solvent and presents two possible interaction sites for bound
inhibitors that are related by a rotation about 1. These sites consist of residues 179180 on one side and
residues 238240 on the other.
The S3' binding cavity opens up out of the canyon and consists almost entirely of surface interactions.
The side chain of P3' interacts with residues 210 and 240, which are mostly conserved tyrosine residues.
Additional interactions could be formed with some of the additional residues found in the insertions
after residue 242.
VI. Structure-Based Design
Structure-based drug design is an iterative process that starts with a lead compound, a structural model
of the target, and a structure-activity relationship
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(SAR) model. The lead compound can come from compound screening, previously discovered
inhibitors, or it can be based on a known substrate. The model can be obtained from x-ray
crystallography, high-field nuclear magnetic resonance spectroscopy, or from homology-based model
building. Inhibitor structures are developed and docked into the model of the binding site of interest,
typically the active site of the enzyme. The interactions of the inhibitor-enzyme complex are evaluated
and ranked. The most promising compounds are then synthesized and tested. Based on the results of the
testing, additional enzyme-inhibitor structures are determined, the SAR model is updated, and the
process beings again.
As a model case of structure-based drug design for MMPs we will look at the design of a right-handed
inhibitor based on the x-ray structures of HFC and HNC.
VII. Zinc-Binding Group
The design of active-site inhibitors based on the natural substrate of the collagenases has produced a
variety of zinc-binding groups to anchor the inhibitor to the catalytic zinc. These group include
hydroxamates, thiols, phosphorous acid derivatives (phosphinate, phosphonate, phosphoramidate), and
carboxylates. The selection of a suitable zinc-binding group has been studied in depth [3740]. The most
potent zinc-binding group found for the collagenases to date is the hydroxamate.
The structural comparison of hydroxamate, carboxylate, and sulfodiimine in matrilysin provided
information on the contribution of the zinc ligand to the overall potency of the inhibitor [19]. The
potency of the zinc-bind group can be directly related to the number of bonds in which it is involved for
this instance. The hydroxamate is the perfect bidentate ligand to the zinc with both oxygens being within
2.2 of the zinc. The hydroxamate group also is involved in hydrogen bonds with Glu219 and the
carbonyl oxygen of Ala182. The carboxylate group is also a bidentate ligand to zinc, however the
oxygens are not equidistant from the zinc. The carboxylate forms only one additional hydrogen bond
with Glu219 of the enzyme. The sulfodiimine bound to matrilysin is a monodentate zinc ligand and the
weakest of the zinc-binding groups. The comparison of several inhibitors with both carboxylate and
hydroxamate zinc-binding groups demonstrates this property in fibroblast and neutrophil collagenases as
well. While the potency of inhibitors with different zinc-binding groups maps directly to the number of
bonds formed by the zinc-binding group, some of the increase in potency of the hydroxamate group over
charged groups most likely is a result of the decreased energetic cost of the desolvation of the neutral
hydroxamate.
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VIII. S1' Interactions

Matrix metalloproteinase structural studies of the P'-side inhibitors to date show a common set of
inhibitor-enzyme interactions. This can be attributed primarily to the strong directional zinc-binding
forces. Further stabilizing forces from the backbone hydrogen-bonding patterns common to a sheet
allow for minor adjustments due to the zinc interactions to be made while maintaining a common
pharmacophore.
The fairly rigid constraints of binding based on the known hydroxamate inhibitors allows the use of
computer-aided modeling to play a useful role in the design of specific MMP inhibitors. Exploration of
the S1' pocket was carried out by docking the P1' group within the cavity followed by rounds of energy
minimization. In order to maintain the integrity of the MMP structure several limitations were used. All
MMP atoms that are greater than 8 from the docked inhibitor were frozen. The C atoms of all
residues within 8 of the inhibitor were initially constrained to their original position by a 20 kcal/mol
2 force constant that was gradually relaxed to 1 kcal/mol 2. Strong initial constraints were also placed
on the conserved hydrogen bonds and zinc-ligand interactions.
This method has several advantages and disadvantages over the common static treatment of target
structures. The advantages are that it more closely approximates the actual dynamic state of protein
structure and is not computationally prohibitive. The disadvantages include the increased computational
cost over a static enzyme target and the fact that gross structural rearrangements can still not be
accounted for.
The structural similarity of the active site of the MMP family allows structure-based drug design to
effectively be used for those enyzmes whose structures have not been determined yet. Examination of
the S1' cavities of HFC and HNC clearly indicates a path for designing inhibitors that bind preferentially
(Figure 5). The cavity of HFC is mostly filled by the leucine side chain of the preferred substrate, while
the S1' pocket of HNC [26] and HFS [30] remains unfilled. The sequence similarities of the gelatinases
with HNC indicate that they too can accommodate a much larger P1' group.
A series of compounds was designed to explore filling the long S1' tunnel of gelatinase B [41, 42]. The
optimum length for binding to gelatinase B was found. There was an increase in affinity for the phenolic
ethers versus the benzylic ethers of the same overall length for binding to gelatinase B, but there was no
preference seen in binding to HFS. Surprisingly the phenolic ethers also showed potent binding to HFC.
The size of the bottom of the S1' pocket and the differences in the preferred torsion angle for the bond to
the aromatic ring both play a role in this differential binding.
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IX. S2' Interactions

The interactions at the S2' site display an interesting structure-activity relationship. While the
interactions do not include any hydrogen bonds, favorable stacking interactions do play a significant role
in binding. A glycine residue at P2' results in a loss of three orders of magnitude in potency for
otherwise identical inhibitors [41]. There is a preference for an aromatic ring at this position in natural
peptide substrates [43]. Structural considerations also allow the placement of a t-butyl group here. The
potency of a t-butyl glycine P2' is less than that of a phenylalanine (Table 1), but the expected gain in
bioavailability brought about by shielding the amide bond from solvation effects should compensate for
the loss in potency.
X. Conclusions
High resolution x-ray crystallographic structure determination is an essential step in structure-based drug
design. The need for high resolution structural data
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Figure 6
(a) The pocket of HFC with a leucine residue in the S1' pocket.
(b) The volume of the S1' pocket of HFC can change when there are
favorable interactions. The binding of the (CH2)4OPh can cause
Arg214 to move, thereby making room for the extended side chain.
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to develop an appropriate SAR is demonstrated in the case of the inhibitors shown in Table 1. The
unusual potency of a benzylic ether for HFC was unexpected and would not have been predicted with
standard docking and minimization studies (Figure 6).
The differences in the potency of the various 4-substituted analogs of inhibitor 10 against HFC suggest interactions are the driving force for the displacement of arginine 214. The electron-withdrawing Cl
substitution decreases the affinity for HFC while increasing the affinity for HFS. The leading 4-pentyl
group can not effectively interact with arginine 214 in HFC; therefore, the rearrangement does not
occur. The open channel that is present in HFS and gelatinase B presents no such impediment to binding
and the affinity is essentially equal to the unsubstituted form.
Not all structure-based design experiments are successful. Attempts to displace the arginine residue that
caps the S1' pocket of HFC by forming a salt link with a P1' carboxylate or hydroxyl moiety were
unsuccessful [42]. However, these failed attempts offer some redeeming features in the refinement of
parameters that can be used to evaluate the energetic potentials for displacing buried water molecules as
well as the inherent desolvation energies for polar compounds.
The outlook for structure-based drug design is good. The advancement in both x-ray area detectors and
computer hardware will make the determination of a series of compounds bound to a target enzyme for
use in SAR development commonplace in drug-discovery efforts. The continued explosion of structural
studies will lead to an increased understanding of the dynamics of protein interactions, which will, in
turn, lead to better docking algorithms. The combination of structural information and greater
computational power will also make more accurate predictions of proteinligand interactions possible.
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7
StructureFunction Relationships in Hydroxysteroid Dehydrogenases
Igor Tsigelny and Michael E. Baker
University of California, San Diego, La Jolla, California
I. Introduction
Steroid hormones regulate a multitude of physiological processes in humans. Androgens and estrogens
regulate sexual development and reproduction; glucocorticoids are important in the response to stress;
vitamin D is important in bone growth; progestins are important for a viable fetus during pregnancy;
mineralocorticoids regulate sodium and potassium balance to maintain normal blood pressure.
Moreover, the growth of some breast and prostate tumors depends on steroids. With this multitude of
medically important steroid-dependent actions, much research has gone into understanding their mode
of action, with most of the effort concerned with the receptors that mediate the actions of steriods.
A. High Blood Pressure and 11-Hydroxysteroid Dehydrogenase
It is only in the last decade that the role of hydroxysteroid dehydrogenases (Figure 1) in regulating the
actions of steroids has been appreciated [16]. This mechanism for regulating steroid hormone action
was uncovered in several laboratories studying various aspects of high blood pressure. One source was
the study in the 1970s that identified the syndrome, Apparent Mineralocorticoid Excess (AME), a
genetic disease that results in high blood pressure in children [710]. Also important is the work from
laboratories investigating paradoxes in the mechanism of action of aldosterone in the kidney [14,9,10].
These studies identified 11-hydroxysteroid dehydrogenase (11-HSD) as a key enzyme.
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Figure 1
Reactions catalyzed by 11-hydroxysteroid and 17-hydroxysteroid
dehydrogenases. (a) 11 -hydroxysteroid dehydrogenase type 1, an NADPH-dependent
enzyme, catalyzes the conversion of the inactive steroid, cortisone, to cortisol, which is
the biologically active glucocorticoid. 11-hydroxysteroid dehydrogenase type 2, an
NAD+-dependent enzyme, catalyzes the reverse direction. (b) 17-hydroxysteroid
dehy-drogenase type 1, an NADPH-dependent enzyme, catalyzes the reduction of
estrone to estradiol. Type 2, an NAD+-dependent enzyme, catalyzes the oxidation of
estradiol to estrone. Type 3, an NADPH-dependent enzyme, catalyzes the reduction of
androstene dione to testosterone. Type 4, an NAD+-dependent enzyme, catalyzes the
oxidation of estradiol to estrone, and androstenediol to dehydroepiandrosterone.
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The enzyme 11-HSD interconverts the active glucocorticoid cortisol and cortisone, an inactive
metabolite (Figure 1a). By the oxidation of cortisol to cortisone, 11-HSD prevents glucocorticoids
from deleterious actions in certain cell types. For example, excess glucocorticoids in Leydig cells inhibit
testosterone synthesis [3,11]. Expression of 11-HSD in Leydig cells prevents this effect of
glucocorticoids. In this way, 11-HSD is important in androgen action. The enzyme 11-HSD is also
important in aldosterone action in the distal tubule of the kidney. Glucocorticoids have high affinity for
the mineralocorticoid receptor [12] and can stimulate the mineralocorticoid responseuptake of sodium
from urineone effect of which is to increase blood pressure. Local expression of 11-HSD in the
distal tubule prevents this effect of glucocorticoids. The steroid aldosterone, which is not metabolized by
11-HSD, can bind to the mineralocorticoid receptor and regulate sodium and potassium balance. Thus,
11-HSD has an important role in regulating the biological actions of both glucocorticoids and
mineralocorticoids.
As would be expected, interference with 11-HSD activity due to a mutation [13,14] or by an inhibitor
such as licorice (Figure 2) [13,15] has a variety of physiological effects including high blood pressure
due to mineralocorticoid actions of glucocorticoids in the kidney's distal tubule. Thus, studies to unravel
genetic hypertension in children and the actions of aldosterone in the kidney yielded the general insight
that, at specific times, altered expression of 11-HSD in specific tissues is an important mechanism for
regulating glucocorticoid, mineralocorticoid, and androgen action.
A similar mechanism has been found for 17-hydroxysteroid dehydrogenase (17-HSD), the enzyme
that regulates the concentrations of estradiol and testosterone in human [5,16,17] (Figure 1b). Genetics
diseases associated with mutations in this enzyme lead to developmental abnormalities [18]. Enzymes
that regulate the concentrations of retinoids [19] and prostaglandins [20] may also have a similar role
[6].
B. Multiple Divergent 11-Hydroxysteroid and 17-Hydroxysteroid Dehydrogenases
The cloning and sequencing of 11-HSD [2125] and 17-HSD [1618,26] revealed two 11-HSDs and
four 17-HSDs with very different sequences
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Figure 2
Structure of licorice and carbenoxolone. Glycyrrhizic acid, a constituent of
licorice extract, is found in the root of Glycyrrhiza glabra. The glycosidic group at C3 is
cleaved by bacteria in the small intestine to form glycyrrhetinic acid, the compound that
inhibits 11-hydroxysteroid dehydrogenase. Carbenoxolone, a water soluble synthetic
analog of glycyrrhetinic acid, is widely used to regulate 11-HSD in vitro and in vivo.
(Figure 3, Table 1). This was surprising, as one would expect the two 11-HSDs to be similar because
they recognize the same substrates. Instead, the two 11-HSDs have less than 20% sequence identity,
after including gaps in the alignment (Table 1). The same degree of sequence divergence is found in the
four 17-HSDs [6]. This sequence divergence is reflected in differences in their catalytic properties. For
example 11-hydroxysteroid dehydrogenase-type 1 (11-HSD-1) is an NADPH-dependent enzyme that
converts cortisone to cortisol, and 11-hydroxysteroid dehydrogenase-type 2 is an NAD+-dependent
enzyme that oxidizes cortisol to cortisone. The enzyme 17HSD-1 is an NADPH-dependent enzyme
that converts estrone to estradiol, and 17HSD-2 is an NAD+-dependent enzyme that oxidizes estradiol
to estrone and testosterone to androstenedione.
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Figure 3
Alignment of 11-and 17-hydroxysteroid dehydrogenases. As seen in this
Figure and Table 1, the sequences of the two 11-HSDs and four 17-HSDs are very
divergent. Boxes denote sites where either 5 or 6 residues are conserved, which are
likely to be functionally important.
There is considerable interest in understanding the structural bases for these differences because this
information would be very useful in designing steroids and other compounds to selectively regulate the
activity of one or more steroid dehydrogenases as a means of treating hormone-responsive diseases.
There is precedent for this kind of treatment in the use of licorice extract from the root of the plant
Glycyrrhiza glabra [15,27] to treat Addison's disease,
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Figure 3
(Continued)
which is characterized by insufficient levels of cortisol. Licorice inhibits 11-HSD-2, which raises the
circulating levels of cortisol and provides some relief from the symptoms of Addison's disease. This use
of licorice is an example of a plant-derived compound having important uses in mammalian steroid
hormone physiology and indicates another reason why elucidation of the structure of steroid
dehydrogenases is of medical interest. Plants contain a wide variety of compounds, many of which have
been purified and had their structures determined; however, we don't know which of these compounds
inhibit steroid
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Table 1 Percent Identity Between Hydroxysteroid Dehydrogenase Sequences Shown in Figure 3
11-HSD-2
11-HSD-2
17-HSD-2
17-HSD-3
17-HSD-1
11-HSD-1
17-HSD-4
0.00
46.1
20.3
28.6
20.6
18.1
0.0
21.1
21.2
17.7
18.9
0.0
19.1
19.6
17.5
21.1
20.4
0.0
17.1
17-HSD-2
17-HSD-3
17-HSD-1
11-HSD-1
0.0
17-HSD-4
0.0
dehydrogenases. Knowledge of structure-activity relationships for the binding site on steroid

dehydrogenases will be helpful in identifying novel compounds from plants and other sources that could
be useful in regulating steroid dehydrogenases.
At this time, we are just beginning to work on this ambitious goal. Structural information is limited. The
3-D structure of 17-HSD type 1 has been determined [28], but without the steroid or cofactor in the
binding site. Fortunately, 11-HSD and 17-HSD belong to a large family of enzymes that are called
short-chain alcohol dehydrogenases [2931] or sec-alcohol dehydrogenases [32]. The structures of
dehydrogenase homologs in bacteria, plants, and animals have been determined [3337] and we used
them as templates for modeling 11-HSD and 17-HSD [38,39]. There also is information about the
effects of mutations on catalytic activity in 11-HSD-1 [40] and 17-HSD-1 [41] and in homologs,
especially for Drosophila alcohol dehydrogenase (ADH) [4248]. Together they enable us to begin to
understand the relationship between structure and function in hydroxysteroid dehydrogenases, as we
discuss in this chapter.
II. Methods
A. Molecular Modeling
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Important for the validity of the models that we constructed is the evidence from models of other
proteins indicating that two proteins can have as little as 20 to 25% sequence identity and still have very
similar 3D structures, especially in helices and stands [4952]. Variation is found in the loops and
coiled structures. A relevant example for this chapter is the comparison of the tertiary structure of rat
dihydropteridine reductase [33] and Streptomyces hydrogenans 20-hydroxysteroid dehydrogenase [34].
As noted by Varughese et al. [33], despite less than 20% sequence identity between dihydropteridine
reductase and
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Figure 4
Amino acids important in cofactor and catalysis in human 11b-hydroxysteroid
dehydrogenase types 1 and 2. (a) 11b-HSD type 1. Preference of 11b-HSD type 1
for NADPH resides in lysine-44 and arginine-66, which have positively charged side
chains that stabilize the binding of the 2'-phosphate on NADPH. These residues also
counteract the repulsive interaction between glutamic acid 69 and the phosphate group.
(b) 11b-HSD type 2. Preference of 11b-HSD type 2 for NAD+ is due to favorable bonds
with aspartic acid-91, serine-92, and threonine-112. Moreover there is a coulombic
repulsion between aspartic acid-91 and NADP+, which destabilizes binding of NADP+
11b-HSD type 2 lacks a nearby amino acid with a positively charged side chain that
could diminish the repulsive interaction between NADP+ and aspartic acid-91. Also
shown are threonine residues that could hydrogen bond
to nicotinamide's carboxamidemoiety.
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20-hydroxysteroid dehydrogenase, the root mean square deviation for the two tertiary structures is 2
over 160 C carbon atoms.
We aligned human 11-HSD-1 [21] and 11-HSD-2 [22] with S. hydrogenans 20-hydroxysteroid
dehydrogenase and Escherichia coli 7-hydroxysteroid dehydrogenase [37] for 3D modeling. Human
11-HSD has extra segments at the amino terminus and carboxyl terminus. Previously reported
alignments [30,31,53] were used to find the core structure consisting of about 225 residues that are
structurally similar to the template. The first 190 residues of the 255 residues are reasonably well
conserved among the hydroxysteroid dehydrogenases. Alignment of the C-terminal 65 residues is less
certain as this part contains gaps and insertions. Fortunately, the core 190 residues contains the catalytic
site and the cofactor binding site. We also superimposed the two 11-HSD structures on mouse carbonyl
reductase [37]. The 11-HSD 3D structures superimpose nicely on helices E and F and other helices
and strands that are important in binding of cofactor and substrate. Then, we extracted NADPH from
carbonyl reductase and NAD+ from 7-hydroxysteroid dehydrogenase and inserted the cofactors into
the structures of the two 11-HSDs.
The helix F in 17-HSD-1 [16], 17-HSD-2 [17], 17-HSD-3 [18], and porcine 17-HSD-4 [26] was
constructed by modeling on helix F in 20-hydroxysteroid dehydrogenase. Comparisons with other
3D structures [3337] have demonstrated that this helix is highly conserved. The modeled dimers
were not minimized as a dimer complex to avoid the artifactual adjustment of helix F side chains.
The Homology program (Biosym Technologies, Inc., 1995) was used to model a 255-residue segment of
11-HSD and helix F of 17-HSD on the S. hydrogenans 20-hydroxysteroid dehydrogenase
template. To produce the final model (Figures 4 and 5) this program finds an optimal configuration of
the residues when arranged in the template structure by minimizing unfavorable interactions between
amino acid side chains. The side chains of each monomer were then minimized (1,000 iterations of the
conjugate gradient) using the Discover program (Biosym Technologies Inc., 1995).
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Figure 5
Structure of helix F interface of human 11-hydroxysteroid dehydrogenase types 1
and 2. The helix F part of the dimer interface on 11-HSD-1 and -2 is shown along
with side chains of the highly conserved tyrosine and lysine residues and other residues
that are oriented into the cavity that binds substrate and nucleotide cofactor.
III. Results and Discussion

A. NADPH Binding Site on 11-Hydroxysteroid Dehydrogenase Types 1 and 2
Several lines of evidencesequence analysis, mutagenesis studies, and the solved 3D structure of
homologs of 11-HSDindicate that the nucleotide binding site in these enzymes has many similarities
to that in other classes of dehydrogenases. For many dehydrogenases, the nucleotide binding domain
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consists of a strand helix, strand in a fold that provides a hydrophobic pocket for the adenosine
monophosphate (AMP) part of the nucleotide cofactor [51,54,55]. In short-chain alcohol
dehydrogenases, this fold is at the amino terminus. The turn between the first strand and the
helix is a glycine-rich segment of the form Gly-X-X-X-Gly-X-Gly. This glycine-rich segment forms a
hydrophobic pocket that allows close association of the AMP part of the cofactor.
However, this glycine-rich segment has other functions in short-chain alcohol dehydrogenases. Tanaka
et al. [37] and our 3D modeling [60] indicate that this glycine rich segment has an important role in
cofactor specificity and binding of the nicotinamide moiety to 11-HSD.
B. 11-HSD-1 Preference for NADPH
Figure 4 shows our 3D model of human 11-HSD types 1 and 2. These models identify residues
important in preference of 11-HSD-1 for NADPH and 11-HSD-2 for NADH. In 11-HSD type 1,
lysine-44 and arginine-66 have favorable coulombic interactions with the 2'-phosphate on NADP+ that
stabilize binding (Figure 4a). Moreover, their positively charged side chains compensate for the negative
interaction between glutamic acid-69 and the 2'-phosphate group. Tanaka et al. [37] found a similar
function for lysine-14 and arginine-39 in the preference of mouse carbonyl reductase for NADPH.
C. 11-HSD-2 Preference for NAD+
The 3D structure of 11-HSD-2 shows that NAD+ has stabilizing interactions between the ribose
hydroxyl and aspartic acid-91, serine-92, and threonine-112. Replacement of NAD+ with NADP+
reveals a coulombic repulsion between the 2'-phosphate group and aspartic acid-91. However, 11-HSD
type 2 lacks a nearby amino acid with a positively charged side chain that could compensate for the
negative charge on aspartic acid-91. This explains the preference of 11-HSD-2 for NAD+.
D. Amino Acids Important in Binding the Nicotinamide Ring and Carboxamide Moiety
Both 11-HSD types 1 and 2 contain residues in the C-terminal half that interact with the nicotinamide
ring and carboxamide moiety to limit rotations about the N-glycosidic bond. These intersections are
important in positioning the cofactor for proS hydride transfer at C4.
In 11-HSD-1, cysteine-213 stabilizes the nicotinamide ring; threonine-220 and threonine-222 stabilize
the carboxamide moiety. In 11-HSD-2, there are more interactions: proline-262, phenylalanine-265,
threonine-267, serine-
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269, and valine-270 are close to either the nicotinamide ring or the carboxamide moiety. In addition, the
face of the side chain of phenylalanine-94 is below the nicotinamide ring and its carboxamide group.
This latter interaction is unusual because phenylalanine-94 is between the two canonical glycine
residues in the fold, which is usually thought of as interacting mainly with the AMP part of the
cofactor. In 11-HSD-2, there is an interesting configuration of amino acids with aromatic side chains
that are below the nicotinamide ring and which provide a hydrophobic cushion for NAD+.
E. Catalytic Site
Comparison of 11-HSD-1 with homologs identifies tyrosine-183 and lysine-187 as being highly
conserved residues. Mutagenesis of these residues [40] and the homologous tyrosine and lysine in 17HSD-1 and Drosophila alcohol dehydrogenase [44,45] shows that these residues are important for
catalytic function. The 3D model of 11-HSD-1 presented in Figure 4 shows that tyrosine-183 is 3.6
from the nicotinamide C4, where hydride transfer occurs. Similarly, in 11-HSD-2, tyrosine-232 is 4
from C4 on NAD+. Their positions support the notion that tyrosine is the catalytically active residue.
However, a problem with this model is that the pKa of tyrosine is about 10, which would make this
residue a poor nucleophile at neutral pH. To resolve this problem for the homologous tyrosine in
Drosophila alcohol dehydrogenase, Chen et al. [44] proposed that the pKa of tyrosine is lowered by a
nearby positively charged lysine. The 3D structure of the two 11-HSDs shows that lysine-187 and
lysine-236 are close to the proposed catalytically active tyrosine residues, which supports the hypothesis
of Chen et al. [44].
F. Dimer Interface and the Catalytic Site
Most short-chain alcohol dehydrogenases are active as either dimers or tetramers. Analysis of rat
dihydropteridine reductase by Varughese et al. [33] indicates that the dimer interface consists of helix
E and helix F from each monomer arranged in a four helix bundle, a structure in which the
hydrophobic surfaces on the helices form a core that yields very stable structure in a wide variety of
proteins [5659]. A four-helix bundle also appears to stabilize S. hydrogenans 20-hydroxysteroid
dehydrogenase, a tetrameric enzyme [34]. The helix F contains the conserved tyrosine and lysine
residue, which adds a constraint to changes in the sequence of this helix. It has at least two functions:
stabilizing the dimer and orienting tyrosine and lysine and other residues for optimal interaction with
substrate and nucleotide cofactor.
The role of a specific site on the outer hydrophobic surface of helix F in dimerization was suggested
recently when a Drosophila ADH mutant that does
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not form stable dimers was sequenced [48]. This ADH mutant has alanine-159 replaced with threonine.
A 3D model of Drosophila ADH shows alanine-159 on the opposite surface of helix F from tyrosine153 and lysine-157 [48]. Alanine-159 along with alanine-158 form a hydrophobic anchor that stabilizes
the dimer interface. These two residues of ADH and the homologous residues in other short-chain
alcohol dehydrogenases have been overlooked in sequence analyses because they are not absolutely
conserved. In fact, at least five amino acids are found in these positions among the different sec-alcohol
dehydrogenases.
G. Dimer Interface in 11-and 17-Hydroxysteroid Dehydrogenases
Because helix F at the dimer interface also contains the catalytic tyrosine and the nearby lysine
residue, any structural analysis of the catalytic site must also consider the structure of this part of the
dimer interface. For this reason, we modeled helix F on 11-HSD-1 and -2 and 17-HSD-1, -2, -3,
and -4 to gain an insight into stabilizing interactions and how they may affect the catalytic site.
H. Human 11-HSD-1
Figure 5 shows the modeled structure for the helix F interface in human 11-HSD-1, in which
phenylalanine-188 and alanine-189 form an anchor. Alanine-189 is 3.5 and 4.7 from alanine-189
and alanine-185, respectively, on the other subunit. The phenylalanine-188 side chain is 3.2 from
glycine-192. There is a hydrogen bond between serine-185 and serine-196, which are 3.2 apart.
Alanine-185 is 4.7 from phenylalanine-193. There also is a hydrophobic interaction between
phenylalanine-193 and alanine-181, which are 3.9 apart.
This web of interaction between side chains on the outer surface of helix F on each subunit influences
residues that have side chains oriented to the interior where catalysis occurs. Alanine-185, which is
stabilized by interactions with phenylalanine-193, and serine-184, which interacts with serine-196, are
between the conserved tyrosine-183 and lysine-187. Phenylalanine-193 is adjacent to phenylalanine194, which is positioned into the catalytic site.
I. Human 11-HSD-2
Figure 5 shows the helix F interface of human 11-HSD-2. Alanine-237 is about 3 from leucine241; alanine-238 is about 3.7 from both alanine-238 and threonine-234. Threonine-234 has a
stabilizing hydrophobic interaction
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Figure 6
Structure of helix F interface of mammalian 17-hydroxysteroid dehydrogenases.
The helix F part of the dimer interface on 17-hydroxysteroid dehydrogenases is
shown along with side chains of the highly conserved tyrosine and lysine residues
and three other residues that are oriented into the cavity that binds substrate and nucleotide
cofactor. (a) Modeled structure of human 17-hydroxysteroid dehydrogenase type 1.
(b) Modeled structure of human 17-hydroxysteroid dehydrogenase type 2. (c) Modeled
structure of human 17-hydroxysteroid dehydrogenase type 3. (d) Modeled structure of
porcine 17-hydroxysteroid dehydrogenase type 4.
with leucine-242, which is 4.2 distant. Threonine-245 is 4.2 from the C carbon of glycine-230.
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The type 1 and type 2 enzymes preferentially catalyze the reduction of the 11-keto group and the
oxidation of the 11-hydroxyl group, respectively, on glucocorticoids. The chemistry of the side chains
on methionine-243 in the type-2
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enzyme and of phenylalanine-194 on 11-hydroxysteriod dehydrogenase type 1 is quite different; it may

be important in the different catalytic properties of these two enzymes.
J. Human 17-HSD-1
Figure 6a shows the modeled helix F interface in human 17-hydroxysteroid dehydrogenase type 1 in
which phenylalanine-160 and alanine-161 form an anchor. Both residues have important stabilizing
interactions across the dimer interface. Alanine-161 is 4.1 from alanine-161 on the other subunit.
Alanine-161 has a hydrophobic interaction with alanine-157, which is in the segment between the
conserved tyrosine-155 and lysine-159. There is a hydrophobic
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interaction between alanine-157 and leucine-165, which are about 3.8 apart. Phenylalanine-160 is 4
from glycine-164. There also is a hydrogen bond between cysteine-156 and serine-168, which are 3.2
apart. This is an interesting structural property of residues in the segment between the conserved
tyrosine and lysine residues: this segment is important in stabilizing dimers. This pattern is repeated in
the other 11- and 17-hydroxysteroid dehydrogenases suggesting conservation of this stabilizing
structure, although the residues are not as well conserved as the tyrosine and lysine.
K. Human 17-HSD-2
Figure 6b shows the modeled helix F interface in human 17-hydroxysteroid dehydrogenase type 2.
Alanine-237 is 3 from the hydrophobic part of the side chain of methionine-241 on the other subunit.
Methionine-241 is 3.2 from serine-234. Alanine-230 is 3.7 from phenylalanine-242 and 4.5 from
valine-245. Alanine-238, the other anchoring residue, is 4.1 from alanine-238 on the other subunit.
L. Human 17-HSD-3
Figure 6c shows the modeled helix F interface in human 17-hydroxysteroid dehydrogenase-type 3.
Alanine-203 is 3.1 from alanine-207. Phenylalanine-204 is 3.2 from the other phenylalanine-204
and alanine-200. These are the only stabilizing interactions that we find in our analysis. Human 17hydroxys-teroid dehydrogenase type 3 has the weakest interactions across the helix F interface among
the four types of 17-hydroxysteroid dehydrogenases. The conformation of this part of 17hydroxysteroid dehydrogenase type 3 could change upon binding of substrate, leading to other
stabilizing interactions. And, of course other parts of the protein may have intersubunit interactions that
stabilize the dimer. Alternatively, the hydrophobic surface of helix F may interact with another protein
or a membrane surface, a potentially important regulatory mechanism that we discuss later in this paper.
M. Pig 17-HSD-4
Figure 6d shows the modeled helix F interface in pig 17-hydroxysteroid dehydrogenase type 4.
Leucine-169 is 2.9 from glycine-173. Leucine-174 is 4.3 from alanine-162 and 3.3 from alanine166. There also is a hydrogen bond between serine-165 and serine-175, which are 3 apart.
N. Prospects for the Application of Structure-Function Analysis of Steroid Dehydrogenases in
Hormone Therapy
In the last two years there has been impressive progress in understanding the structure of steroid
dehydrogenases that are important in regulating blood pres
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sure and the actions of reproductive hormones. This progress has come from several directions. First, the
cloning and sequencing the dehydrogenases that regulate the actions of aldosterone, cortisol, estradiol,
and testosterone. Second, determination of the 3D structure of 17-HSD-1 and several homologs.
Analyses of their 3D structures confirm a general principle that structural similarity is much higher than
sequence similarity. This supports proposed molecular models of medically important steroid
dehydrogenases using the alignment of their sequences onto the templates of 3D structural homologs.
Models of 11-HSD-1 and -2 are beginning to reveal important properties about these enzymes. We
now have a good picture of the structural basis for specificity for NADPH and NADH in 11-HSD-1
and -2. With this information, we can now turn our attention to modeling cortisol in these two enzymes.
This information will open up the possibility for developing analogs to regulate the actions of these two
enzymes for use in regulating blood pressure and other physiological processes. The development of
carbenoxolone, a water-soluble synthetic analog of glycyrrhetinic acid, shows that chemists can create
compounds that have high affinity for 11-HSD. The next task is to synthesize compounds that are
specific for 11-HSD-1 or 11-HSD-2. Molecular modeling can contribute important information to
solving this kind of problem.
Similarly important information will come from the 3D models of 17-HSD-1, -2, -3 and -4. These
models will be useful in developing compounds to regulate estrogen and androgen action, which have
important application in reproductive medicine and in treating estrogen-dependent breast tumors and
androgen-dependent prostatic tumors. Many compounds in plants have estrogenic and androgenic
activity; some of these compounds are likely to work via inhibition of one of the 17-HSD enzymes
[27]. Analogous to the development of carbenoxolone to regulate 11-HSD, we can seek synthetic
compounds that regulate specific types of 17-HSD, which may be useful in reproductive medicine and
in treating cancers.
Considering the explosive pace of biomedical research and the new developments in computers for
sophisticated structural analyses, the next few years promise to yield important advances in design of
new hormone therapies based on the knowledge of the structure of steroid dehydrogenases.
Acknowledgments
We thank Drs. Tanaka, Nonaka, Nakanishi, Deyashiki, Hara, and Mitsui for providing us with the x-ray
crystallographic coordinates of carbonyl reductase and 7-hydroxysteroid dehydrogenase. The support
of the Supercomputer Center of the University of California, San Diego is gratefully acknowledged.
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8
Design of ATP Competitive Specific Inhibitors of Protein Kinases Using
Template Modeling
Janusz M. Sowadski,* Charles A. Ellis,* Rolf Karlsson*
Madhusudan
Scripps Research Institute, La Jolla, California
I. Protein Kinases and Diseases
The protein kinase family encompasses more than three hundred members of critically important
enzymes, each one with a specific role or function within the cell. These enzymes, ATPphosphotransferases, recognize target proteins and through the phosphorylation of specific sites either
activate or deactivate a particular pathway of signal transduction. Many of these signaling pathways are
associated with cell surface receptors, which are located in the membranes that surround cells. The
difference between the families of protein kinases is that they have different targets and generally fall
into two major classes:
The serine/threonine protein kinases transfer a phosphate from ATP to a serine or threonine residue in
the target protein. This class of enzymes are generally associated with cytoplasmic signaling events.
The tyrosine protein kinases transfer phosphate from ATP to tyrosine residues in the target protein and
are generally associated with receptors that become activated after binding a growth factor or other
ligand.
Protein kinases are significant targets for therapeutic drug development and have been implicated as the
disease causing components of numerous tumor
*Current
affiliation: Tufts University, Boston, Massachusetts.
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viruses. Specifically, it is the deregulation of the activity of protein kinases that leads to disease by
tumor viruses. The importance of this deregulation can be dramatically illustrated by the large number of
viral oncogenes (or cancer causing genes) that encode structurally modified protein kinases. These
deregulated enzymes are able to bypass the normal tightly regulated processes of growth control, leading
to acute malignant transformation. These oncogenes are one of the first examples of the identification of
disease-causing genes. Many of these viral genes have subsequently been implicated in human diseases.
Malignant tissues also share the common characteristic of an acquired independence from controls. The
receptorfor example, PDGF and EGFRcan be stimulated by a ligand coming either from the cell
itself (autocrine) or from nearby tissues (paracrine). Regardless of the mechanism leading to receptor
activity, the resulting kinase activity results in a cascade of signals that turn on cellular proliferation
programs. Therefore, selective inhibition of receptor tyrosine kinase will block tyrosine kinase driven
cell proliferation resulting in antitumor activity. In addition to cancer, a growing number of
nonmalignant proliferative diseases, (e.g., psoriasis, atherosclerosis, restenosis, fibrosis, etc.) or
inflammatory responses (e.g., septic shock, asthma, osteo and rheumatoid arthritis, etc.) involve
dysfunctional signaling pathways. Successful development of drugs that target this class of enzymes will
depend on the discovery of selective inhibitors designed for the appropriate protein kinase within the
family.
In the past several years there has been an explosion of structural studies within the protein kinase
family [18]. These studies, initiated by the crystal structure of Protein Kinase A [912] (CAPK) have
shown that all members of the protein kinase family fold into a uniform three-dimensional catalytic core.
Yet this uniform three-dimensional fold exhibits both different surface charges and at least two major
conformations.
II. Protein Kinase Template
The stereo view of the ribbon diagram of cAPK is presented in Figure 1a. The overall topology of the
core extending from strand 1 through helix h, Figure 1b, is identical (except helix B) with the eight other
structures of protein kinases determined to this point. Furthermore, Figure 2 presents an overall
structural comparison of the catalytic cores of the five kinases, cAPK, CDK2, CDK2-CYCLIN, IR, and
MAP. The N-terminal helix A, which is present only in the cAPK crystal structure, is anchored by
myristic acid in the mammalian bovine heart of cAPK. Myristic acid inserts itself into the hydrophobic
pocket of the lower lobe of the enzyme, which results in the structural ordering of helixA and Ser10[13],
one of the four autophosphorylation sites.
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Figure 1
Diagram of cAPK fold. (a) Stereo MOLSCRIPT diagram. (b) The key loops
are as follows: phosphate anchor located between strand 1 and strand 2,
catalytic loop located between strands 6 and 7, DFG motif located between
strands 8 and 9, activation loop including P+1 site between strand 9 and
helix F. Phosphorylation site Thr197 is indicated by a large circle,
inhibitor PKI (524) is colored in dark, and the P site in the peptide
is shown in the dark circle. This figure has been generated using
MOLSCRIPT [27].
Following the connectivity diagram, helix A is connected to -strand 1, then to the phosphate anchor
encompassing signature motif Gly50XGly 52XXGly55. The -strand 2 is followed by -strand 3
carrying invariant Lys72. Three antiparallel beta strands create the unique fold of the nucleotide binding
site of the protein kinase. The -strands 3 is followed by helix B, which is present only in cAPK, helix
C, and -strands 4 and 5. Helix C shows the largest displacements among many different protein kinase
structures and consists of invariant Glu91, which forms a salt bridge with Lys72. This salt bridge is
absent in the inactive CDK-2 structure [1] but present in the crystal structure of the complex of CDK-2
and its activator-cyclin [7]. Displacement of helix C is perhaps most pronounced in the case of the
insulin receptor tyrosine kinase structure (IRK) [3]. In PKA, Phe185 resides in the hydrophobic pocket
formed by the hydrophobic residues of helix C (upper lobe) and Tyr164 (lower lobe). In the IRK crystal
structure, the hydrophobic residues of helix C, which provide the pocket for invariant Phe185 (of DFG
motif), no longer interact with
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Figure 1
(Continued)
this residue. The DFG motif in IRK occupies the ATP site, which blocks the access of ATP.
The division between the upper and lower lobes of the enzyme is well defined by the two major
conformations of the upper lobe observed in the crystal structures of cAPK. One conformation has been
observed in the orthorhombic crystals of recombinant cAPK [9,10,14] and another in the cubic
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Figure 2
The diagrams of the C trace of the five kinases, cAPK, CDK2, CDK2-CYCLIN,
IR, MAP. The thin line represents crystallographically determined homologous
regions of the five kinases (R. Karlsson and J.M. Sowadski, personal
communication).
crystals of bovine heart mammalian cAPK [15,16]. A comparison between the two structures has shown
that there is rotation of the upper lobe by 15 degrees and a translation of 1.9 in the mammalian
structure, which results in the opening of the nucleotide binding cleft.
The motion of this lobe, which includes His87, one of the ligands to Thr197 [15], indicates that the
phosphorylation of this site will be important for conformational diversity of the upper lobe. This is
confirmed by the varying degrees of displacement of the upper lobe of all structures of the inactive
unphosphorylated protein kinases (see review [17]). The lower lobe of the enzyme starts with helix D,
which is followed by helix E and -strands 6 and 7. The catalytic loop connecting both strands consists
of a critical set of residues with Tyr164 and Arg165 at the beginning of the loop. The Tyr164 residue
forms a hydrogen bond with invariant Asp220. The Arg165 residue, which is present in a great majority
of protein kinases, provides two hydrogen bonds to the oxygens of the phosphate of Thr197. Invariant
Asp166 (the catalytic base) and Asn171 (the ligand to one of the metal sites) are also located within this
loop.
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The catalytic loop is the region of divergence between Ser/Thr and Tyr kinases. In cAPK and all Ser/Thr
Kinases, Lys168 interacts with the phosphate of ATP during catalysis [12]. The role of Lys is replaced
by Arg [9] and the insulin receptor tyrosine kinase structure [3] shows Arg1136 in a similar position as
Lys168 in the active site of cAPK.
The catalytic loop and -strand 7 are followed by -strand 8 and a short DFG conserved motif. This
conserved motif consists of invariant Asp184, a ligand to the metal site, and invariant Phe185. The DFG
motif is followed by -strand 9, an activation loop that includes Thr197. The activation loop differs
considerably among unphosphorylated kinases, CDK-2, ERK-2, and insulin receptor kinase.
Furthermore, two crystal structures, twitchin protein kinase [2] and phosphorylase kinase [5], both lack a
phosphorylation site in this region. In the first structure, twitchin protein kinase, Thr197 and Arg165, are
replaced by hydrophobic residues, Val6098 and Leu6062 respectively [2]. This was predicted using
modeling [18] and subsequently confirmed by the three-dimensional structure. In the second structure,
phosphorylase kinase, the Thr197 is replaced by Glu182, which interacts with Arg148 [5]. Hence, in
both structures the regulatory function of the phosphorylation site is replaced by a stable scaffold
secured by either hydrophobic or electrostatic interactions. Since it has been shown that this loop
provides a stable template for PKI(524) binding in cAPK, the status of phosphorylation of the
activation loop critically affects the substrate binding. This is demonstrated in c-Src, a homolog of the
Rous Sarcoma virus oncogene by mutation of Arg385, which is predicted to interact with the phosphate
of Tyr416, and results in loss of activity toward the exogenous substrate [19].
The activation loop is followed by a P+1 loop which accommodates the P+1 site of the substrate. The
P+1 loop is followed by invariant Glu208, which forms a salt bridge with invariant Arg280. This
conserved pair plays a structural role and as the structure of CK-1 [16] shows, it can be replaced by
other charged residues that maintain the same fold of the lower lobe. The P+1 loop and Glu 208 are
followed by helix F consisting of invariant Asp220, followed by helices G, H, and I. The helix J and the
C-terminal tail of cAPK, which are absent in other protein kinases, undergo a large motion during the
cleft opening.
The opening of the cleft results in loss of hydrogen bonds provided by the phosphate of ATP and the
peptide that would bridge the lower and upper lobe of the enzyme. The motion of the upper domain
increases the accessibility of the ATP binding site and one can envision that in the open conformation
ATP binds. Yet, in the closed conformation ATP and its phosphate are positioned for a nucleophilic
attack on the substrate. The motion of this lobewhich includes His87, one of the ligands to Thr197
[15]indicates that the phosphorylation of this site will be important for conformational diversity of the
upper lobe. This is confirmed by the varying degrees of displacement of the upper lobe
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of all structures of the inactive unphosphorylated protein kinases (see review [17]).
The various displacements of the conserved upper domain of the catalytic cores of various kinases
documented by crystallographic work suggest that this is the important underlying mechanism of
catalysis. Analysis of crystal contacts of various kinases is however required to define the extent of
displacement due to the lattice forces. In the case of cAPK, the displacement as observed for mammalian
cAPK in the cubic crystal form is due to the intermolecular interaction in the lattice [20]. Analysis of the
two crystal structures of the cell cycle-controlling kinases clearly shows two binding modes of ATP. In
the inactive state without cyclin, ATP binding of its triphosphate moieties is different from that in the
active form with cyclin bound. The major difference is the re-arrangement upon cyclin binding of the
conserved Lys33-Glu51 pair, which is responsible for the binding of the and phosphates of ATP.
III. Crystallographic Analysis of Substrate Specificities of Individual Kinases
The most important contribution of subsequent crystallographic studies has been the confirmation of the
structural homology extending through the members of this family of enzymes. The crystal structures of
CDK-2 [1], ERK-2 [5], twitchin [2], insulin receptor kinase [3], phosphorylase kinase, CK-1 [6], along
with structure of calcium/calmodulin-dependent protein kinase I [8] provide solid proof for the structural
conservation of the catalytic core in the family. This is further confirmed by the recent structure of the
active complex of CDK2/cyclin, which shows that Lys33-Glu51 pair is at a distance of 3.0 [7] as
predicted in the model of CDK-2 based on the cAPK structure [21]. The structure of the complex has
also confirmed the binding of cyclin to helix C and to the upper lobe, demonstrating the mechanism of
activation by cyclin that results in bridging the invariant residues into the common network of distances
required by structural homology of the protein kinase catalytic core.
The crystallographic analysis of the structural homology of protein kinases can now be carried out using
structures of various kinases to find a common search model to be used in molecular replacement
methods (J.M. Sowadski and R. Karlsson private communication). The structures of various kinases
have been used as search models to solve the structure of the cAPK using cAPK diffraction data. The
best search model consists of fragments of the catalytic core excluding the activation loop, inserts, and
upper lobe due to rotational motion observed in each structure. A structure solution has been found for
several protein kinases using this selected model as shown in Figure 2. One of the most critical aspects
of this analysis is the presence of the structurally
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conserved substrate-binding cleft as observed in the crystal structure of cAPK:PKI complex (Figure
la,b). This finding allows the charges within this cleft to be predicted using the amino acid sequence of
any given kinase.
In the analysis of the structural data of other protein kinases, it is noted that only cAPK has been
crystallized with its specific peptide inhibitor. Nevertheless, three other structures of protein kinases
compared with the structure of the cAPK-PKI complex provide substantial evidence for the conservation
of the substrate binding cleft. The substrate binding cleft of the phosphorylase kinase structure has been
analyzed in detail and it is clear that all amino acids of the known specific substrate can be built into the
PKI model and all required corresponding charges can be found in the cleft of the phosphorylase kinase
structure. In the CK-1 structure determined without a peptide, the requirement of the peptide specificity
resides on the P-3 site, which has to be phosphorylated. An analysis of the surface charges of the cleft of
the CK-1 structure reveals the exact correspondence of the residues required to interact with a
phosphorylated substrate at this site.
Finally, the tripeptide of the pseudosubstrate site of IRK consisting of the Asp-Tyr-Tyr motif has
corresponding charges in the structure of the enzyme's substrate cleft and confirms the data obtained
from the degenerated peptide library for the unique sequence motifs of nine tyrosine kinases [22]. It is
becoming increasingly clear that the wealth of structural data of protein kinases with cAPK as a
prototype provides evidence for two important features concerning substrate binding. First, the substrate
binding cleft is structurally conserved and second, the surface charges of this cleft and hydrophobic
cavities on the surface are very diverse and correspond to the specificity requirement of the substrate for
individual protein kinases (see Figure la). It is now possible to use the structural conservation of the
substrate binding cleft to predict the charges and hydrophobic residues of the cleft to define substrate
specificities for individual kinases.
IV. Crystallographic Analysis of the ATP Binding Site Reveals Distinct Differences Utilized for the
Further Design of Specific Inhibitors
The diagram elucidating detailed interactions of ATP with the enzyme is presented in Figure 3a,b. Six
out of nine invariant residues of the catalytic core of protein kinase are involved in ATP binding and
catalysis. The key residues that hold the and phosphates in position are the phosphate anchor, the
metal sites, and Lys168. The amides of the residuesPhe54, Gly55, and Ser53are essential for the
position of the and phosphates. The metal site coordinated by invariant Asp184 is also sequestered
by the and phosphates and the metal
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Figure 3
(a) Ternary complex of MnATP with the inhibitor peptide PKI (524).
Glu121 and Val123 of the conserved linker region of the protein kinase catalytic core
form the bidentate hydrogen bond with the 6-amino group and N1 nitrogen of the purine
base. Thr183, non-conserved, forms a hydrogen bond with the N7 position of purine.
2'-OH of ribose interacts with the side chain of Glu127 of helix D and P-3 Arg of the
specific inhibitor while the 3'-OH interacts with Glu170 of the catalytic loop. (b) The
local environment of serine nucleophile at P site (left site) and local environment of
phosphorylated P site serine (right side). The side chain of the catalytic base is at
hydrogen bond distance from-OH of the Ser nucleophile which defines the conserved
substrate binding P site.
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site coordinated by invariant Asn171 is also sequestered by the and phosphates. The residue Lys168
is at hydrogen-bond distance from the phosphate. The postulated in-line mechanism of
phosphotransfer in cAPK [23] can be examined through an analysis of the MnATP and PKI (524), Ser
substrate peptide and ADP complexes [24]. A comparison between cAPK and IRK structures indicates
that Ser versus Tyr specificity is obtained by displacement of the substrate binding site in such a way
that the hydroxyl of nucleophiles of both Ser and Tyr fall in the same point in the active site facing
corresponding catalytic bases Asp1132 and Asp166 for IRK and cAPK respectively.
The purine base of ATP is anchored to the enzyme by three hydrogen bonds, two of them involve the 6amino group and N1 nitrogen, which interact with the backbone atoms of Glu121 and Val123. the 6amino group and N1 nitrogen of the purine base form the hydrogen bonds also in the structure of CK-1
and in the structure of phosphorylase kinase. Yet, in the structure of inactive CDK-2, the purine base
forms only one hydrogen bond via its N6 position. While N7 nitrogen interacts directly in cAPK with
the side chain of Thr183 in CK-1, this nitrogen interacts directly with Glu55 and Tyr59 via two
hydrogen-bonded water molecules and in phosposhorylase kinase via one water molecule. Hence, the
N7 nitrogen and its interaction with the enzyme is a region for potential modification of ATP
competitive inhibitors. Ribose is held by both enzyme (Glu127 and Glu170) and inhibitor (P-3 Arg).
While the side chain of Glu127 interacts with 2'-OH of ribose in cAPK, in CK-1 the 2'-OH interacts via
two water molecules with Ser91 and Asp94. In ERK-2, 2'-OH interacts with Asp109. This region has
been utilized to design ATP-based specific inhibitors by modifications of the ATP-competitive
nonspecific inhibitor staurosporine [25], see Figure 4.
In the model cAPK with bound staurosporine inhibitor, the lactam amide group of the inhibitor functions
as a bidentate hydrogen bond donor-acceptor
Figure 4
Chemical structures of staurosporine inhibitor, left, and
CGP52411.
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Figure 5
(a) Staurosporine molecule docked in the ATP binding site of PKA.
hydrogen bonds anchoring staurosporine molecule in the active site of PKA
consists of the 6-amino group and N1 nitrogen and carbonyl of Glu121 and
the amide hydrogen atom of Val123. This bidentate hydrogen bond formation
has been observed in all complexes of protein kinases and ATP solved so far.
(b) Inhibitor of CGP 52411 and ATP docked on the active site of PKA.
Residues of Glu127 and Glu170 are also shown and these are not
conserved in the EGFR kinase.
(Figure 5a). This key observation is supported by chemical data of lactam amide derivatives, which
provide a plausible model of staurosporine inhibition. This is in the protonated boat-type conformation
found to fit in the ATP binding cleft with minimal steric hindrance. In this model, the 4-amino group
forms hydrogen bonds with the backbone carbonyl of Glu170 and the carboxylate group of Glu127 of
cAPK. A model of the EGFR kinase shows that Glu127 and 170 are replaced by Cys and Arg,
respectively (Figure 5b). Replacement of Glu127 by Cys is critical according to the model and explains
the several-fold decrease of potency of staurosporine inhibitor toward the EGFR kinase (IC50=630 nM
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Figure 5
(Continued)
versus IC50=15 nM for cAPK). Yet enhancement of specificity utilizing the inhibitor CGP52411 (Figure
4), whose selectivity originates in the occupancy by one of the anilino moieties of the inhibitor in the
region of the enzyme cleft that normally binds the ribose ring of ATP, is considerable. The inhibitor
CGP52411 inhibits the EGFR tyrosine kinase with an IC50 value of 300 nM while it is less active by at
least two orders of magnitude on a panel of protein kinases including cAPK, phosphorylase kinase,
casein kinase, protein kinase C (most isoforms), and v-abl, c-lyn, c-fgr tyrosine kinases.
Hence, the three-dimensional model of EGFR tyrosine kinase rationalizes the specificity of the
CGP52411 inhibitor. This model suggests that analysis of the putative regions of the ribose binding of
ATP in other kinases through template modeling would provide the required chemical modification of
pharmacophores to enhance their selectivity. In modeling, the use of the bidendate hydrogen bonding
provided by the linker region (Figure 3a) of the conserved
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protein kinase core is essential. Yet to predict the region of the structure of the inhibitor that will form
these strong Watson-Crick hydrogen bonds remains difficult.
The use of the 6-amino group and N1 nitrogen of the purine base to model the pharmacophore, as seen
with staurosporine, is not possible in two other adenine-type inhibitors. Both isopentenyl adenine, a
nonspecific inhibitor of protein kinases, and olomoucin, a more specific inhibitor of Ser/Thr protein
kinases, are modified only at the 6-amino group position. Thus, bidentate hydrogen-bond formation as
seen in the ATP purine base is not possible. Furthermore, there are inhibitors that do not contain the
chemical structure of adenine, for example des-chloro-flavopyridol, a potent inhibitor of cdc-2 cell cycle
kinase.
In crystallographic analysis of the binding of three inhibitors, olomoucin (OLO), isopentenyl adenine
(ISO) and des-chloro-flavopyridol (DFP) to inactive CDK-2 cell cycle protein kinase, Kim and coworkers [26] have provided additional insight into the binding of adenine-and nonadenine-based
inhibitors. Inhibitors with purine rings (OLO and ISO) bind in relatively the same area of the binding
cleft as the adenine ring of ATP. Relative orientation of each purine ring with respect to the protein is
different for all three ligands. This is most likely due to the fact that the 6-amino group of adenine in
ATP is replaced by an isopentenylamino group in ISO and by the bulky benzylamino group in OLO. In
the case of the third inhibitor, which is not an adenine derivative, the benzopyran ring occupies
approximately the same region as the purine ring of ATP. The two ring systems overlap in the same
plane but benzopyran is rotated about 60 degrees relative to the adenine of ATP. In this orientation, two
strong bidendate hydrogen bond are formed with the oxygens in the 4th and 5th positions of the
inhibitor. Furthermore, these bonds are the same ones formed by the 6-amino group and N1 nitrogen of
the adenine ring.
Crystallographic analysis has shown that both the substrate and ATP-binding clefts are structurally
conserved yet differ in the surface charges between individual protein kinases. The structural template of
the protein kinase family as discovered in the structure solution of cAPK predicts these differences.
Template modeling provides a rational basis for the design of specific inhibitors for protein kinases
based on the ATP binding site [25]. This is the first significant step in the design of specific inhibitors
targeted at the ATP site.
Recent work has now shown the conformational diversity of inhibitors binding in the interdomain ATPbinding cleft [26]. Although the residues of the protein kinase catalytic core that form the bidentate
donor-acceptor bond with inhibitors are identical throughout different structures, the residues of the
inhibitors vary greatly. All inhibitors use this common bidentate bond yet the specificity lies in several
other bonds formed between the inhibitor and specific regions of the individual protein kinases.
Furthermore, it is difficult to model
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the bidentate bond-forming residues of the inhibitor and it is through protein crystallography that these
are determined. Template modeling can then be used to determine the specific surface charges of the
modeled kinase that can be exploited by the inhibitor for its specificity. Modeling and focused
combinatorial chemistry are the tools to achieve the goal of inhibitor specificity.
Acknowledgments
This work was supported by CTR grant 4237. We thank Drs. Furet, P. Traxler, and N. Lydon of Ciba for
their drawings presented in Figure 5. We also thank Dr. Nikola P. Pavletich for the coordinates of the
CDK2-CYCLIN complex used in Figure 2.
References
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11. Zheng J-H, Trafny EA, Kninghton DR, Xuong N-H, Taylor SS, Ten Eyck LF, Sowadski JM. 2.2
refined crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with
MnATP and a peptide inhibitor. Acta Cryst 1993; D49:362365.
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14. Knighton DR, Bell S, Xuong N-H, Ten Eyck LF, Taylor SS, Sowadski JM. 2.0 refined crystal
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15. Karlsson RF, Zheng J-H, Xuong N-H, Taylor SS, Sowadski JM. Crystal structure of the mammalian
catalytic subunit of cAMP-dependent protein kinase and an inhibitor peptide displays an open
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16. Zheng J-H, Knighton DR, Xuong N-H, Taylor SS, Sowadski JM, Ten Eyck LF. Crystal structures of
the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed
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9
Structural Studies of Aldose Reductase Inhibition
David K. Wilson and Florante A. Quiocho
Baylor College of Medicine, Houston, Texas
J. Mark Petrash
Washington University School of Medicine, St. Louis, Missouri
I. Introduction
Aldose reductase (ALR2; EC 1.1.1.21) is an ~36 kDa enzyme that catalyzes the reduction of a wide
range of carbonyl-containing compounds to their corresponding alcohols. It is a member of an extensive
aldo-keto oxidoreductase enzyme family, a collection of structurally similar proteins expressed in both
animals and plants. Most members of the enzyme family possess similarities in molecular mass, pH
optimum, coenzyme dependence, and demonstrate overlapping specificity for many substrates and
inhibitors.
While no essential physiological function has been established for ALR2, extensive experimental
evidence suggests that it plays an important role in the development of diabetic complications affecting
the visual, nervous, and renal systems [1]. The linkage between ALR2 and pathogenesis of diabetic
complications lies in the polyol pathway of glucose metabolism (Figure 1). In hyperglycemic tissues
such as in diabetes mellitus, the capacity of hexokinase to shunt glucose to glycolysis and other major
pathways of glucose metabolism is exceeded. Consequently, enhanced flux of glucose through the
polyol pathway occurs. The enzyme ALR2 catalyzes the first step in this pathway, producing sorbitol, an
active osmolyte. The polyol pathway is completed by the NAD+-dependent oxidation of sorbitol to
fructose, mediated by sorbitol dehydrogenase.
Extensive evidence exists to suggest a linkage between the pathogenesis of diabetic complications and
enhanced glucose metabolism via the polyol pathway. The polyol pathway functions in all tissues
susceptible to clinically
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Figure 1
Schematic of the polyol pathway showing the NADPH-dependent reduction of open
chain D-glucose to sorbitol, which is catalyzed by ALR2. This step is followed by the
NAD+-dependent oxidation of sorbitol by sorbitol dehydrogenase to yield D-fructose.
significant diabetic complications. Transgenic animals overexpressing ALR2 in target tissues of diabetic
complications are more prone to development of experimentally induced diabetic complications [2,3].
The most extensive body of evidence linking ALR2 to the pathogenesis of diabetic complications comes
from numerous successes in the treatment of experimental animals with a variety of ALR2 inhibitors
(ARI) [4]. Many of these studies demonstrated that ARIs substantially delay or in some cases prevent
the onset of complications.
Clinical trials of ARIs have yielded encouraging results in alleviating painful symptoms of diabetic
complications. However, unacceptable side effects related to toxicity or inadequate pharmocokinetic
profiles have rendered most of the drug candidates undesirable. Nevertheless, several ARIs are
commercially available in some countries and more appear to be in the pipeline. The therapeutic
rationale for treatment of human diabetics with ARIs to delay or prevent onset of diabetic complications
is compelling. Animal models with experimentally induced hyperglycemia develop complications that
are morphologically and functionally similar to that seen in the human diabetic patient. Many
structurally
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diverse ARIs have been shown to substantially delay or completely prevent the onset of such
complications in experimental animal models. While some studies indicate that ALR2 may play a
functional role in osmotic homeostasis in the kidney, evidence from animal studies suggests that it is
metabolically dispensible.
Long-term complications exact a terrible toll of morbidity and mortality on patients with diabetes
mellitus. For example, patients with diabetes have about a 25-fold increased risk for becoming blind
over that of the general population. Diabetic retinopathy is one of the most common causes of visual loss
and accounts for about 12% of new cases of blindness each year in the United States alone [5].
II. Drug Design Prior to Structural Data
Many inhibitors have been developed over the past two decades without the advantage of a structural
understanding of the enzyme [4,6,7]. Significant improvement has been made since the discovery of the
first such orally active compound to show in vivo activity, alrestatin (Figure 2), which had an IC50 in the
low micromolar range [8]. Many high-affinity inhibitors with IC50s in the low nanomolar range are now
under study.
Recent drug-design efforts have yielded compounds usually with one of two chemical motifs:
carboxylates or spirohydantoins. A number of these compounds such as tolrestat [9], ponalrestat [10],
epalrestat [11], sorbinil [12], and zopolrestat [13] have progressed to the point of clinical trials.
Unfortunately, clinical ineffectiveness and/or unacceptable side effects have limited the usefulness of
most of those that had been shown to be effective in vitro. The latter problems may be associated with a
lack of specificity since many aldose reductase inhibitors inhibit both ALR2 and aldehyde reductase
[14]. For this reason, ALR2 as well as the other members of the aldo-keto reductase family have been
the subject of crystallographic studies with the hope of determining a structural basis for inhibitor
specificity and ultimately to provide a basis for enhancing binding affinity.
III. Structural Studies of Aldose Reductase
The first crystal structures available for ALR2 were those of the porcine form complexed with the
NADPH analog 2'-monophosphoadenosine-5'-diphosphoribose [15] and the human enzyme complexed
with the NADPH cofactor [16]. Further studies have been conducted on mutants of the human enzyme
[17] and ternary complexes of the human enzyme with an inhibitor [18]. All of these
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Figure 2
A number of ALR2 inhibitors that have entered clinical trails.
structures show the protein to fold into a (/)8 barrel (Figure 3). This fold has emerged as the most
common enzyme motif [19] although most of the proteins adopting this structure share no sequence
homology. The ALR2 enzyme is, however, the first NAD(P)H binding protein to adopt this fold. It
contains an extra hairpin preceding the first strand, which caps the N-terminal end of the barrel. It
also has two helices that are not part of the regular barrel. One precedes 7 and the other follows 8.
A. Cofactor Binding
The NADPH cofactor is bound in an extended conformation across the C-terminal end of the barrel.
The catalytically active nicotinamide moiety is located
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Figure 3
C trace of the ALR2 holoenzyme looking down the (/)8 barrel. The NADPH
cofactor is seen bound across the carboxy-terminal end of the -barrel with the active
nicotinamide moiety in the center. Figure produced using the MOLSCRIPT
program [48].
at the center of the barrel while the adenosine extends away to bind between 7 and 8. A belt
composed of residues 213 to 227 folds over the pyrophosphate of the NADPH to sequester a large part
of the cofactor from the solvent. It is fastened to the other side of the NADPH binding site via Asp216
on the loop that forms bifurcated salt links with Lys21 and Lys262. The dominant interactions holding
the coenzyme in place are directional hydrogen bonds and salt links from positively charged side chains
to the phosphates. The interaction between the 2' phosphate on the NADPH and the side chains from
Lys262 and Arg268 account for the enzyme's preference for NADPH over NADH. Earlier biochemical
studies had shown that it is the 4-pro-R hydride that is transferred from the nicotinamide to the substrate
[20]. This is ensured by a hydrogen-bonding network using side chains from Ser159 and Asn160 and the
main chain of Gln183 to orient the amide. It is also determined by the stacking interactions with Tyr209,
which is adjacent to the 4-pro-S side of the nicotinamide.
B. Mechanism
The catalytic site was unambiguously identified using the location of the nicotinamide moiety of the
NADPH cofactor in the holoenzyme structure. The region surrounding the catalytic site is a 12- deep
groove that measures
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approximately 7 by 13 and is lined primarily with hydrophobic side chains. This is entirely consistent
with earlier experiments showing that the enzyme has a marked preference for lipophilic substrates
versus polar substrates such as sugars [21].
The catalytic site also suggested a model for the enzyme's chemical mechanism, which is substantially
similar to most other NAD(P)H-dependent oxido reductases. Upon binding of the reduced cofactor, the
enzyme is able to form a ternary complex with the substrate. The pro-R hydride from the C-4 of the
nicotinamide is transferred to the carbonyl carbon of the substrate, which in turn causes the carbonyl
oxygen to abstract a proton from a general acid, which is presumably located on the protein, to form the
alcoholic product. Three proton-donating side chains are located within 6 of the C-4 atom in the
NADPH cofactor that could potentially fulfill this role: Tyr48, His110, and Cys298. Since it is not
conserved in other members of the aldo-keto reductase family that exhibit enzymatic activity (Figure 8)
Cys298 was unlikely as a candidate proton donor. The histidine is surrounded by several hydrophobic
residues including Val47, Trp79, and Trp111, which would serve to lower the pKa of the side chain,
making it less effective as a proton donor at physiological pHs. The tryrosine, which ordinarily has a pKa
of approximately 11 engages in an interaction with the charged ammonium group of Lys77, which in
turn charge-pairs with Asp43. This network serves to depress the pKa of the phenolic oxygen, increasing
the exchangability of the proton.
Subsequent activity studies involving site-directed mutants support this model [17,22]. The Tyr48
rarrow.gif Phe mutation shows a complete lack of activity while the Asp43 rarrow.gif Asn, Lys77
rarrow.gif Met, His110 rarrow.gif Asn, and Cys298 rarrow.gif Ser showed losses in catalytic
efficiency of approximately 100-, 1000-, 106-, and 10-fold respectively when compared with the wildtype enzyme. These results correlate well with the functions predicted for each residue with the
exception of the histidine.
The structure of the ALR2 holoenzyme showed that the catalytic site was situated atop the nicotinamide
moiety of the NADPH cofactor. The substrate binding site, which would determine the enzyme's
specificity and also presumably bind inhibitors, appeared to be composed of a deep cleft (Figures 3 and
4). It extended away from the catalytic site towards the loop composed of residues between 4 and 4
and the last 20 residues of the carboxy-terminal meander. This hypothesis was supported by the
appearance of poorly resolved density that occupied this region, which suggested the presence of an
endogenously bound substrate or inhibitor in the structure of the holoenzyme [16]. Subsequent studies
indicate that this electron density may be a citrate molecule, one of the components included in the
crystallization mixture. Activity studies indicate that citrate is indeed one of the many inhibitors of the
enzyme with a Ki in the millimolar range [23].
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Figure 4
Surface representation of the ALR2 holoenzyme in an orientation similar to Figure 3.
The nicotinamide moiety that defines the active site of the enzyme is seen in the center.
The groove that extends down from it is highly hydrophobic and was initially assumed
to be the inhibitor binding site. Figure prepared using the GRASP program [49].
IV. Aldose Reductase Complexed with Inhibitor

While a large number of high-potency inhibitors for ALR2 have been developed [4], a structural
understanding of the exact molecular features that foster this affinity have been only vaguely
understood. Several general chemical motifs such as hydrophobic ring systems, a spirohydrantoin group
or carboxylate group are seen repeatedly when examining a list of known inhibitors (Figure 2) but little
was known about the specific role for each in inhibitor binding.
The structure of the ALR2/NADPH/zopolrestat ternary complex [18] has provided some answers about
the mode of binding of zopolrestat (Figure 2), a high-affinity, carboxylate-containing compound
developed by Pfizer, Inc. [13]. While the overall structure was preserved, the inhibitor binding induced a
conformational change of the enzyme. This change, which involved the movement of several loops in
the active site of the molecule, was large enough to cause a change in crystal packing relative to the
holoenzyme. As a consequence of the shifting of the active-site loops, a cavity is created inside the
protein in which the benzothiazole ring is seated and the groove that was implicated in substrate and
inhibitor binding by the holoenzyme structure vanishes. This illustrates the unpredictability of
conformational changes within a protein in response to substrate or inhibitor binding. It also implies that
modeling compounds
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in the active site of an enzyme or drug-design algorithms such as inhibitor docking may be inappropriate
in some cases if they do not adequately model considerable plasticity in the binding site.
A closer look at the binding of zopolrestat shows that it is dominated by extensive hydrophobic contacts
between the protein and the inhibitor. These include side chains from Trp20, Tyr48, Trp79, Trp111,
Phe115, Phe122, Trp219, Ala299, Leu300, Tyr309, and Pro310 (Figure 7). This is not surprising given
the apolar nature of the enzyme's active site as determined by the holoenzyme structure. What is
surprising is that the inhibitor created the part of its own binding site that the benzothiazole rings occupy
by burrowing into the hydrophobic core of the protein to carve out a region with very good steric
complementarity to this moiety. It does this rather than binding in the solvent-exposed hydrophobic
binding groove that is seen in the holoenzyme structure.
The remaining interactions involving hydrogen bonds and salt links also appear to be very important in
inhibitor binding. With the exception of one of the fluorine atoms, all atoms that are able to engage in
hydrogen bonding do so. The carboxylate, which is seen in so many aldose reductase inhibitors, is saltlinked to His110, which is located very near the catalytic site (Figure 7). Presumably, the carboxylate in
the other inhibitors plays the same role and could be used as an anchor when modeling these into the
active site.
Inhibition studies involving ALR2 have indicated noncompetitive inhibition for virtually all compounds
examined to date when the forward (reduction) reaction is monitored. This mode of inhibition is often
interpreted as meaning that the inhibitor binds to a site on the enzyme that is independent of the catalytic
site. Kinetic and competition studies have both led to this conclusion in the case of ALR2 [24,25]. The
crystal structure of the enzyme complexed with both the NADPH cofactor and zopolrestat, however,
clearly shows the inhibitor occupying the region directly above the nicotinamide of the NADPH and,
therefore, the active site (Figures 5, 6, and 7).
Most previous inhibition studies reported noncompetitive and/or uncompetitive inhibition patterns when
aldose reductase inhibitors were examined in the forward direction, i.e. inhibition of NADPH-dependent
aldehyde reduction. With the finding that the overall rate-limiting step in the direction of aldehyde
reduction is at the level of structural isomerization following alcohol product release [26,27], it is not
surprising that lack of competitive inhibition would be observed in such standard double reciprocal
plots. To further complicate matters, many aldose reductase inhibitors were not recognized in previous
studies as tight-binding inhibitors and were inappropriately evaluated using Michaelis-Menten kinetics.
Thus, noncompetitive or uncompetitive inhibition patterns were previously reported for inhibitors that
were subsequently shown to bind directly at the active site. Recent structure-function and kinetic studies
have revealed important details concerning the structural basis for the catalytic
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Figure 5
Schematic representation of the ALR2/NADPH/zopolrestat ternary complex. The
NADPH is bound across the enzyme from the center to the right while the zopolrestat
binds atop the nicotinamide and extends to the lower left. Conformational changes are
seen in the C-terminal loop below the zopolrestat in the picture and the loop to the right
of the inhibitor between 4 and 4.
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Figure 6
A surface representation of the ternary complex as seen in Figure 5. Note that the
inhbitor creates part of its binding site by burrowing into the protein rather than
binding entirely in the groove seen Figure 4.
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Figure 7
Stereo of zopolrestat binding to the active site of ALR2. The salt link made by the carboxylate of the inhibitor and
hydrogen bonds are depicted with dashed lines. The remainder of the interactions are apolar with the residues shown.
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and inhibition mechanisms and provided a clarification for the mechanism by which inhibitors of both
the carboxylate [28] and spirohydantoin [29] classes bind at the active site of ALR2.
V. Aldo-Keto Reductase Family
A. Effects of Sequence on Drug Design
The problem of structure-based drug design for ALR2 and the drug-design effort in general is
compounded by the fact that this enzymes is a member of a large family of aldo-keto reductases with
overlapping substrate specificity. In humans at least three such enzymes have been found: ALR2 [30],
aldehyde reductase (ALR1) [30], and chlordecone reductase [31]. Other members of the family that have
been isolated in other species include rat 3-hydroxysteroid dehydrogenase [32], murine fibroblast
growth factor induced protein [33], bovine prostagladin F synthase [34], murine vas deferens protein
[35], frog -crystallin [36], the P100/11E gene product in Leishmania major [37], and Corynebactium
diketogluconate reductase [38]. This large number suggests that there may be more such enzymes to be
found in humans. The similarities between the proteins with respect to both the sequence and substrate
specificity implies that the nature of the substrate binding sites are similar across the family. This has
indeed been the case in all the structures determined from this family to date (see below).
While detailed binding studies of various inhibitors with all the different enzymes have not been
conducted, it is likely that drugs intended for ALR2 are likely to cross react with many of the other
enzymes within the family. One such case that has recently been studied both crystallographically and
biochemically is the murine FR-1 protein described below.
The binding sites of all of these enzymes are characterized by their large size and hydrophobicity
suggesting that ideal substrates may be steroids or molecules of a similar size and nature. Sequence
comparisons of all the proteins, including those whose structure has not yet been determined, show that
there is a large amount of similarity involving residues implicated in substrate binding (see Figure 8).
One region that diverges somewhat is the 15-amino acid segment at the carboxy terminus of the protein.
This segment is likely to be responsible for what little differences in substrate specificity exhibited by
the enzymes. It is the same segment that is seen adopting a different conformation upon zopolrestat
binding to ALR2. It may then be possible that it is not only the chemical nature of this loop thatin
making positive and negative interactions with the substrate/inhibitormodulates specificity, but also
the flexibility conferred by the amino acid sequence. Such a difference is seen when contrasting the
structures of ALR2 and FR-1 bound to zopolrestat.
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Figure 8
Sequence alignment of several members of the aldo-keto reductase family. Abbreviations used
are HALR2, human aldose reductase (30); HALR1, human aldehyde reductase [30]; 3-HSD,
3-hydroxysteroid dehydrogenase [32]; FR-1, murine FR-1 [33]; BPGFS, bovine
prostaglandin F synthase [34]; CCDR12, human chlordecone reductase [31]; CDGR,
Corynebacterium diketogluconate reductase [38]; MVDP, murine vas deferens protein [35],
JFRC, Japanese frog crystallin [36].
B. Structures
Structures of several other members of the aldo-keto reductase family have also been determined. These
include aldehyde reductase [39,40], FR-1 [41] and 3-hydroxysteroid dehydrogenase [42]. Since each of
these proteins retain a large amount of sequence identity and homology with human ALR2, it is not
surprising to note that the overall tertiary structures are very similar. Root-meansquare C deviations
between the human ALR2 holoenzyme structure and the rest of the family are in the range of 12
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Closer examination of the active site shows that the residues involved in catalysis (most notably the
residues analogous to Asp43, and Tyr48, and Lys77 in ALR2) are structurally conserved among each of
these proteins (Figure 8), suggesting that the mechanism is also conserved throughout the family. Many
of the residues found in the binding site (defined as those making contact with the zopolrestat in the
ternary complex) are also largely conserved with the exception of a number of residues in the carboxy
terminus of the protein. This is indeed where the most structure variation appears to be concentrated
among the proteins. These residues compose a loop that is the same loop that shifts upon binding of
zopolrestat in ALR2. Inhibitors with improved specificity will very likely take advantage of the subtle
structural differences that are introduced by the variation in sequences in this area.
VI. Future Design of Aldose Reductase Inhibitors
The availability of structural data for ALR2 in its holoenzyme and different ternary forms is likely to
lead to improvements in the affinity of future generations of inhibitors. As the architecture and plasticity
of the binding site are better understood, increasingly potent inhibitors may be designed to occupy it.
Although these structures provide a positive target for drug design, there are a number of negative
targets. Increased in vivo potency is likely to be derived from the specific inhibition of ALR2 that would
entail the avoidance of other members of the aldo-keto reductase family. Determination of the structures
of other members of the family may increase the specificity of compounds by providing structures of
targets to avoid. While the incorporation of the negative targets in the drug-design process relies on the
determination of other structures and is likely to be complicated, conventional computational techniques
may be applied to the problem of the positive target. Two such methods that may hold promise are
docking [43] and computational thermodynamic perturbation [44].
A. Inhibitor Docking to the Enzyme
Our initial efforts to exploit the ALR2 holoenzyme structure for drug design utilized the program DOCK
[43]. This program is capable of finding depressions on the surface of the enzyme that could serve as
binding sites for substrates or inhibitors. Once the correct area is defined, the program rotates structures
of candidate compounds within this space and scores each compound based upon its steric
complementarity with the binding site. However, the program does not include the potential polar
interactions between the inhibitor and protein when scoring. The search was further constrained by the
inability to include conformational variations both in the test compounds as well as the protein, due to
computational limitations.
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This method was used to screen a significant portion of the contents of the Cambridge Structural
Database [45] against the ALR2 holoenzyme binding site (D.K. Wilson, J. M. Petrash, and F. A.
Quiocho, unpublished data). Among the approximately 30 highest scoring compounds were several
aromatic aldoximes that had inhibition constants in the micromolar range. These were similar to
aldoximes such as benzaldoxime, which has been previously observed to have similar inhibition
constants [46].
A disappointing result was that this search did not rediscover any of the known high-affinity ALR2
inhibitors that were contained in the search library. Before the determination of the ternary complex of
the enzyme with zopolrestat, this was interpreted as meaning that these compounds bound to the enzyme
in a conformation somewhat different than the one adopted in the crystal structure used for the search.
The structure of the ternary complex showed this to be a wrong assumption; it was the protein that
changed conformation upon inhibitor binding, creating a pocket that did not exist in the holoenzyme
structure. When bound to the protein, zopolrestat is actually quite similar in conformation to its small
molecule x-ray structure. It is therefore very possible that different ALR2 inhibitors and substrates may
cause the enzyme to flex in different ways, creating binding sites that may be different in size and
chemical nature.
B. Computational Thermodynamic Perturbation
Computational thermodynamic perturbation is a powerful, albeit computationally expensive, group of
techniques that are designed to estimate relative binding affinities of two closely related drugs, given the
structure of at least one of them complexed with the target protein [44]. This approach has the potential
to assay candidate compounds in the computer for improvements in inhibitor binding, thereby removing
the necessity to sythesize and assay these compounds in the lab.
For a number of reasons, ALR2 promises to be a good system for the application of this technique and
the experimental verification of the results. The structure is very well determined in complex with
zopolrestat, a high-affinity inhibitor. A number of zopolrestat derivatives with various functional groups
decorating the compound have been sythesized and partially characterized with respect to ALR2
inhibition [13,47]. These compounds could serve as a sort of basis set of controls for the theoretical
calculations. If parameters used in these calculations can be selected such that the computationally
derived binding energies agree even qualitatively with the experimentally determined binding energies,
serious consideration should be given to new compounds that are predicted to bind with enhanced
affinity. Since ALR2 is crystallizable with zopolrestat bound, there is every reason to believe that
crystals of the enzyme complexed with similar compounds will be obtainable. Such structures could
provide the basis for further rounds of drug improvement.
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VII. Conclusions
The observation of a protein undergoing a conformational change when binding to an inhibitor, as seen
with ALR2 and zopolrestat, illustrates a common problem associated with structure-based drug design.
It is tempting to view proteins as static structures since their crystal structures are static. Attempts to
design drugs to fit the apparent active site of an enzyme may fail when the plasticity of the protein is not
taken into account. While the disorder associated with amino acid side chains can be modeled with a
moderate computational effort, larger conformational changessuch as the loop movement seen in
ALR2are virtually impossible to predict. Until this becomes possible, x-ray crystal structures of
complexes will continue to be indispensible.
Finally, it can be easy to forget that a compound's affinity for the protein is not the only consideration
when designing inhibitors of enzymes from a structural point of view. The structures of aldose reductase
and the FR-1 protein complexed with the drug zopolrestat, a compound with a very high affinity for
ALR2, can serve as a reminder of how specificity can also be a very important factor. This is
particularly true when a protein is a member of a family of proteins that share sequence homology and
are apt to have overlapping specificities. Structure may then play a key role in the determination of
features that are unique to the target protein and therefore prime considerations when designing
inhibitors.
Acknowledgments
We thank T. Reynolds who assisted with the production of the figures. This work was supported by a
grant from Research to Prevent Blindness, Inc. and grants EY05856, EY02687, and DK20579 to J. Mark
Petrash. Florante A. Quiocho is an investigator of the Howard Hughes Medical Institute.
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10
Structure-Based Design of Thrombin Inhibitors
Patricia C. Weber and Michael Czarniecki
Schering-Plough Research Institute, Kenilworth, New Jersey
I. Roles of Thrombin in Hemostasis and the Therapeutic Utility of Thrombin Inhibitors
Thrombin is a serine protease that plays critical roles in both blood clot formation and anticoagulation.
In the penultimate step of the coagulation cascade, thrombin cleaves soluble fibrinogen to form
insoluble fibrin. Thrombin also activates other coagulation factors including Factor XIII, the enzyme
responsible for crosslinking fibrin to further stabilize the thrombus. Additional clot-promoting functions
include stimulation of platelet aggregation by cleavage of the thrombin receptor. In contrast to its roles
in clot formation, thrombin participates in anticoagulant functions. For example, thrombin-mediated
activation of protein C, a protease involved in anticoagulation, is enhanced when thrombin is complexed
with thrombomodulin, and in this complex, thrombin can neither cleave fibrinogen nor activate platelets.
The interrelationship among thrombin's many roles in hemostasis is complex and presents several
mechanisms for inhibition of thrombus formation. For recent reviews see References 1 through 5.
Most drug discovery efforts focus on thrombin inhibition as a means to prevent the serious
consequences of thrombus formation in myocardial infarction and stroke. Thrombin inhibitors may also
prevent clot formation in patients prone to deep vein thrombosis or repeat heart attack. In combination
with thrombus dissolution therapies, thrombin inhibitors may decrease the incidence of reocclusion due,
in part, to the release of active clot-bound thrombin.
In this article, recent examples of small molecule inhibitors interacting at the fibrinogen primary
specificity pocket and with residues of the catalytic triad
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are given. Inhibitors designed to make more extended interactions with thrombin are also presented.
II. Structure of Thrombin
Thrombin consists of two polypeptides, an A chain of 36 residues and a 259-residue B chain, linked by a
disulfide bond. The crystallographic structure of thrombin reveals a globular protein organized about
two barrels with the overall folding pattern of the chymotrypsin serine protease family [6,7]. The
catalytic triad and nearby oxyanion hole are located roughly between the barrels and adopt the
geometric arrangement required for serine-protease-assisted, peptide bond cleavage (Figure 1).
Thrombin's multifunctionality and regulation of activity are achieved by specialized subsites on the
enzyme's surface (Figure 2). Fibrinogen cleavage, for example, involves interactions at the primary
specificity pocket, the extended fibrinogen recognition exosite, and an additional specificity pocket.
Subsite interactions differ for cleavage of other thrombin substrates including the thrombin receptor and
protein C. Additional and overlapping subsites exist for thrombin effector molecules including heparin,
antithrombin III, and heparin cofactor II [8,9].
Figure 1
Stereoscopic view of the crystallographic structure of thrombin complexed with
N-acetyl-(D-Phe)-Pro-boroArg-OH. Helical regions are represented in the standard
way and arrows indicate regions of sheet. Solid lines show the thrombin
bound conformation of N-acetyl-(D-Phe)-Pro-boroArg-OH (taken from Reference 10).
Active-site residues, His57 and Ser195, are shown with a ball-and-stick representation.
The authors thank Dr. C. L. Strickland for the drawing.
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Figure 2
Schematic representation of Subsite Utilization in Thrombin Complexes (after
Reference 8). Fibrinogen interacts with three thrombin subsites (here thrombin is
represented by a large oval and the interconnected subsites by an irregular
three-armed shape). Physiological effectors of thrombin and thrombin inhibitors form
distinct interactions at these subsites. Additional subsites, such as the
heparin-binding site, exist on the thrombin surface and are not indicated here.
The catalytic triad is represented by three circles at the vertices of a triangle.
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III. Thrombin Inhibitors Directed at the Fibrinopeptide a Binding Pocket

The majority of synthetic thrombin inhibitors interact at the fibrinopeptide A binding pocket, which
includes the catalytic residues Ser195 and His57, hydrogen-bonding capabilities within the oxyanion
hole, peptide backbone functional groups that hydrogen bond with the peptide backbone of the substrate,
and residues involved in amino acid recognition (Figure 3). Many of these binding determinants are
utilized by N-acetyl-(D-Phe)-Pro-Arg-chloromethylketone (PPACK [7]) and its boronic acid analog
(DUP714 [10]). The crystallographic structures of these molecules complexed with thrombin have both
served as starting points for structure-based drug design and as reference structures for comparison of
binding modes of other inhibitors.
The use of arginine boronate esters as transition-state mimetics results in potent peptidyl thrombin
inhibitors. These inhibitors, however, exhibit significant affinity for other serine proteases that have in
common a specificity for substrates with basic residues at P1 (e.g. trypsin, Factor Xa, and plasmin).
Earlier work demonstrated that neutral side chains of P1 boronate esters impart greater selectivity for
thrombin. The boropeptide shown in Figure 4 was investigated as the prototype of neutral side chain,
tripeptide thrombin inhibitors [11]. It had a Ki against thrombin of 7 nM and shows selectivity relative to
other trypsin-like plasma proteases. Since these inhibitors have a neutral residue at the P1 site, Deadman
and coworkers [11] sought to demonstrate the mode of binding to thrombin in the absence of a salt
bridge with Asp189.
Figure 3
Schematic diagram of binding determinants within
the fibrinopeptide A binding pocket of thrombin and
their utilization by N-acetyl-(D-Phe)-Pro-boroArg-OH.
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Figure 4
Schematic representation of the active-site orientation of a
neutral P1 boronic acid thrombin inhibitor.
Boron-11-NMR, a sensitive probe of the chemical environment around boronate esters, can distinguish
between trigonal and tetrahedral forms of boron. The 11B-NMR spectrum of this inhibitor complexed
with thrombin showed a single peak at -17 ppm that remained constant for 7 hours. The chemical shift
suggests boron adopts a tetrahedral geometry on binding to thrombin and is consistent with the
orientation of the inhibitor in the active site shown in Figure 4. While the 11B-NMR revealed an
interaction within the catalytic site, it could not distinguish between bonding with Ser195 or His57.
Kahn and coworkers [12] recently investigated the application of synthesized peptidomimetics as novel
inhibitors of thrombin. Fibrinogen peptide A mimetic (FPAM, Figure 5) incorporates a bicyclic
peptidomimetic within the turn region of fibrinogen peptide A. The bicyclic peptidomimetic confers
conformational stability to the turn region as suggested by x-ray crystal structures of fibrinogen peptide
complexes as well as complexes of BPTI with thrombin.
X-ray crystallographic studies of FPAM complexed with thrombin (Figure 5) showed that the S1 subsite
is occupied by the arginine guanidinium [12]. The Val group of FPAM makes extensive hydrophobic
contacts within the S2 apolar binding site. The Gly at P3 interacts with thrombin via a -sheet-type
hydrogen bond with the carbonyl group of Gly216 and appears to be important in the positioning of the
bicyclic ring corresponding to the bend. This bicyclic ring, although not aromatic, forms an edge-toface contact with Trp215. One of the phenyl rings shows hydrophobic contact with lle174, while the
other shows no significant interactions with thrombin. By comparison to other inhibitors complexed to
thrombin, FPAM appears to have a new binding mode that differs from that of substrate, or hirudin, or
argatroban.
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Figure 5
Schematic representation of the principal intermolecular
interactions of a fibrinogen peptide A mimetic within the active site
of thrombin.
Obst et al. [13], departing from the peptide template, designed and synthesized novel nonpeptide
inhibitors of thrombin. They began with a cyclic template having attachment sites for three side chains
that would be complementary to the S1, S2, and S3 sites in thrombin. Important to the design was a rigid
template that would avoid hydrophobic collapse of the side chains and loss of conformational degrees of
freedom upon complex formation with thrombin. Using computational approaches (Insight
II/Discover/CVFF force field), possible templates were modeled within the active site of thrombin.
These studies resulted in the synthesis of thirteen analogs that shared a common template.
The most active molecule (Ki = 90 nM, 8-fold selective versus trypsin) was studied further by x-ray
crystallography (Figure 6). The positively charged benzamidine binds into the S1 pocket of thrombin
forming a bidentate hydrogen bond with Asp189. The proximal carbonyl of the rigid template acts as a
hydrogen-bond acceptor for the amide NH of Gly216. The methylene dioxybenzyl group at P3 interacts
with thrombin in two ways. An edge-to-face interaction was observed with Trp215, and an oxygen of
the methylenedioxy group acts as an acceptor for a hydrogen bond with the OH hydrogen of Tyr60A.
Recent communications from Bristol-Myers Squibb [14,15] describe peptidomimetic inhibitors (Figure
7) that were designed to bind thrombin with an N- to C-polypeptide chain sense opposite that of the
substrate and form interactions similar to those made by the first three residues of hirudin (lle1, Thr2,
Tyr3). In the x-ray crystal structure of BMS-183507 (Ki = 17.2 nM) with thrombin [15], the N terminus
is facing the catalytic site while the methyl ester is
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Figure 6
interactions of a nonpeptide bicyclic inhibitor within the
active site of thrombin.
exposed to solvent. No specific interactions were observed with the catalytic triad. A bound water
molecule hydrogen bonded to the Ser195 hydroxyl.
The complex is stabilized by a network of hydrogen bonds as well as hydrophobic interactions. The
Phe1-O and the Phe3-NH form hydrogen bonds with Gly216, and the Phe1-NH hydrogen bonds to the
backbone carbonyl of Ser214. The Phe1 phenyl group occupies the S2 site, while Phe3 interacts within
the S3 site.
The retro-inhibitors contain a 4-guanidinobutanoyl group that extends into the S1 specificity site. Rather
than forming two hydrogen bonds between the guanidine and Asp189 in a manner similar to PPACK,
BMS-183507 forms only one, with the second hydrogen bond being directed to the carbonyl oxygen of
Gly219. Binding affinity, as evidenced by loss of more than two orders of magnitude in affinity on
addition of one or two methylene groups, was sensitive to chain length at this position.
The allo-Thr hydroxyl oxygen accepts a hydrogen bond from the backbone NH of Gly219. This
additional interaction accounts, at least in part, for the increase in affinity when compared to the
inhibitor with Leu in this position. Comparison of the crystal structures of thrombin complexed with
BMS-183507 and with hirudin reveals that the hirudin residue, Thr2, and the allo-Thr of BMS-183507
interact differently with thrombin. The hirudin Thr2 binds at S2,
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Figure 7
Schematic representation comparing the principal
inhibitor-to-thombin interactions of related inhibitors with either
Leu (7a) or allo-Thr (7b) at P3.
whereas the allo-Thr sidechain is oriented toward the protein exterior and is partially exposed to solvent.
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Cyclotheonamide A (CtA), a macrocyclic marine natural product derived from the Japanese sponge,
Theonella sp., inhibits thrombin with an IC50 value of
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Figure 8
interactions of cyclotheonamide A within the active site of
thrombin.
100 nM and represents a novel structural class of serine protease inhibitors. An x-ray crystal structure of
CtA complexed with thrombin was used to determine the molecular basis for this inhibition (Figure 8
[16]). The Arg-Pro unit binds to the S1 and S2 sites in a manner similar to the Arg-Pro of PPACK. The
Arg guanidinium group forms a bidentate hydrogen bond with Asp189 while the Pro establishes a sheet interaction with the Ser214-Gly216 backbone. The -ketoamide acts as a transition-state mimetic
forming a tetrahedral hemiketal with the hydroxyl of Ser195. Within the complex, CtA adopts a
relatively open conformation with the Pro orthogonal to the macrocycle and confined by a hydrophobic
pocket defined by Tyr60A, Trp60D, and Leu99. Two aromatic residues are involved in stacking
interactions with Tyr60A and Trp60D. Cyclotheonamide A, however, does not effectively match the S3
interactions provided by the D-Phe group found in PPACK. In CtA, the formamide group is too polar to
effectively complement the S3 site adjacent to Trp215. The authors note that the complex of CtA with
thrombin does not appear optimal and suggest that synthetic analogs could significantly improve both
potency and selectivity.
Starting with the known thrombin inhibitors Argatroban and N(2-naphthyl-sulfonyl-glycyl)-DL-pamidinophenylalanyl-piperidine (NAPAP), a group at Roche initiated a medicinal chemistry program to
develop thrombin inhibitors with reduced toxicity and an improved hemodynamic profile [17].
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The discovery program proceeded in four iterative phases which are shown in Table 1. Initial screening
of low molecular weight organic bases led to the discovery of 1-amidinopiperidine (11) as a new
surrogate for the guanidine and amidine functionality in Argatroban and NAPAP, respectively. A
distinct advantage of 1-amidinopiperidine is its intrinsic selectivity for thrombin over
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trypsin. Application of three-fold iterative strategy of design involving synthesis, x-ray crystallography,
and molecular modeling, this group elaborated the 1-amidinopiperidine from structures that inhibited in
the micromolar range to some inhibiting in the picomolar range. In doing so significant improvements in
the selectivity of thrombin relative to trypsin were also achieved. In the case of the D-amino acid series
(12), a second inhibitor binding mode that differed from that of Argatroban was identified. In this
new and unexpected binding mode, the S2 pocket is unoccupied and the napthalenesulfonyl group fills
the S3 site and overlaps the front of the S2 site. The benzyl group of the phenylalanine is oriented
toward the protein surface and is partially exposed to solvent. The Argatroban or inhibitor binding
mode was favored by the more potent L-amino acid series (13 and 14) where the piperidide (13) or Nbenzyl (14) binds to the S2 site and the aryl groups are found in the S3 site.
IV. Bivalent Thrombin Inhibitors Directed at the Fibrinopeptide a Binding Pocket and the
Fibrinogen Recognition Site
A strategy to prepare highly selective thrombin inhibitors involves linkage of molecules capable of
interacting at distinct subsites. This approach should result in inhibitors more specific for thrombin:
while serine proteases possess common structural features related to catalysis and some serine
proteasesincluding the coagulation enzyme Factor Xaalso exhibit primary substrate specificity for
positively charged residues, only thrombin possesses recognition subsites for fibrinogen and effector
molecules such as thrombomodulin. Nature has used this strategy in the evolution of hirudin, the
anticoagulant protein produced by the medicinal leech. When this effective anticoagulant binds
thrombin [1820], the N-terminal domain blocks the primary specificity pocket while the C-terminal
residues adopt an extended conformation and make multiple interactions within the fibrinogen
recognition exosite.
Guided by structural and biochemical information, small molecules capable of simultaneous interactions
with both the primary specificity pocket and the fibrinogen recognition exosite were designed and
synthesized. These bivalent inhibitors are composed of three regions: a group to block the primary
specificity pocket, a sequence to bind the fibrinogen recognition site, and a chemical linker. The bivalent
inhibitor approach was first executed with peptides [2122]. In 1990, DiMaio et al. (33 [22]) used the
peptide sequence from hirudin to link (d-Phe)-Pro-Arg-Pro, known to bind at the primary specificity
pocket [23], with hirudin C-terminal residues, known to bind at the fibrinogen recognition site.
Polyglycine linkers were also used to connect these sequences (Maraganore
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et al. [21]). Among these hirudin analogs, the tetraglycine linker appeared optimal (38, Ki = 2.3 nM,
Table 2).
Most of the peptide-based bivalent inhibitors were slowly cleaved by thrombin. Incorporation of a
ketomethylene pseudo peptide bond (34) resulted in a noncleavable bivalent inhibitor that retained high
thrombin affinity [24]. Decreased proteolysis in bivalent inhibitors increasingly nonpeptide in character
continues to be observed.
Chemically simpler linkers were made using multiple methylene-containing glycine variants [25]. The
dependence of affinity on placement of amide linkage within linkers containing the same number of
atoms indicated some specific thrombin-to-linker interactions (313,14,15,16). This was confirmed in
the crystal structure of hirutonin-6:thrombin complex (326 [26]) where continuous electron density was
observed for the entire bivalent inhibitor including the linker region.
The extended nature of the fibrinogen recognition site complicates attempts to reduce inhibitor
molecular weight while maintaining affinity. Although of similar molecular weight, substitution of the
sequence -Asp-Tyr-Glu-Pro-lle-Pro-Glu-Glu-Ala-cyclohexylalanine-(D-Glu) for -Asp-Phe-Glu-Glu-llePro-Glu-Glu-Tyr-Leu-Gin increases affinity an order of magnitude (compare 317 and 318). Within a
series of bivalent inhibitors, inclusion of sulfated tyrosine, the naturally occurring residue of hirudin,
increases affinity 5 to 6 fold (38 compared to 311, and 31 to 32). Only seven residues are present in
one of the smallest bivalent inhibitors (326).
Increasingly nonpeptide substituents have been incorporated into the primary specificity pocket binding
portion of the bivalent inhibitors. Higher affinity for thrombin was achieved by replacement of the (DPhe)-Pro-Arg with either dansyl-Arg-(D-pipecolic acid) (317, [27]) or 4-tert-butylbenzenesulfonyl-Arg(D-pipecolic acid) (318, [27]). While the arginine side chain of these and the (D-Phe)-Pro-Argcontaining inhibitors make similar interactions with the aspartic acid within the S1 specificity pocket,
the dansyl-Arg-(D-pipecolic acid) inhibitors bind in a nonsubstrate mode [27]. This initial result suggests
that other nonpeptide thrombin inhibitors may be successfully incorporated into bivalent inhibitors.
Recently, a pyridinium methyl ketone bivalent inhibitor capable of forming a reversible covalent
complex with thrombin was synthesized (326, [28]). Crystallographic analysis of its complex with
thrombin showed the ketone carbonyl becomes tetrahedrally coordinate by bonding to the side chain of
thrombin's active site residue, Ser195. Substitutions of cyclohexylalanine for phenylalanine (34
compared to 35) and the cyclohexylalanine-containing fibrinogen recognition peptide for the hirudin
sequence (317 compared to 318) also contribute to the increased affinity of this bivalent inhibitor.
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V. Inactivated Thrombin as an Inhibitor of Clot Formation

A means to selectively inhibit thrombin's role in coagulation while preserving its anticoagulant functions
involves site-directed mutagenesis of thrombin itself. By introduction of a single mutation, Gibbs et al.
[29] altered thrombin's relative specificity for fibrinogen and protein C. The engineered thrombin's
increased activation of protein C over fibrinogen cleavage offers the possibility of inhibiting clot
formation with a modified human protein, a molecule likely to exhibit few side effects.
VI. The Role of Structural Information
The discovery of thrombin inhibitors has benefited from available protein structural information. Models
of the thrombin overall structure and its active site geometry, constructed from available structures of
related serine proteases [30], aided in the design of the mechanism-based inhibitors such as PPACK [31]
and its boroarginine analog [10]. The unexpected, nonsubstrate binding mode of early thrombin
inhibitors such as NAPAP was revealed by x-ray crystallographic analyses [32]. Iterative structurebased design methods have been critical in the optimization of bivalent inhibitors and inhibitors directed
at the primary specificity pocket. Structures of inhibitor:thrombin complexes are essential for the
optimization of substitutents forming interactions within the aryl-binding site of the primary specificity
pocket. In some cases (e.g. Table 1), seemingly minor alterations of the inhibitor can result in dramatic
changes in the inhibitor's overall interactions with thrombin [17].
Drug discovery efforts have also been strongly influenced by results of structural studies of thrombin
complexed with effectors and substrate peptides. For example, recently the structures of thrombin
complexed with fibrinopeptide A [33] and human prothrombin fragment F1 [34] have been determined.
In addition to their role in design of high-affinity inhibitors, these structures provide valuable insights
for design of drugs specific for the various subsites and conformational states of thrombin.
VII. Conclusion
Discovery of therapeutically effective thrombin inhibitors involves issues such as affinity and
selectivity, bioavailability, and formulation. In addition to these relatively common concerns, the
complex in vivo mechanisms designed to
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balance its pro- and anticoagulant activities present additional challenges in the discovery of
therapeutically effective thrombin inhibitors.
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11
Design of Antithrombotic Agents Directed at Factor Xa
William C. Ripka
Corvas International, Inc., San Diego, California
I. Introduction
Serine proteases have long been recognized as important players in a number of biochemical processes
and their specific and selective inhibition provides multiple therapeutic opportunities [1]. In particular,
the blood coagulation process is the result of an amplified cascade of proteolytic events in which several
specific zymogens of serine proteases in blood are activated sequentially by selective cleavages to
produce active enzymes [2,3]. This process, in pathological circumstances, may lead to the formation of
a thrombusan insoluble matrix of fibrin and platelets. Thrombosis is a serious medical problem in the
United States and Europe as exemplified by the fact that half the people who die each year die of
cardiovascular related problems. While much recent work in antithrombotic therapeutic approaches has
focused on inhibition of thrombin, the central role that Factor Xa plays in the coagulation response to
vascular injury also makes it an ideal pharmacological target for antithrombotic drug development. The
recent report of the x-ray crystal structure of native Factor Xa [4] allows, for the first time, a wellfounded structure-based drug design approach for inhibitors. A number of reviews describing the
biology [510] and chemistry [11,12] of Factor Xa inhibitors have appeared.
II. Coagulation Cascade
In the coagulation cascade (Figure 1), a highly amplified process leads to the formation of thrombin,
which is the primary mediator for the conversion of fibrinogen to fibrin, as well as activation of platelets
through the thrombin
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Figure 1
The coagulation cascade.
receptor. Thrombin generation is itself, however, the result of Factor Xa in complex with Factor Va on a
phospholipid surface (the prothrombinase complex) acting on prothrombin. Vascular injury is the
initiating event in the coagulation process, causing the activation of Factor Xa by the Factor VIIa/tissue
factor complex. Factor Xa is, therefore, a central and crucial enzyme directly leading to the production
of thrombin and its inhibition should be effective in blocking thrombogenesis. As a consequence of its
key role early in the coagulation cascade process Factor Xa represents a potentially valuable therapeutic
target for potent and specific inhibition.
III. Proof of Principle for a Factor Xa Inhibitor
In recent years the method by which certain hematophageous organisms maintain blood flow during
feeding has been determined. Interestly, several of these organisms utilize Factor Xa inhibitors to
prevent coagulation [1315]; the tick anticoagulant peptide (TAP), a small protein isolated from the
Ornithidoros moubata tick [13], and antistasin isolated from the Haementeria officinalis leech [14] are
both potent and selective inhibitors of Factor Xa. As expected, these molecules are effective
antithrombotics in several animal models of thrombosis (Table 1) and provide an important proof of
principle with regard to the potential effectiveness of Factor Xa inhibitors as therapeutic anticoagulants.
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Table 1 Factor Xa Inhibitors (TAP, Antistasin) in Experimental Models of Thrombosis
Rat and rabbit models of venous thrombosis [25]
Canine model of high shear, coronary arterial thrombosis [6,26]
Canine model of femoral arterial thrombosis [27,28]
Rhesus monkey model of acute disseminated intravascular coagulation [29,30]
Baboon model of platelet dependent arterial thrombosis [9,31,32]
In an alternate approach, it has been shown that a covalently blocked, activesite-modified Factor Xa
(DEGR-Xa) [16] as well as a catalytically impaired recombinant form [17] can be effective
anticoagulants in models of deep-vein thrombosis [18] and in canine arterial thrombosis models [8]. In
these examples, the active-site-inactivated Factor Xa competes with the active form for incorporation
into the active prothrombinase complex since the binding of Factor Xa to Factor Va in this complex is
independent of the active site [20]. These studies with Factor Xa inhibitors suggest that inhibiting earlier
in the coagulation cascade, as well as inhibiting the production of thrombin by inactivating Factor Xa in
the prothrombinase complex, may have certain therapeutic advantages.
IV. Factor XaStructure and Function
Factor Xa is a 59 kilodalton protein synthesized in the liver and secreted into the blood as an inactive
zymogen (Figure 2) [21]. Prior to secretion the singlechain molecule undergoes co- and posttranslational modifications including removal of a signal sequence [2224], gamma carboxylation of
several glutamic acids (Gla) in the N-terminus [33], beta hydroxylation of Asp63 [34], N-glycosylation at
two sites [35], and cleavage at two sites, Arg139 and Arg142, to give a two-chain molecule [36]. The
mature form of Factor X consists of a light chain (139 amino acids) and a heavy chain (303 amino acids)
held together by a single disulfide (Figure 2). The Gla residues are responsible for calcium and
phospholipid binding and the second EGF domain is thought to mediate binding to Factor VIIIa and
Factor Va [37,38]. The heavy chain contains the catalytic domain with the prototypic serine protease
active site triad, His226, Asp279, and Ser376. During coagulation, Factor X is converted to the active
protease, Factor Xa, by a complex of Factor VIIa/tissue factor or a complex of Factor IXa/Factor,
VIIIa/phospholipid, and calcium, both of which cleave a specific Arg-Ile bond to release an activation
peptide (Figure 2) [39]. Similar to the activation of chymotrypsin, trypsin, and thrombin, the newly
formed N-terminal Ile folds into the interior of the protein to form an ion pair at the active site with
Asp]375 [39,40]. In the presence of calcium ions the newly formed Factor Xa associates with Factor Va
on a phospholipid membrane surface to form the
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Figure 2
Factor Xa structure. Residues of the catalytic triad (His226, Asp279, Ser376) are circled.
prothrombinase complex that rapidly converts prothrombin (PT) to thrombin by cleavage at two sites in
PT, Arg271Thr272 and Arg320Ile321 [41]. The x-ray structure of human des(145) Factor Xa at 2.2
resolution has now been reported [4] (Figure 3).
V. Natural Inhibitors of Factor Xa
Several small, potent, and naturally occurring Factor Xa inhibitorstick antico-agulant protein
(TAP)[3], tissue factor pathway inhibitor (TFPI) [42], antistasin (ATS) [43], Ecotin [44,45]have been
isolated and characterized (Table 2). All but TAP apparently inhibit the enzyme in the extended
substrate conformation referred to as the standard mechanism of inhibition (Figure 4) [47]. In this
standard mechanism the inhibitor presents a conformationally constrained binding loop with a partial
beta sheet motif to the target enzyme that mimics the required substrate conformation and, after binding
to the enzyme, can undergo a reversible proteolytic hydrolysis at the reactive site peptide bond (P1P1').
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Figure 3
Stereo ribbon diagram of Factor Xa [4]. Residues of the catalytic triad are shown (His57, Asp102, Ser195) as well as residues
of specific interest for the binding of small molecules to the active site: Glu192; S4 pocket residues Tyr99, Phe174, Trp215; and
S1 pocket residue, Asp189. Residues are designated with the chymotrypsin numbering.
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Table 2 Naturally Occurring Inhibitors of Factor Xa
Inhibitor
Source
Ki
Structural information
TFPI
Human
3 pM [49]
90 nM [50]
Ecotin
Escherichia coli
50 pM [44]
TAP
Ornithidoros moubata (tick)
Antistasin
Haementeria officinalis (leech)
61 pM [51]
(X-ray in progress) [52]
AcAP5
Ancylostoma caninum
43 pM [19]
homology to Ascaris lumbricoides

var.suum [79,80]
135 pM [13]
X-ray; complex with trypsin [46]

2D-NMR [57,58]
Studies of these natural inhibitors can be useful in defining the active site requirements for Factor Xa
inhibition, and importantly, can indicate the level of inhibition that may be necessary for an effective
Factor Xa inhibitor, recognizing that TAP and antistasin have evolved to yield functional, in vivo
antithrombotics. Table 3 shows the reactive-site sequences of these substratelike inhibitors as well as the
cleavage site sequences recognized by Factor Xa in the activation of prothrombin (PT), Factor VII, and
Factor V.
A. Tissue Factor Pathway Inhibitor (TFPI)
The mature tissue factor pathway inhibitor (TFPI) is a 276-residue protein consisting of three tandom
domains with homology to the Kunitz-like protease
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Figure 4
Extended binding modes for substrates and inhibitors. Sites in the enzyme (S) and in the
inhibitor (P) are designated by the Schecter-Berger notation [48].
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Table 3 Active Site Sequences for Factor Xa Substrates and Inhibitors
Substrates
P4
P3
P2
P1
P'1
P'2
P'3
P'4
PT271,272
Ile
Glu
Gly
Arg
Thr
Ala
Thr
Ser
PT320,321
Ile
Asp
Gly
Arg
Ile
Val
Glu
Gly
FVII
Pro
Gln
Gly
Arg
Ile
Val
Gly
Gly
FV
Lys
Lys
Tyr
Arg
Ser
Leu
His
Leu
Antistasin
Val
Arg
Cys
Arg
Val
His
Cys
Pro
Ecotin
Val
Ser
Thr
Met
Met
Ala
Cys
Pro
TFPI-II
Gly
Ile
Cys
Arg
Gly
Tyr
Ile
Thr
AcAP5
Cys
Arg
Ser
Arg
Gly
Cys
Leu
Leu
AcAP6
Cys
Arg
Ser
Phe
Ser
Cys
Pro
Gly
Inhibitors
inhibitors [42]. A potent inhibitor of both Factor VIIa and Xa as well as trypsin, TFPI does not,
however, have significant activity against leukocyte elastase, urokinase, activated Protein C, tissue
factor plasminogen activator, thrombin, or kallikrein [53,54]. The second Kunitz domain from the Nterminus of TFPI has been identified as primarily responsible for the Factor Xa inhibition while both the
first and second domains contribute to inhibition of Factor VIIa [42] (Figure 5). The proposed
mechanism for this Factor-Xa-dependent inhibition of FVIIa/tissue factor involves the formation of a
quarternary FXa-TFPI-FVIIa/TF complex [42]. The recombinant, isolated second domain, TFPI-II, has
a Ki for Factor Xa of 90 nM [50] compared to 3 pM [49] for the intact protein. The sequence of the
P4P5' region of the Factor Xa inhibitory second Kunitz domain (Table 3) has been incorporated into a
prototypic Kunitz inhibitor, bovine pancreatic inhibitor (BPTI), to produce potent and selective Factor
Xa inhibitors [75,76].
B. Antistasin (ATS)
Antistasin is one of several anticoagulants isolated from the Mexican leech, Haementeria officinalis
[14]. It is a 119-amino-acid cysteine-rich protein with a primary structure that shows a two-fold
sequence symmetry suggesting the molecule possesses two separate and distinct domains [51].
Mutagenesis studies have shown that ATS binds to Factor Xa in a substratelike manner in the
P3(Arg32P'3(Cys37) regions and is cleaved only in the first domain [55].
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While the x-ray structure of antistasin has not been reported, it is known that a Factor-Xa-induced
cleavage occurs between Arg34 and Val35 suggesting this peptide loop conforms to the conformationally
rigid substratelike conformation suggested by other known protein inhibitors of serine proteases [55]. A
hallmark of this mode of inhibition is the rigid structure around the cleaved
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Figure 5
Predicted secondary structure of tissue factor pathway inhibitor (TFPI) showing the Factor
Xa and Factor VIIa inhibitory domains. The arrows point to the P1 sites.
bond, often imposed by cysteine crosslinks constraining the two ends of the cleavage site to be in close
proximity even after cleavage [47]. Antistasin has cysteines at the P2(Cys33) and P'3 (Cys37) positions.
C. Tick Anticoagulant Peptide (TAP)
The tick anticoagulant peptide (TAP) is a 60-amino-acid polypeptide isolated from the soft tick
Ornithodorus Moubata and is a potent (Ki = 2200 pM) and selective inhibitor of Factor Xa, both as the
free enzyme and in the prothrombinase complex [13]. The TAP anticoagulant does not inhibit trypsin or
other trypsinlike serine proteases and, importantly, is not cleaved by Factor Xa. The mechanism by
which TAP inhibits Factor Xa appears to be unique and it apparently does not utilize the substratelike
binding modes characteristic of antistasin and the Kunitz inhibitors. Mutagenesis studies have shown
that the primary interaction of TAP with Factor Xa occurs at the N-terminous where Arg3 appears to
play a key role [56]. The solution structure of TAP has been deter-
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Figure 6
Stereo diagram of the NMR solution structure of the tick anticoagulant peptide. The crucial
N-terminus Arg3 is indicated along with the pattern of cysteine bonds.
mined by 2D-NMR studies [57,58] (Figure 6). With the exception of the region neighboring the
Cys15Cys39 bond in TAP, these studies support the originally proposed idea that there are significant
structural similarities between TAP and Kunitz proteinase inhibitors [10]. Nevertheless, it is clear that
TAP inhibitors FXa by a fundamentally different, and as yet, not fully understood mechanism.
D. AcAP's
In addition to ticks and leeches, other hematophagous organisms such as hook- worms have also evolved
potent and selective Factor Xa inhibitors as anticoagulant strategies. Two such proteins, AcAP5 and
AcAP6, have been isolated from the Ancylostoma caninum hookworm [19]. The AcAP5 protein is a 77amino-acid polypeptide with 10 cysteine residues. It inhibits the amidolytic activity of Factor Xa with a
Ki of 43 5 pM. Incubation of rAcAP5 with its target enzyme Factor Xa results in partial cleavage of
the Arg40Gly41 peptide bond suggesting the sequence around this cleavage site can adopt the restricted
conformational requirements of substrates [47].
The AcAP6 protein is a 75-amino-acid polypeptide, also with cysteines, with a Ki for Factor Xa
inhibition of 996 65 pM. Alignment of the sequences of AcAP5 and AcAP6 suggest the P1 residue in
AcAP6 is Phe38 and not the basic residue usually associated with Factor Xa specificity [19]. Substitution
of Phe38 in AcAP6 with Arg resulted in a mutant that inhibited Factor Xa with a potency similar to
rAcAP5. Both AcAP6 and ecotin suggest that an Arg or
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Lys in the P1 position is not an absolute requirement for potent and selective activity.
E. Ecotin
Ecotin, a protein isolated from Escherichia coli, is a promiscuous protease inhibitor that potently inhibits
kallikrein, urokinase, Factor XIIa, granzyme B, trypsin, chymotrypsin, and elastase (reviewed in
Reference 46). As with most protein inhibitors (hirudin [59] and TAP [10] being the exceptions) ecotin
presents a baitlike substrate sequence to the target protease resulting in a reversible cleavage of the
P1P1' peptide bond. However, unlike other Factor Xa inhibitors that require basic residues such as Arg
or Lys in the P1 position, ecotin is cleaved between two hydrophobic amino acids, Met84 and Met85 [44].
Three preliminary crystal structures of ecotin with the serine proteases chymotrypsin, trypsin, and
fiddler crab collagenase have been described [46]. The conformation of the sequence around the reactive
site is similar to the bovine pancreatic trypsin inhibitor (BPTI) but differs in that the Cys at P' (not P2 as
in BPTI) provides the rigidifying function for the reactive-loop sequence. Interestingly, antistasin has
cysteines at both the P2 and P3' positions. The Pro at P4' is commonly found in FXa inhibitors including
antistasin.
In its complexes with the serine proteases for which structures are available [46] there is a sub van der
Waals contact between Met84-C and the enzyme Ser195-O. The Met84-O faces the oxyanion hole and
forms hydrogen bonds with Ser195 and Gly193. In the trypsin structure the Met84 side chain extends into
the S1 site in a manner similar to Lys15 in the BPTI-trypsin complex. Ecotin also forms both beta sheet
hydrogen bonds to the enzyme Gly216, Ser82-N and O to Gly216 O and N.
VI. Small Molecule Inhibitors of Factor Xa
While the x-ray structure of native Factor Xa has been reported [4] the nature of its crystal packing,
specifically the fact that the active site of one Factor Xa molecule is blocked by the N-terminus of a
second resulting in a continuous polymeric structure, apparently has precluded diffusing inhibitors
into the preformed crystals to obtain complexes. Complexes with inhibitors cocrystallized with Factor
Xa also have not been reported [81]. Thus, efforts to do structure-based design with this enzyme have
relied on molecular modeling.
Since, to date, it has not been possible to directly obtain x-ray structures of inhibitor complexes with
Factor Xa, the substantial information available with respect to how serine proteases, particularly
thrombin, bind inhibitors can
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be utilized to model known Factor Xa inhibitors using the x-ray coordinates of native Factor Xa.
Preliminary modeling of the several inhibitors described below into the Factor Xa active site was
accomplished by the author [69] by first superimposing the backbone atoms (N, Ca, C) of the catalytic
triads (His57, Asp102, Ser195 in the benzamidine:thrombin and PPACK:thrombin x-ray structures on the
corresponding residues in Factor Xa. This allowed an excellent fit of the P1 basic groups,
arylbenzamidine in the case of benzamidine and arginine for PPACK, into the S1 pocket of Factor Xa.
These groups were then used as templates for positioning the appropriate P1 basic groups of the various
synthetic inhibitors [69]. Holding these docked P1 groups fixed, the remaining rotatable bonds were
manipulated to allow a reasonable and complementary fit of the inhibitor atoms to the solvent accessible
surface of the Factor Xa active site. In those cases where it was possible, hydrogen bonds, particularly to
Gly216, were formed. To fit extended peptide sequences such as that for antistasin and the antistasinderived peptides described below, the backbone atoms of the residues around the cleavage site (e.g.,
P4P4') were positioned using the corresponding BPTI backbone atoms as a template. This was done
after first aligning the trypsin catalytic triad backbone in the BPTI:trypsin x-ray complex (vida infra) to
Factor Xa. Where the side chains of the bound peptide segments differed from BPTI, their orientation
was either modeled for maximum complementarity to the Factor Xa molecular surface or set by an
algorithmic approach [78]. In some cases the structures obtained were energy minimized initially by
steepest descent followed by conjugate gradient minimization.
Recently, compounds based on a bisamidine motif (e.g., DX-9065a) have been reported as potent and
selective Factor Xa inhibitors (1, DX-9065a) [6063].
The position of the amidino group makes little difference to the Factor Xa potency of these compounds
but, interestingly, has a dramatic effect on the selectivity towards thrombin [62]. It was also observed
that one carboxylic acid isomer (CX-9065a) was 7 times more potent on Factor Xa than the other. A
second set of analogs shows a similar SAR [60].
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In both cases the acids are much more selective for Factor Xa over thrombin. An initial modeling study
using a homology-built Factor Xa structure proposed a fit of DX9065a to Factor Xa in which the
amidinoary1 group occupies the S1 pocket and the acetimidoyl group is directed out of the S4 pocket
[60].
Lin et al. [64] have provided a more systematic study of possible fits of compound 2 and DX9065a
using the recently available Factor Xa coordinates [4]. After aligning the His57, Ser195, and Asp102
backbone atoms for Factor Xa and thrombin (in the benzamidine:thrombin x-ray structure [65]) the
arylamidino group of 2 was superimposed on the benzamidine template and a systematic conformational
search was performed on the rotatable bonds of inhibitor 2. Energetics and complementarity to the
Factor Xa surface determined a saved set, about 300 low-energy conformations, for further study. The
final result was an optimized structure in which the acetimino group of 2 fits into
Figure 7
Molecular modeling fit of compound 2 with the arylamidino group positioned in the S1
pocket and the acetimino group in the S4 cation- site [69].
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the S4 pocket formed by the aromatic residues Trp215, Tyr99, and Phe174 (Figure 7). It was proposed that
this collection of aryl residues forms the basis for a cation- interaction that has recently been well
documented in other cases [66]. Interestingly, this cation- site is absent in thrombin's equivalent aryl
binding site where the corresponding residues are Trp215, Leu99, and Ile174residues that cannot
provide the -electrons necessary for stabilization of the cation in the inhibitor. The apparent preference
for FXa inhibitors to have cations in the P3,P4 position may be directly related to the ability of the
Factor Xa S4 site to stabilize these cations via a cation- interaction. Factor Xa appears to be unique
among the coagulation factors in providing this electron-rich S4 pocket (Table 4).
The initial discovery of bisamidine structures as potential Factor Xa inhibitors was actually made much
earlier with the finding that compounds such as 4 showed an almost 300-fold preference for Factor Xa
over thrombin with a Ki of 13 nM (FXa) [67],
As with the DX-9065 analogs and compounds 2 and 3, the Factor Xa potency was relatively insensitive
to the positioning of the amidino groups (4,4' versus 3,3') while replacing the 7-membered cycloalkyl
ring with the 5 or 6 membered ring analogs reduced potency by about 10 fold. Model building
compound 4 and docking into Factor Xa [69], again using the x-ray benzamidine:thrombin complex [65]
as a template, shows that the second aryl amidino group can be positioned into the S4 aromatic pocket of
Factor Xa in a conformation closely related to the mode of binding proposed for DX9065a (Figure 8)
[64].
In an effort to compare the relative efficacy of thrombin versus Factor Xa inhibitors, Markwardt et al.
[67] synthesized a set of amidinoaryl compounds with moderate potency as Factor Xa inhibitors (5).
Table 4 S4 Residues in Selected Serine Proteases
Residue Positiona
Factor Xa
Thrombin
Factor VIIa
Trypsin
99
Tyr
Leu
Thr
Leu
174
Phe
Ile
Pro
Gly
215
Trp
Trp
Trp
Trp
aChymotrypsin
numbering system. Sequence alignments by comparison of x-ray structures (sequence

for Factor VIIa).
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The n=3 chain length was optimal while the nature of the tosyl group on the - nitrogen was relatively
nonspecific, with tosylGly and N--naphthylsulfonyl- Gly being of similar potency. While the phenyl
group on the amide nitrogen was best, other groups were also tolerated. Although the authors did not
speculate on how these compounds were bound to Factor Xa, it is reasonable to suggest that the amidino
phenyl group fits in the S1 pocket similar to the orientation determined by x-ray crystallography for
benzamidine in the benzamidine:thrombin x-ray crystal structure. If the aryl amidino group of 5 is
matched to that of benzamidine after alignment of the catalytic triad backbone atoms (N,C,C) for
thrombin and Factor Xa, a proposed fit of 5 to Factor Xa can be made (Figure 9) [69]. This mode of
binding is consistent with the structure activity relationships observed but does not suggest the reasons
for the observed small preference for Factor Xa over thrombin.
Figure 8
Molecular modeling fit of compound 4 in the Factor Xa active site [69]. The carbonyl of the
cycloheptanone makes a hydrogen bond (3.12 ) to N-Gly216.
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Figure 9
Molecular modeling fit of compound 5 in the Factor Xa active site.
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Figure 10
Proposed model of dansyl-Glu-Gly-Arg-chloromethyl ketone in Factor Xa [4,69].
Tulinsky and coworkers [4] have proposed a model for the complex of dansyl-Glu-Gly-Argchloromethyl ketone using the thrombin-PPACK crystal structure as a template for the fit to Factor Xa
(Figure 10).
Using antistasin as a starting point, Ohta et al. [68] have synthesized a series of cyclic peptides based on
the antistasin sequence. Three of these peptides are shown below and represent the most potent in the
series.
Ki (FXa)
ATS29-47
NH2-Ser-Gly-Val-Arg-Cys*-Arg-Val-His-Cys*-Pro-His-Gly-Phe-Gln-ArgSer-Arg-Tyr-Gly-OH
ATS29-40
NH2-Ser-Gly-Val-Arg-Cys*-Arg-Val-His-Cys*-Pro-His-Gly-OH
11.8 M
dR-ATS32-38
NH2-dArg-Cys-Arg-Val-His-Cys-Pro-OH
0.96 M
(The Cys*-Cys* are joined in disulfide bonds to form cyclic structures)
0.035 M
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These molecules are cleaved by Factor Xa suggesting they bind in a similar manner to antistasin itself.
Assuming the sequence around the cleavage site occupies the FXa active site locally in a manner similar
to BPTI in the BPTI:trypsin complex, a modeled structure of the complex can be constructed
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Figure 11
dArg-ATS32-38 modeled into the active site of Factor Xa utilizing the BPTI: trypsin x-ray structure as a template [69].
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using BPTI residues 1119 as a template [69]. The modeled dR-ATS32-38:FXa complex is shown in
Figure 11. Interestingly, these peptides do not inhibit trypsin even at 1000-fold higher concentrations
than their FXa inhibitory concentrations. Antistasin, on the other hand, inhibits trypsin with a Ki of 10
nM.
Preliminary reports have appeared describing a pentapeptide that is a potent (Ki=3 nM) and selective
inhibitor of Factor Xa (SEL2711) [70,71].
Two possible modes of binding can be envisioned for this compound with either the methylpyridinium
group occupying the S1 site in a substratelike mode or the p-amidinophenyl group in the S1 pocket,
which would require a reversed binding reminiscent of the hirudin-thrombin interaction [59]. Figure 12
shows the case for the amidinophenyl group in the Factor Xa S1 pocket [69]. In this mode of binding the
methylpyridinium group easily fits the S4-aryl binding site and is well positioned for a -cation
interaction.
Of interest from a drug-design viewpoint is the finding that cyclotheonamide, a compound isolated from
a marine sponge and originally reported as a thrombin inhibitor, has been found to also inhibit Factor Xa
with a Ki of 50 nM [72]. Cyclotheonamide possesses a novel -ketoamide transition state functionality
and x-ray structures of cyclotheonamide with trypsin [73] and thrombin [74] provide templates for
modeling this inhibitor into Factor Xa [69]. In the resulting fit (Figure 13) cyclotheonamide does not
project functionality into the S4-cation- site and would not be expected to show Factor Xa selectivity.
VII. Defining the Requirements for Factor Xa Inhibition by Mutagenesis of BPTI
It has been known for some time that many examples of naturally occurring Kunitz inhibitors exist, both
isolated and as domains in larger proteins, which inhibit a variety of serine proteases [47]. This strongly
suggests that this molecular framework is compatible with inhibition of this general class. The contact
region between these inhibitors and their protease targets is known from a
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Figure 12
Modeled fit of SEL2711 in Factor Xa with the arylamidino group positioned in the S1 pocket and the methylpyridinium
group in the cation- S4 site.
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Figure 13
Cyclotheonamide modeled into Factor Xa utilizing the x-ray structure of cyclotheonamide:trypsin [73] and
cyclotheonamide:thrombin [74] as templates [69].
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number of x-ray structures of complexes. Appropriate site-specific and random mutagenesis, particularly
of the P3-P4' residues of a prototypic Kunitz inhibitor, bovine pancreatic trypsin inhibitor (BPTI), has
been shown to result in potent and selective Factor Xa inhibitors [75,76].
A potent inhibitor of trypsin, kallikrein, and plasmin, BPTI does not inhibit Factor Xa. It binds to serine
proteases such as trypsin in an extended substrate mode from residue 13 (P3) through 17 (P'2) [47]. A
second loop from BPTI also extends into the active site bringing residues 34, 39, and 46 into contact
with the protease-active site. In terms of spatial proximity of residues three clusters can be defined:
cluster 1 (13,39); cluster 2 (11,17,19,34); and cluster 3 (16,18,20,46). While residue 39 is approximately
in the same region of space as residue 13 (9.4 CB-CB) the CA rarrow.gif CB vectors are directed in
different directions and substitution at 39 would not be expected to have a cooperative effect with
residue 13. Residue 34 on the other hand is in a key position. It is centrally located between residues
11,17, and 19 with CB-CB distances of 5.6, 5.7, and 6.5 respectively, and its CA rarrow.gif CB
vector converges with the corresponding vectors from these residues to a common point in space. This
residue is therefore expected to have a substantial cooperative effect with the other residues of cluster 2.
Finally residue 46 is close to residue 20 (CBCB of 6.5 ) although the CA rarrow.gif CB vectors are
approximately parallel and cooperative effects are expected to be minimal. The BPTI residues
11,12,13,1520, 34,39, and 46 were therefore the focus of the site-directed and random mutagenesis
studies. Residue 14 is Cys in BPTI and was not modified in the mutants since it is required for structural
reasons.
A. Site Specific Mutagenesis
As a starting point for the design of BPTI-based Factor Xa inhibitors, the second domain of TFPI (TFPIII) was used as a template [75,76]. Table 5 shows the results of site-directed mutagenesis of BPTI.
Mutant 50cl is a direct analog of TFPI-II with the exception of the Lys at position 46. The finding that
4c2 and 4c10 are essentially equivalent in potency (Ki 2.8 versus 1.8 nM) and are identical
Table 5
Site-Directed BPTI Mutants with Factor Xa Inhibition

Ki(nM)
12
13
14
15
16
17
18
19
20
34
39
46
r-TFPI-II
90
Gly
Ile
Cys
Arg
Gly
Tyr
Ile
Thr
Arg
Lys
Leu
Glu
50cl
205
Gly
Ile
Cys
Arg
Ala
Tyr
Ile
Thr
Arg
Lys
Leu
Lys
4c2
2.8
Gly
Ile
Cys
Arg
Ala
Tyr
Ile
Thr
Arg
Val
Leu
Glu
4c10
1.8
Gly
Ile
Cys
Arg
Ala
Tyr
Ile
Thr
Arg
Val
Leu
Lys
57c1
1.6
Gly
Ile
Cys
Arg
Ala
Tyr
Ile
Ile
Arg
Val
Leu
Lys
Document
w.t.
BPTI
>1 mM
Gly
Pro
Cys
Lys
Ala
Arg
Ile
Ile
Arg
Val
Arg
Lys
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cal in sequence with the exception of the Glu46 Lys switch, suggests this residue is of minor importance.
Therefore, the potency of 50cl (within a factor of 3) is consistent with r-TFPI-II. On the other hand, the
incorporation of Val for Lys at position 34 leads to a dramatic increase in potency (100 fold; cf. 4c10
and 4c2 with 50c1). From cluster 2 above this suggests the importance of P5, P'2,P'4 for potency. The
changes in wild type BPTI that result in a potent Factor Xa inhibitor are Lys15 rarrow.gif Arg15, Arg17,
rarrow.gif Tyr17, and Arg39 rarrow.gif Leu39.
B. Random Mutagenesis
11Libraries of mutant BPTI were created by inserting mutagenic cassettes in the BPTI gene of
filamentous phage PIII coat proteins [76]. These libraries produced large numbers of mutants (~106)
with randomized amino acids in positions 11, 13, 16, 17, 18, 19, 20, 34, and 39. The mutants were
panned against Factor Xa, which was affixed to a solid support by a nonneutralizing antibody and the
most potent inhibitors were separately expressed as soluble proteins. By this process it was possible to
determine consensus sequences at the reactive sites and to define the pharmacophore requirements of
inhibitors of Factor Xa in both a functional and conformational sense from the P4 to the P5 positions.
Inhibitor amino acid preferences from both site directed and random mutagenesis studies are shown in
Table 6.
VIII. Positional Requirements of Factor Xa Inhibitors (Table 6)
Examination of models of BPTI-mutants bound to Factor Xa show the L-amino acids in the P3 position
project into solvent. In the Factor Xa cleavage sites in thrombin these residues are polar and acidic (Glu,
Asp); they are polar and basic in antistasin (Arg), and polar and neutral in Ecotin (Ser). The exception is
TFPI- II, with this position occupied by Ile. The BPTI random mutant results are consistent with the
TFPI-II case and show a preference for aliphatics or aromatics in this position. There is a hydrophobic
pocket in the enzyme, formed by Trp215, Tyr99, and Phe174, that would be accessible to a D-residue in
this position.
The accessibility of the S2 pocket of the enzyme by P2 groups would be expected to be influenced by
the orientation of Tyr99. As the x-ray structure shows its position this residue puts severe limitations on
the size of the P2 group. Consistent with this is the observation that Gly is the sole residue in the
FXa:thrombin cleavage sites. Little information is available from the BPTI mutants, TFPI-II, or
antistasin, which all have a structural requirement for Cys at this position. Synthetic compounds show,
however, that in potent inhibitors large bulky aromatics are, in fact, allowed at this position, a situation
that requires Tyr99 to move out of the way [75].
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Additionally, the binding of BPTI mutants also requires Tyr99 to move because of potential severe
interactions with the Cys14Cys38 bridge of the BPTI mutants. While larger groups can be
accommodated, structural requirements are fairly rigid in agreement with the limited mobility expected
of the Tyr99. The apparent requirement for a P2-Gly in the larger substrates may suggest that in these
cases extended binding occurs that does not permit movement of Tyr99.
In the P1 position, as expected, a basic group is preferred. Interestingly, of the two naturally occuring
basic amino acids, arginine is preferred over lysine. This is seen in both the BPTI mutants as well as the
Arg rarrow.gif Lys switch in antistasin. This may be due to Ala190 in the S1 pocket of FXa, which
cannot orient and stabilize the BPTI lysine analog as Ser190 does in trypsin. An interesting exception to
the need for a basic group is in Ecotin where a methionine occupies this site. The x-ray of Ecotin with
trypsin clearly shows this neutral residue in the P1 pocket, aligned very closely to that seen for lysine in
the BPTI:trypsin complex, and proximal to the charged Asp189 [77]. Apparently, extended binding over
the rest of the site compensates for this energetically unfavorable situation.
In the P'1 position, the natural cleavage sites use Thr and Ile while Ecotin has Met. In contrast to
thrombin, FXa lacks the 60-insertion loop and can accommodate large groups at this position. The BPTI
mutants, however, are forced to use a small residue (Ala) because of steric hindrance from the Cys58Cys42 group residue 61 in the enzyme. The inhibitor TFPI-II has a Gly at P'1.
The BPTI mutants, TFPI-II, and antistasin all show a preference for aromatic groups at P'2. In the BPTI
panning experiments Tyr was selected more than 80% of the time at this position. It can be seen from
Table 5 that the Arg to Tyr change at position 17 is one of three significant changes that converts wild
type BPTI from a non-Factor Xa inhibitor to a 1.6 nM inhibitor. There is a possible hydrogen-bond
interaction between Tyr17 of the inhibitor and Gln192 of the enzyme, which may explain the strong
preference.
While models suggest the P'3 residue is directed at solvent and the FXa thrombin cleavage sites have
polar residues at this position (Thr, Glu), the BPTI mutant results show a clear preference for a
hydrophobic group. It is possible that aromatic groups can pack to Phe41 of the enzyme. Mutant results
show Ile is favored over Phe, His, which in turn is selected over Tyr.
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IX. Conclusions
Factor Xa is clearly an important component of the coagulation process and inhibition of this enzyme
can lead to potent anticoagulant effects. Recently, a number of naturally occurring anti-Factor-Xa
polypeptides have been isolated from several hematophagous organisms including ticks, leeches, and
hook- worms. With the exception of TAP, these molecules appear to bind to Factor Xa by the standard
mechanism of inhibition proposed earlier [49]. The sequence information derived from these inhibitors
as well as the natural cleavage sites of substrates of Factor Xa can be used along with the conformational
constraints imposed by the proposed substrate-like binding to define the pharmacophore requirements of
the active site of Factor Xa. The structurally rigid BPTI mutants, which have been found to be potent
Factor Xa inhibitors, also provide important conformational information particularly with regard to the
specific binding interactions on the P' side of the Factor Xa active site. A number of small molecule
inhibitors have also recently been reported which appear to take advantage of a unique cation- S4-site
available in Factor Xa to achieve good selectivity with moderate potency. The availbility of the X-ray
structure of native Factor Xa has allowed molecular modeling approaches to suggest possible fits of
these inhibitors to the Factor Xa active site.
Note Added in Proof
After this review was written, the x-ray structure of Factor Xa with DX-9065a was reported [81].
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12
Polypeptide Modulators of Sodium Channel Function as a Basis for the
Development of Novel Cardiac Stimulants
Raymond S. Norton
Biomolecular Research Institute, Parkville, Victoria, Australia
I. Introduction
Cardiovascular diseases remain one of the major causes of premature death in western societies. Chronic
congestive heart failure (CHF) in particular is a common disease with a poor prognosis, median survival
times after the onset of heart failure being 1.7 years in men and 3.2 years in women [1]. Current
treatment relies on diuretics to reduce fluid volume, vasodilators to decrease the work load of the heart,
and positive inotropic agents to increase cardiac contractility [2]. The most commonly prescribed of the
positive inotropes is the cardiac glycoside digoxin (Figure 1) [3]. Although this drug has been in
therapeutic use for over two hundred years, its efficacy in patients with a sinus rhythm has remained
controversial, and evidence for its beneficial effects is quite recent [35]. It is also possible that these
beneficial effects are not due solely to the positive inotropic activity of digoxin and that its
neurohormonal effects may also be important [2, 57] Nevertheless, digoxin remains a widely used drug
[3] and it follows that a suitable replacement or adjunct would find access to a significant market
worldwide.
The incentive to develop such a replacement follows from the low therapeutic index of digoxin [8,9] and
the relatively common occurrence of side effects due to digitalis toxicity. In the 1960s and 1970s,
2030% of patients receiving digitalis experienced serious toxicity and about one quarter of this group
died [6, 10]. Digitalis toxicity is manifest in CNS side-effects such as
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Figure 1
Structures of the positive inotropes digoxin [3,4], DPI 201-106 [15], and BDF 9148
[1517]. In digoxin the R group is (O-2,6-dideoxy--D-ribo-hexopyranosyl-(1 rarrow.gif 4)-O-2,
6-dideoxy--D-ribo-hexopyranosyl-(1 rarrow.gif 4)-2, 6-dideoxy--D-ribo-hexopyranosyl)oxy. In
DPI 201-106 the configuration at the hydroxyl-bearing carbon influences cardiac
activity.
fatigue, visual disturbances, and anorexia, and in cardiac side-effects that depend on the nature and
extent of the underlying heart disease [3]. Careful monitoring of digoxin serum levels and bioavailability
have reduced the incidence of digitalis toxicity [3] and the recent introduction of digoxin-binding
antibodies or antibody fragments has provided an effective means of treating severe digitalis toxicity
[3,7]. Nevertheless the quest continues for a substitute for the cardiac glycosides in the treatment of
chronic CHF.
Positive inotropic compounds can be classified into three groups: cAMP generators, intracellular
calcium regulators, and modulators of ion channels or pumps [11]. The cAMP generators such as
dopamine, dobutamine, and milrinone (a phosphodiesterase inhibitor) may worsen ischemia, cause
arrhythmias, and increase mortality [2,6]. Intracellular calcium modulators have not reached clinical use,
possibly because of additional effects such as vasoconstriction,
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whereas, calcium sensitizers such as EMD 57033 may be useful positive inotropic compounds, even in
the diseased myocardium [12].
Ion channel modulation represents another approach to positive inotropy [13]. Sodium channel
modulators increase Na+ influx and prolong the plateau phase of the action potential; sodium/calcium
exchange then leads to an increase in the level of calcium available to the contractile elements, thus
increasing the force of cardiac contraction [13,14]. Synthetic compounds such as DPI 201-106 and BDF
9148 (Figure 1) increase the mean open time of the sodium channel by inhibiting channel inactivation
[15]. Importantly, BDF 9148 remains an effective positive inotropic compound even in severely failing
human myocardium [16] and in rat models of cardiovascular disease [17]. Modulators of calcium and
potassium channel activities also function as positive inotropes [13], but in the remainder of this article
we shall focus on sodium channel modulators.
II. The Anthopleurins
Two decades ago drugs from the sea were the subject of high expectations and a good deal of effort in
various centers around the world. The number of therapeutically useful compounds to have emerged
from that effort has been rather limited, but with the advent of high-throughput screening it is likely that
useful new leads will be found, even from species investigated previously. Notwithstanding, some
valuable leads did emerge from work carried out in the 1970s, amongst which were the polypeptide
cardiac stimulants known as the anthopleurins. These were isolated from sea anemones, where they are
components of the animal's venom and are believed to have a function in defense and the capture of
prey. The work that led to the isolation and characterization of these and related polypeptides from sea
anemones is covered in earlier reviews [18,19] and will not be reiterated here.
The best characterized of the anthopleurins is anthopleurin-A (AP-A), which was isolated from the
northern Pacific sea anemone Anthopleura xanthogrammica and consists of 49 residues cross-linked
by three disulfide bonds [18,20]. It is active as a cardiac stimulant at nanomolar concentrations in vitro,
making it some 200 fold more potent on a molar basis that digoxin. Its positive inotropic activity is not
associated with any significant effects on heart rate or blood pressure [21], and in conscious dogs its
therapeutic index is 7.5, which is about three-fold higher than that of digoxin [8]. Anthopleurin-A is
active under conditions of stress and hypocalcaemia [18,22], as well as in ischemic myocardium where
many other positive inotropes give equivocal results [23]. The profile of activity for AP-A suggests that
it is a potentially valuable lead in the development of an alternative positive inotrope to digoxin
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for use in the treatment of chronic CHF. This chapter describes how this development is being tackled
using the approach of structure-based drug design.
A. Related Sea Anemone Toxins
Anthopleurin-A is a member of a family of sea anemone polypeptides [24] (Figure 2) that is steadily
increasing in number. These polypeptides have been classified into two groups, designated Types 1 and
2 [27], which are similar with respect to the locations of their disulfide bridges and a number of residues
thought to play a role in biological activity or maintenance of the tertiary structure [24,27], but are
distinguishable on the basis of sequence similarity (>>60% within each type but <30% between the two
types) and immunological cross-reactivity. Figure 2 shows the amino acid sequences of the Type 1
toxins, which have much stronger affinities for cardiac tissue than the Type 2 toxins characterized to
date [24,27].
Apart from AP-A, the best characterized of these polypeptides with respect to its biological activity is
Anemonia sulcata toxin II (ATX II) [19]. This molecule is also cardioactive [28], as would be expected
from its similarity to AP-A. Renaud et al. [29] have compared the activities of a number of sea anemone
and scorpion toxins on isolated rat atria and found that anthopleurin-B (AP-B, also known as Ax II) had
the highest potency and the greatest margin between the concentrations necessary for maximal inotropic
activity and for provoking arrhythmias (0.3 versus 10 nM). It was also found that sodium channels of rat
cardiac cells in culture, which have a low affinity for tetrodotoxin (TTX), have a particularly high
affinity for Type 1 anemone toxins [29], whereas Type 2 toxins [30] and scorpion toxins [31] had
similar affinities for TTX-sensitive and TTX-insensitive channels in rat neuroblastoma cells and skeletal
myotubes, respectively.
The polypeptide ATX II has been found to have class III antiarrhythmic activity, indicating its potential
in the management of cardiac arrhythmias [32]. A positive inotropic drug that was also effective as an
antiarrhythmic might offer significant advantages therapeutically [33,34].
B. Site of Action
The sea anemone polypeptides act by delaying inactivation of the myocardial voltage-gated sodium
channel [35], thereby prolonging the action potential. Binding sites for a number of different agents,
mainly toxins, on the sodium channel have been identified [3537], the sea anemone toxin binding site
being designated site 3. Other polypeptides that also bind to site 3 (or a partially overlapping site) are the
short anemone polypeptides [19,24] such as ATX III [38] and PaTX [39], which are neurotoxic to
crustacea, the Anemonia sulcata
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Figure 2
Amino acid sequences of Type 1 sea anemone polypeptides. The residue numbering system is based on the sequences of AP-A and
AP-B. Literature references are from Norton [24], except in the case of recently published sequences for Bc III [25], Bg II and Bg III
[26]. Identical residues are shaded in grey and conserved residues are boxed. The sequences of Bg II and Bg III around residue 28
could also be aligned to bring the Gly-Cys sequence into register with the other toxins if the Arg were treated as an insertion. Toxins are
named in this figure according to their genus and species; common names relevant to the text are Ax I = AP-A; Ax II = AP-B; As Ia =
ATX Ia.
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polypeptides BDS I and II [40,41], which were claimed to have antihypertensive and antiviral activity,
and the -scorpion toxins [24,27,42]. As discussed below, we may expect to obtain some useful
information from comparisons of the molecular surfaces of these different classes of polypeptides, but
the utility of this approach depends on the extent to which their binding sites are identical and not just
partially overlapping, as well as the issue of channel sub-type specificity of the different toxins.
The synthetic agent DPI 201-106 has been evaluated extensively as a potential replacement for digoxin
[43]. It is a potent positive inotrope that also acts by delaying inactivation of the sodium channel, but its
binding site appears to be distinct from that of ATX II [44]. Moreover, it exerts antihypertensive and
local anesthetic effects and may also antagonise the calcium channel [43]. At present we are not aware
of any low molecular mass compound that binds to the same site as the anthopleurins. This offers the
prospect that a mimetic based on the anthopleurins might have a pharmacological profile distinct from
other positive inotropes.
The structure of the receptor for the anthopleurins, the -subunit of the voltage-gated sodium channel, is
known only in schematic form [3537]. As illustrated in Figure 3, it contains four homologous domains,
each consisting of six transmembrane regions (assumed to be helices) designated S1 to S6. The S4
segments are thought to act as the voltage sensors of the channel. All four
Figure 3
Schematic of the -subunit of the voltage-gated sodium channel, based on its amino
acid sequence [3537]. The transmembrane segments S1S6 in each domain are
thought to form heliceswith the positively charged S4 segment acting as a
voltage sensorand the S5S6 loop of each domain is thought to contribute to the
transmem-brane pore. Site 3 includes regions of the S5S6 loops of domains I
and IV, and the inactivation gate (h) is located on the intracellular segment linking
domains III and IV.
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domains contribute to formation of the transmembrane pore, which is believed to be lined by short
segments from the loops linking S5 and S6 in each domain. Binding site 3 is located on the extracellular
surface of the channel and involves regions of the loops between S5 and S6 in domains I and IV [37,45].
The binding sites for several modulators of sodium-channel activity, including the blocker tetrodotoxin,
have been mapped quite precisely onto the sequence (and thus the structural model of the channel) by
combining information derived from comparisons of naturally occurring variants of the channel and
from site-directed mutagenesis [37]. A similar approach could be applied to the definition of site 3, but
in the absence of a crystal structure for the -subunit the three- dimensional structure of this site will
remain speculative. Therefore current attempts to design a low molecular mass analogue of the
anthopleurins must be based on the structure of the ligand rather than the receptor.
C. Development of a New Positive Inotrope
Being polypeptides, the anthopleurins have limited therapeutic potential in their own right, as they are
not active following oral administration and are antigenic in experimental animals [18]. Recent advances
in the field of peptide mimetics, however, lend credence to the concept of harnessing the favorable
cardiotonic properties of the anthopleurins in a low molecular mass, nonpeptide, synthetic compound.
The goal of such a development would be to retain the activity of the parent polypeptides in a molecule
that had good bioavailability and was not antigenic. Ideally, it might also be possible to increase the
cardiac selectivity of such a compound.
In order to achieve this goal, a knowledge of the three-dimensional structures of the anthopleurins and
their structure-function relationships is essential. Significant progress has been made towards these goals
over the past few years and we are now in a position to commence analogue design and synthesis. The
following sections in this chapter summarize our knowledge of the cardioactive pharmacophore of the
anthopleurins and the prospects for mimicking this in a nonpeptide moiety. Aspects of the
pharmacological profile which such a compound would need to display in order to be useful in CHF
therapy are also discussed.
III. 3D Structure
The structures in aqueous solution of both AP-A [46] and AP-B [47] have been solved using highresolution 1H NMR data. Structures have also been determined for the Type 1 toxin ATX Ia [48] and the
Type 2 toxin Sh I [49,50] from NMR data. The main secondary structure element in each of these
structures is a
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Figure 4
Richardson-style diagram of the polypeptide backbone of the
individual structure of AP-A [46] that is closest to the average over the
whole molecule. The locations of the sulfurs in the three disulfide bonds
(446, 636, and 2947) are shown in CPK format. The locations of
reverse turns found in more than half the NMR-derived structures
(69, 2528, and 3033) are indicated by darker backbone shading.
The diagram was generated using MOLSCRIPT [51].
four-stranded, antiparallel -sheet linked by three loops, as illustrated for AP-A in Figure 4. The first of
these loops, spanning residues 816 in AP-A and 817 in AP-B, is the largest and least well defined in
solution (Figure 5), although it contains several residues that are essential for activity, as described in the
next section.
Differences between the structures of AP-A, ATX Ia, and Sh I have been noted [46] but the overall
picture that emerges is one of similar backbone folds for all three molecules, making it likely that
differences among the potencies and species-specificities of these toxins are due to the presence or
absence of particular side chains rather than significant structural differences. Given that ATX Ia and Sh
I have weak or negligible activities on mammalian nerve and heart tissue [24,27], we have focused on
the anthopleurins in an effort to define the structure of the cardioactive pharmacophore of the sea
anemone polypeptides. There are several challenges in this endeavor. One is that the structures are based
on NMR data that, because of the paucity of NOE restraints for surface residues compared with those in
the core of the structure, do not define the locations of the solvent-exposed side chains very precisely
(although it should be borne in mind that this may be a more accurate picture of the actual structure in
solution than one in which the side chains are fixed in a single
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Figure 5
Stereo views of 20 structures of AP-B [47] superimposed over the backbone heavy
atoms (N, C, C) of residues 27 and 1749. The three disulfide bonds are shown in
lighter shading. The lower view is related to the upper one by an approximately 180
rotation about the vertical axis.
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location). Another is that both the backbone and the side chains of residues 8 16 are less well defined
than the bulk of the structure due to a lack of medium- and long-range NMR restraints between residues
in this loop and the rest of the molecule, as illustrated in Figure 5. These problems are exacerbated in the
case of AP-A and AP-B by the presence of multiple conformers in solution, one cause of which is cistrans isomerism about the Gly40-Pro41 peptide bond [52]. The additional peak overlap caused by these
conformers limited the number of NOE restraints that could be obtained from the spectra. Finally, the
structures available at present are for the free ligand and we have no information on how much these
structures might change upon binding to the sodium channel.
One way of addressing the issue of a lack of precision in the locations of functionally important side
chains is to determine the range of conformational space available to them in different ligands. To this
end, we undertook a detailed comparison of the structures of AP-A and AP-B in solution [47]. This
proved to be a useful exercise both in terms of defining the positions of side chains known to be
important for cardiotonic activity and identifying neighboring residues which might also be involved
[47].
Models have been described in the literature for AP-B [53] and Bunodosoma granulifera toxins II (Bg
II) [26]. The AP-B model was derived from the structure of Sh I using energy minimization and the Bg
II model from that of BDS I using energy minimization and 10 ps of dynamics. In both cases the
calculations appear to have been carried out for the molecule in vacuo without the use of a distancedependent dielectric, under which conditions the positions of the charged side chains on the surface are
likely to be distorted. Visual comparison of the model of AP-B [53] with the experimentally determined
solution structure [47] indicates significant differences in side-chain orientations.
IV. Residues Essential For Cardiotonic Activity
Information about which residues are essential for the cardiac stimulatory activity of the sea anemone
toxins has been obtained from selective chemical modification and proteolysis studies, comparisons
among naturally occurring sequences, and, most recently, site-directed mutagenesis. Although there are
some discrepancies among the inferences drawn from different studies and different techniques, a
consensus is emerging regarding the location of the cardioactive pharmacophore. Ideally, only effects on
cardiac tissue should be considered, but doing so would exclude some useful data on activity against
mammalian nerve preparations. However, data obtained on the Type 2 toxins or on the activity of Type
1 toxins on nonmammalian tissues will not be discussed in detail.
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A. Chemical Modification
A number of chemical modification studies have been carried out on the sea anemone toxins. As
discussed previously [24], the results of these studies have to be interpreted with some caution because
of less than rigorous characterization of the reaction products in many cases. Nevertheless, in the Type 1
toxins it appears that one or both of the Asp7 and Asp9 carboxylates are required for activity, as well as
one or both of the Lys37 and Lys48 -ammonium groups [24,27,54]. The C-terminal carboxylate
appears not to be essential, whereas the N-terminal ammonium appears to have some role, although the
various studies give a confusing view of its importance. There are also conflicting data on the
importance of His34 and His39 but it seems that at least one of them might be important. Both are
located in the vicinity of other residues found to be necessary for activity, but His39 is in close contact
with Asp7 and Lys37 and on this basis may be expected to be the more important.
Although the available evidence points to a role for one or both of the Asp7 and Asp9 carboxylates in
cardiotonic activity, it has not been established that either residue makes contact with the sodium
channel. As indicated above, the carboxylate of Asp9 participates in a hydrogen bond to the backbone
amide of Cys6, so its role may be structural. The carboxylate of Asp7 is close to the side chains of
Lys37 and His39 and is exposed to the solvent, making it a more likely candidate for direct interactions
with the sodium channel. The only evidence for its importance, however, is indirect, coming from the
observation that its replacement by Asn in synthetic Sh I abolished toxicity to crabs [55].
Considerable confusion has surrounded the role of Arg14, which is conserved throughout the Type 1 and
Type 2 toxins. A recent study has shown, however, that modification of Arg14 in AP-A with 1,2cyclohexanedione under conditions where the positive charge is maintained did not affect positive
inotropic activity [54]. This study also showed indirectly that any contact the Arg14 side chain makes
with the sodium channel must be relatively loose: although the adduct is active, it is no longer
susceptible to tryptic proteolysis, indicating that the modified side chain cannot be accommodated in the
substrate binding site of the protease. The conclusion that the positive charge on Arg14, but not its exact
spatial location, might be important for activity is consistent with the results of site-directed mutagenesis
experiments discussed below.
B. Selective Proteolysis
When AP-A was treated with trypsin only the Arg14 to Gly15 peptide bond was cleaved [56]. The
resulting derivative lacked cardiotonic activity but its binding affinity for the rat brain sodium channel
was reduced by less than an order of magnitude (Llewellyn LE et al., unpublished results). Its overall
structure, as
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monitored by NMR, was unchanged, although it should be noted that the structure of the loop containing
Arg14 was not well defined in either native or trypsinised AP-A. That the backbone structure was
largely unaffected was confirmed by the observation that the NH and CH chemical shifts were
unaltered except in the immediate vicinity of the cleavage (which is the site of a reverse turn in AP-A
[46]) and the N-terminal regions of the loop and the second strand of the sheet. It is possible that
perturbations of functionally important groups near the start of the loop may be responsible for the lack
of activity of the trypsinised derivative, and now that a high-resolution structure is available for AP-A a
more detailed comparison with the cleavage product would be useful. Other possible explanations for
the lack of activity are that the position of the Arg14 side chain relative to other key residues in the
molecule is more important than suggested by chemical modification and site-directed mutagenesis data,
or that the introduction of additional charges associated with the new termini affects activity.
Endoproteinase LysC cleaved AP-A between Lys37 and Ala38 to yield a derivative with cardiotonic
activity an order of magnitude lower than that of the parent molecule (Monks SA and Norton RS,
unpublished results). This reduction in activity could be a consequence of local conformational
perturbations. Treatment of AP-B with carboxypeptidase B removed Lys49, resulting in only a two-fold
reduction in cardiotonic activity (Monks SA and Norton RS, unpublished results).
C. Sequence Comparisons
Among the Type 1 toxins shown in Figure 2, the Bc and Bg toxins (from the genus Bunodosuma) form a
subgroup with characteristic differences from the Anemonia and Anthopleura toxins at residues 5, 1213
and 3742. In addition, the Bg toxins also have Asp7 rarrow.gif Lys and Gly27 rarrow.gif Arg
substitutions. A potent toxin in mice [26], the cardiac stimulatory activity of Bg II has not been reported.
The potent activity of Bg II was ascribed to its abundance of positive charges [26]. Ignoring the
histidines, which at least in AP-A and ATX II would be predominantly in their neutral forms at
physiological pH [57], Bg II has six positively charged side chains and only one negatively charged side
chain, whereas, AP-A has three and two respectively, and AP-B has five and two. As discussed below,
however, it may be that an abundance of positive charge is associated with a lack of discrimination
between the neuronal and cardiac sodium channels, as found for the scorpion -toxins.
Comparison of the activities of Af I and Af II is useful because of their close similarity. Differences
between Af I and Af II are Ala3 rarrow.gif Pro, Asn12 rarrow.gif Ser, Thr21 rarrow.gif Ile, and an
additional Gly at the N-terminus. Inspection of the sequences in Figures 2 shows that the identity of
residue 3 correlates with that of residue 21, Ala or Ser at 3 co-occurring with Thr21, and Pro 3 with
Ile21
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(the exception is ATX Ib [19,24], which has Pro 3 and Thr21, but it is not known if this has the same
local structure). Residues 3 and 21 are juxtaposed in the sheet [4648] and the effect of switching
Ser3/Thr21 to Pro3/Ile21 can be assessed by comparing the structures of AP-A [46] and AP-B [47]. It
appears that this leads to a distortion of the sheet between the second and third residues but causes no
significant perturbations to the overall structure. In the calculated structures of AP-A a Va12 NH
rarrow.gif Leu22 CO hydrogen bond was found but this was not present in the AP-B structures,
presumably as a result of the local differences. Comparing Af I and Af II, the combination of the
Ala3/Thr21 to Pro3/Ile21 switch plus the addition of an extra Gly at the N-terminus of Af II would be
expected to alter the local conformation at the N-terminus. The fact that the cardiac stimulatory activities
of the two are the same [58] therefore implies that the N-terminus is not important functionally. This
inference is conditional on the effect on activity of the only other difference between Af I and Af II,
Asn12 rarrow.gif Ser, being minimal. In the Type 1 toxins, Ser is found more often than Asn at position
12, while in the Type 2 toxins, replacement of Asn12 by Tyr increases toxicity in mice by a factor of
two [24,27]. Thus, it is possible that the presence of Ser in Af II slightly favors cardiac activity while the
changes at the N-terminus might reduce it slightly; the main conclusion to be drawn, however, is that
neither change has a significant effect. The lack of effect of changes at the N-terminus is consistent with
the results from expression of AP-B [59], where a Gly-Arg extension at the N-terminus had no effect on
activity.
At seven locations AP-B differs from AP-A. Two of these, Ser3 rarrow.gif Pro and Thr21 rarrow.gif
Ile, were discussed above. Two more, Leu24 rarrow.gif Phe and Thr42 rarrow.gif Asn, are
conservative changes located in or at the start of loops that are not in the immediate vicinity of the
sodium channel binding surface and would not be expected to have a direct effect on activity. This
leaves Ser12 rarrow.gif Arg, Val13 rarrow.gif Pro, and Gln49 rarrow.gif Lys, which do have
functional significance, as discussed in the following section.
It is important to note that there are several residues that are common to all of the long toxins and serve
to maintain the biologically active conformations of these molecules. The clearest examples of residues
in this category are the six half-cystines, although we believe that the Gly10-Pro11 sequence may
influence the structure and flexibility of the Arg14-containing loop [46] (at the other end of this loop
Thr17 and/or Ser19-Gly20 may also be important). Also conserved, Trp33 is probably important
structurally even though its surroundings are different in the Type 1 and Type 2 toxins.
It is also interesting that in ATX Ia, which is a potent crustacean neurotoxin but a poor mammalian
cardiac stimulant, Lys37 and both histidines are missing, suggesting that one or more of these side
chains may be important in promoting specificity for the mammalian cardiac sodium channel.
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D. Site-Directed Mutagenesis
The complete synthesis of AP-A has been reported [60] but so far this approach has not been pursued to
generate analogues. More productive has been the analysis of analogues produced by site-directed
mutagenesis, following the successful cloning and expression of AP-B [59]. A series of single-site
mutations [61,62] showed that R14A, K48A, and K49A had affinities for the cardiac sodium channel
that were, respectively, only 3.2-, 2.9-, and 2.4-fold lower than that of native AP-B, while R12A had an
8.5-fold lower affinity. These results suggested that amongst the cationic side chains only Arg12 was
significant for activity. Recent data on double mutations [53] indicates that the situation is not quite that
simple. For example, the R12S-R14Q double mutant had a 72-fold lower affinity for the neuronal
sodium channel whereas the R12S and R14Q mutants individually had 5.9- and 2.4-fold lower affinities,
respectively, which should combine to produce only a 14-fold effect. It appears that the presence of one
cationic side chain in the Arg14 loop may be sufficient for activity and that its exact location can vary
somewhat. It would be interesting to know if the same applies to the C-terminus, where Lys48 and
Lys49 may be able to compensate for one another. However, the proposal that the cationic side chains of
residues 12, 14, and 49 form a cluster [53] is not consistent with the solution structure of AP-B [47].
A similar situation appears to exist in the -conotoxins, which possess 57 net positive charges.
Substitution of individual cationic groups had a relatively minor effect on affinity for the voltage-gated
calcium channel, whereas replacement of several had a significant effect, greater than that expected from
the sum of the individual effects [63,64].
A further outcome of analyses of the double mutants was that the cationic side chains at positions 12 and
49 in AP-B seem to favor binding to the neuronal over the cardiac sodium channel. Thus, the R12SR49Q double mutant, in which residues 12 and 49 in AP-A, had a 37-fold lower affinity for the neuronal
channel but only a 5-fold lower affinity for the cardiac channel relative to native AP-B [53]. In fact, the
affinity of this double mutant for the cardiac channel was lower than that of AP-A, implying that one or
more of the other five differences between AP-A and AP-B might decrease affinity for the cardiac
channel. It seems, therefore, that while AP-A is slightly less potent than AP-B on cardiac channels, it
has greater selectivity for the cardiac channel when measured in sodium flux experiments. In voltageclamp experiments AP-A and AP-B favor the cardiac channel to similar degrees [53].
V. Other Ligands for Site 3 on the Sodium Channel
A. Sea Anemone Toxins
Apart from the long sea anemone polypeptides that are the main focus of our interest, there are two
other classes of anemone polypeptides that bind at or near
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site 3 on the sodium channel. The first of these are the short anemone polypeptides [19,24] such as ATX
III [38] and PaTX [39], which are neurotoxic to crustacea. The polypeptide ATX III, which consists of
27 residues cross-linked by three disulfide bonds, and PaTX, which has 31 residues and four disulfides,
can be aligned such that 16 residues are identical. The three disulfides in ATX III are linked in a
15/24/36 pattern in the same way as in the long polypeptides [65], but the only other similarity at the
level of primary structure is a GCPXG sequence corresponding to residues 2832 of AP-A. Although
neurotoxic to crustacea, ATX III is inactive as a positive inotrope [28], suggesting that it possesses an
appropriate structural scaffold to interact with site 3 but lacks key side chains required for interaction
with the cardiac channel. Nevertheless, the smaller size of ATX III makes it an attractive candidate for
further study, with the aim of engineering into it the ability to bind to the cardiac channel. The welldefined solution structure for this toxin [65] provides and essential basis for such an effort.
The Anemonia sulcata polypeptides BDS I and II [40], which were claimed to have antihypertensive and
antiviral activity, also bind to site 3 on neuronal sodium channels and have weak negative inotropic
activity [41]. The points of similarity and difference between the solution structures of BDS I [40] and
the long anemone polypeptides have been discussed previously [40,41] and will not be reiterated here;
suffice to say that the overall folds are similar but the Arg14 loop in the long polypeptides is truncated in
BDS I and the molecule lacks several residues that have been shown to be important for activity.
B. Scorpion Toxins
The scorpion -toxins have been shown to bind to site 3 on the voltage-gated sodium channel
[24,27,42]. These polypeptides contain up to 70 residues crosslinked by four disulfide bonds, but show
no sequence similarity to the anemone polypeptides. Possible structural similarities have been discussed
[24], and in a theoretical model of the anemone toxin Bg II, some of the cationic residues were in similar
locations to those in the crystal structure of the scorpion toxin Aah II [26].
It is clear that positively charged residues play an important role in the interactions of sea anemone
toxins and scorpion toxins with site 3 on the sodium channel (as indeed they do with other polypeptide
toxins binding to other ion channels) but this role may be relatively more important for the TTXsensitive sodium channel in nerve and muscle than for the TTX-insensitive channel of the heart. For
example, Bg II, which is more positively charged than AP-A and AP-B (see above), has a higher affinity
for neuronal sodium channels [26], and replacement of Arg12 and Lys49 in AP-B with uncharged
residues favors its binding to the cardiac channel [53]. Similarly, the scorpion -toxins bind more tightly
to the neuronal channel than the anemone toxins but, as with the Type 2
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anemone toxins, do not discriminate between TTX-sensitive and TTX-insensitive channels. In fact the
scorpion toxins are less potent cardiac stimulants than the Type 1 anemone toxins [29]. Thus, while it
will be interesting to compare the sodium channel binding surfaces of the two classes of toxin as these
surfaces become better defined in each case, more useful input to the design of a positive inotrope acting
at site 3 is likely to come from direct studies on the anthopleurins.
VI. Defining the Cardioactive Pharmacophore
Now that high-resolution structures are available for both AP-A [46] and AP-B [47], we can begin to
interpret the results described above in terms of an emerging picture of the cardioactive pharmacophore
of these molecules. It is encouraging that most of the residues that have been shown hitherto to be
important for activity lie on one face of the structures, as shown in Figure 6. Of the residues highlighted
in Figure 6a, evidence to support their role in activity on the neuronal or cardiac channels (or both) has
come from chemical modification or site-directed mutagenesis studies except in the case of Asn35. The
reason for including this residue is that in AP-B it is close to the Asp7/Lys37/His39 region and its side
chain is hydrogen bonded to the backbone carbonyl of Lys37 [47]. Moreover, it is conserved throughout
the Type 1 toxins (Figure 2).
We anticipate that many of the residues highlighted in Figure 6 will participate in interactions with the
sodium channel binding site. One residue which may not is Asp9, the side-chain carboxylate of which
hydrogen bonds with the amide of Cys6 in AP-A and AP-B. Thus, it is possible that this residue has a
structural role rather than a functional one. Other residues in the vicinity of the pharmacophore that
may also have a structural role are Gly10 and Pro11, as discussed above. The side chain of Ser8 is
exposed and on the same surface of the molecule, placing it in a position potentially to interact with the
sodium channel; it is also conserved throughout the Type 1 toxins.
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Figure 6
(a) CPK representation of the individual structure of
AP-B [47], which is closest to the average over the
whole molecule, showing residues thought to contribute
to the cardioactive pharmacophore. The surfaces of
residues 7 and 9 are shaded black, those of 14, 37, 39, and
48 dark grey, and that of 35 lighter grey. As discussed in
the text, the primary functions of Asp9 (the side chain of
which is hardly visible in this view) and Asn35 may be to
maintain the local structure in an active conformation,
but it cannot be excluded that they also interact directly
with the sodium channel. In AP-B the cationic side
chains of Arg12 and Lys49 are also important, but it
appears that their roles can be compensated for by nearby
cationic side chains (Arg14 and Lys48, respectively) and
that they favor binding to the neuronal sodium channel
rather than the cardiac channel [53]. (b) Connolly surface
of AP-B in the same orientation as in part a, with the
charged residues Asp7, Asp9, Arg14, Lys37, and Lys48
highlighted. This figure was generated using Insight
(Biosym Technologies).
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In peptideprotein and proteinprotein interactions the size of the buried surface area ranges from 400
2 to 1400 2 [66]. In the potassium channel blocker charybdotoxin, 58 residues with surface areas of
530850 2 were found to be essential for binding, depending on the type of potassium channel
investigated [67,68]. In the calcium channel blocker -conotoxin GVIA, an alanine scan identified only
two residues, Lys2 and Tyr13, that were important for activity [69]. It is likely, however, that the
number of residues contributing to the binding surface is greater than this, particularly given the high
affinity of this toxin for its receptor. If we consider a larger ligand such as human growth hormone, eight
of the 31 residues in the growth hormone-receptor interface contribute 85% of the binding energy and
more than half make no significant contribution to the affinity [70]. A similar result was found for a
growth hormone-monoclonal antibody complex, where only five residues were critical for binding [71].
In fact, the example of growth hormone may be more relevant to the anthopleurins than those of
charybdotoxin and conotoxin, which function simply as ion channel blockers. Thus, we expect the
essential residues in the anthopleurins to number between five and ten, a total somewhat less than
previously anticipated [24].
If we ignore Asp9 and assume that only one of Arg12 or Arg14 and one of Lys48 or Lys49 are
necessary, then it is likely that the residues highlighted in Figure 6 make a significant contribution to the
sodium channel binding surface of the anthopleurins. They span a larger area on the surface than the
essential residues in charybdotoxin, but it is important to note that the conformation of the Arg14 loop in
solution is not fixed and that conformations in which the Arg14 side chain is closer to the region around
Asp7 could be more representative of the sodium-channel-bound structure. For example, the distance
between the Arg14 guanidino group and the Asp7 carboxylate varies from 13 to 26 over the family of
structures of AP-A and 10 to 22 in AP-B.
By analogy with other proteinprotein interactions, it is likely that the sodium channel binding surface
of the anthopleurins will include side chains that mutagenesis studies will not identify as having a
significant role in binding or activity. As indicated above, alanine scanning of residues in the human
growth hormonereceptor interface indicated that less than a quarter of the contact residues provided
most of the binding energy [70]. Thus, we believe that the residues identified above will contribute to
the sodium channel binding surface of the anthopleurins by virtue of their location on the protein surface
in the immediate vicinity of residues, which clearly are important for activity. Nevertheless, some of
them may make only modest contributions to binding affinity and could be altered without destroying
activity. Other residues, as yet unidentified, may also make significant contributions. By analogy with
other proteinprotein interactions characterized to date, it is reasonable to anticipate that some of these
residues will be hydrophobic, in contrast to the charged
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residues that have been the main focus of attention hitherto. A key requirement now is to identify those
residues that provide the majority of the binding energy and to differentiate these from others whose
main role is to stabilize the binding residues in the active conformation.
VII. Mimicking The Pharmacophore
Significant progress has been made over the past few years in the field of peptide mimetics, although the
most successful examples are those where a small peptide ligand or a linear segment of a larger protein
has been the target [7274]. An alternative approach to de novo design is to optimize a lead compound
obtained by screening chemical libraries on the basis of a knowledge of the conformation of the
polypeptide ligand, as in the case of the endothelin receptor antagonist SB 209670 [75].
The task of mimicking a pharmacophore is simplified where the contributing residues are contiguous in
the amino acid sequence. This is not the case in the anthopleurins, with residues from at least four
different regions of the sequence contributing to affinity. In charybdotoxin the essential residues come
from two or three regions of the sequence, depending on which potassium channel is considered, while
in growth hormone, binding site I is comprised of residues from three different regions of the protein
and site II from two regions. Mimicking the pharmacophore of the anthopleurins therefore represents a
task at least as challenging as those presented by these two examples. Strategies for achieving this goal
include de novo design, conformationally directed data base searching and screening chemical libraries
(synthetic and naturally occurring) for leads, which could then be optimized on the basis of our
knowledge of the structure. Our approach is based on the first two of these.
Initial attempts to mimic the pharmacophore of AP-A were based on linear and disulfide-cyclized
versions of the Arg14-containing loop [76]. At that time, our level of understanding of the
pharmacophore was inadequate and it is clear in retrospect that not enough of the key elements were
present. Nevertheless, conformational analysis of these peptides by NMR was useful in showing that
they retained several elements of local structure observed in the corresponding region of the native
protein, thereby emphasizing the independence of this loop from the rest of the structure in solution.
VIII. Conclusions
In this chapter I have attempted to summarize the current state of our understanding of the structure and
structure-function relationships of the type 1 sea
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anemone toxins and to indicate how the favorable cardiotonic properties of the anthopleurins might be
mimicked in a low molecular weight analog.
Progress in defining the cardiotonic pharmacophore has been hampered by difficulties in determining
high-resolution structures of the anthopleurins in solution due to the presence of multiple conformers,
and by uncertainties concerning the exact location of some of the key residues in the Arg14 loop. Both
of these problems would have been alleviated by isotopic labeling of the molecules in a high-yield
expression system, which would have allowed for better definition of the structures in solution. The
question of how the structure in solution might change upon binding to the sodium channel remains
open and is particularly relevant to residues in the Arg14-containing loop. Conformationally constrained
analogs of the ligand offer one means of addressing this problem. The definitive solution would be
provided by a high-resolution structure for the sodium channel, determined by x-ray or electron
crystallography, but this will not be available in the near future. In the meantime, the approach of
complementary mutagenesis (of both ligand and receptor), which has been very informative in defining
the charybdotoxin-potassium channel interface [68,77], can be employed to produce a crude model of
the binding site.
Once a lead compound is obtained, further development will almost certainly be required to optimize
properties such as bioavailability and stability in vivo. A key requirement will also be good selectivity
for the cardiac over other sodium channels, but the results of mutagenesis studies carried out so far on
the anthopleurins suggest that this should be achievable. Lead compounds will also have to be rigorously
evaluated in terms of their effects on cardiac arrhythmias to ensure that they ameliorate rather than
exacerbate this problem, especially in the failing heart. Finally, the possibility that the beneficial effects
of positive inotropes in vivo may be the result of inotropic and noninotropic activities [6] would need to
be evaluated for mimetics of the anthopleurins. These requirements notwithstanding, there is good
reason to be optimistic that a mimetic of the anthopleurins can be developed and that it may have
significant benefits in the treatment of congestive heart failure.
Acknowledgments
I am grateful to all the colleagues who have contributed to our sea anemone toxin work over the years,
and, in particular, to Steve Monks, Paul Pallaghy, and Jane Tudor for assistance with the figures and for
helpful discussions. I also thank Ken Blumenthal for communicating results prior to publication.
Note Added in Proof
Since completion of this chapter, two additional papers on site-directed mutations of anthopleurin-B
have been published. In the first (Khera PK, Blumenthal
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KM. Importance of highly conserved anionic residues and electrostatic interactions in the activity and
structure of the cardiotonic polypeptide anthopleurin B. Biochemistry 1996; 35:35033507), it was
concluded that Asp7 may be important for folding, Asp9 may be important for protein folding and
interaction with the sodium channel, and Lys37 for channel interaction. In the second (Dias-Kadambi
BL, Drum CL, Hanck DA, Blumenthal KM. Leucine 18, a hydrophobic residue essential for high
affinity binding of anthopleurin-B to the voltage-sensitive sodium channel. J Biol Chem 1996;
271:94229428), Leu18 was shown to be important for binding, with several hundred fold losses in
affinity being associated with its mutation to Val or Ala. This residue is adjacent to the surface
highlighted in Figure 6.
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13
Rational Design of Renin Inhibitors
V. Dhanaraj* and J.B. Cooper**
Birkbeck College, London, England
I. Introduction
There has been much interest in the development of therapies for hypertension and associated heart
failure, which is a major cause of death in the western world. One of the key mediators in primary
hypertension is the plasma octapeptide angiotensin II (AII), which plays a major role by causing
vasoconstriction and stimulating aldosterone release, thereby increasing blood volume by its action on
the kidneys. Angiotensin II is produced by a proteolytic cascadeknown as the renin-angiotensin
systemin which the aspartic proteinase renin catalyses the rate-limiting cleavage of angiotensinogen
produced by the liver to yield the decapeptide angiotensin I (AI). The subsequent removal of the
carboxy-terminal dipeptide from AI by angiotensin-converting enzyme (ACE), yielding AII, is the target
for a number of drugs that are effective for treating hypertension, hyperaldosteronism, and congestive
heart failure [1].
The development of potent low-molecular-weight orally active ACE inhibitors from natural and
synthetic metalloproteinase inhibitors has been rapid, due in part to the relative lack of specificity of this
enzyme. In contrast, renin cleaves only its natural substrate or very close analogs and although inhibition
of an enzyme more specific than ACE may be desirable for reducing side effects in vivo, the selectivity
of renin meant that during the early stages of drug development, potent inhibition required the use of
large peptide-based compounds. These were often poorly absorbed and susceptible to gastric proteolysis
and biliary excretion. Nevertheless, the commercial and clinical success of ACE inhibitors fueled
interest in the search for therapeutic renin drugs. Most inhibitors have been developed by elaboration of
the minimal substrate sequence (residues 613 of angiotensinogen), which exhibits weak competitive
inhibition, and replacement of the scissile bond with various nonhydrolysable surrogates, some of which
may be transition state analogues [2].
Current affiliation: University of Cambridge, Cambridge, England.
Current affiliation: University of Southhampton, Southhampton, England.
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Figure 1
The three-dimensional structures of human (left) and mouse renins (right) showing
oligopeptide inhibitors bound in the active site cleft. The cleft lies between the N- and
C-terminal domains of the enzyme and is approximately perpendicular to the plane
of the page. It can accommodate 910 residues with the substrate/inhibitor bound
in an extended conformation. The catalytic aspartic acid residues (not shown)
are centrally placed at the base of the cleft.
Renin is a member of the homologous group of enzymes known as aspartic proteinases that includes
pepsin and a group of fungal enzymes such as endothiapepsin, penicillopepsin, and rhizopuspepsin.
Their sequences all contain two aspartates (at positions 32 and 215 in porcine pepsin) that are essential
for catalytic activity. The crystal structures of several aspartic proteinases have been solved by x-ray
diffraction at high resolution, revealing a common bilobal structure with a large cleft between the N- and
C-terminal domains that can accommodate up to nine residues of a substrate (Figure 1) [3]. The two
essential carboxyls of Asp 32 and Asp 215 are within hydrogen-bonding distance and are approximately
co-planar due to the constraints of a hydrogen-bonding network involving residues of the two highly
conserved loops that contain the essential aspartates. The three-dimensional structures of the two
domains are related by a topological two-fold axis passing between the catalytic residues where the
pseudosymmetry happens to be strongest. Modeling studies based on the homology with other aspartic
proteinases showed that human renin assumes a tertiary structure that is similar to the other enzymes and
that the homology is greatest for the binding cleft region [4]. Subsequent x-ray analysis of the structure
of renin (described later) revealed the specific interactions made with inhibitors and implicated certain
loop regions covering the active site as being important for tight binding of peptides.
One enigmatic feature of renin is its extreme substrate specificity, its only known natural substrate being
a single Leu-Val peptide bond of angiotensinogen. The minimal synthetic analog is the 613 octapeptide
that encompasses the scissile peptide bond of angiotensinogen between residues 10 and 11 [5]. It has
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been suggested that specificity of proteinases in general is due to rigidity in the binding pockets [6];
broad specificity results from the ability of the pockets to change shape in response to different ligand
side chains. However, renin is known to be inhibited by a wide variety of peptide analogs of different
length and sequence indicating either that the active site may be somewhat flexible or that the strength
of binding of a substrate does not determine the rate of subsequent turnover. This is emphasised by the
existence of substrates that act as competitive inhibitors, e.g., RIP of Haber and Burton [7], which has a
Ki that is lower than the Km. Therefore hydrolysis of bound substrate appears to be more specific than
the binding step. This may be because only certain substrate sequences allow correct positioning of the
scissile bond for hydrolysis. Evidence for this effect was provided by comparison of 21 inhibitor
structures of endothiapepsin [8], where it was shown that for inhibitors with different sequences but with
the same transition state analog, the scissile bond analog can be disposed somewhat differently with
respect to the catalytic carboxyls in each case.
The availability of crystal structures of a number of renin inhibitors complexed with fungal aspartic
proteinases [8, 9] allowed new compounds to be designed and modeled by such techniques as computer
graphics, energy minimization, and molecular dynamics [10]. X-ray crystallographic analysis of aspartic
proteinase inhibitor complexes has made a significant contribution to rationalizing the activity data for
many of these compounds as well as understanding the catalytic mechanism of this class of proteinase.
II. Strategies for Design of Renin Inhibitors
Some of the parameters that have been varied in the search for therapeutically active renin inhibitors are
outlined below.
A. Elaboration of the Transition State Analog
There is no evidence that aspartic proteinase catalysis involves a covalently bound intermediate [11] and
major advances in the design of nonhydrolysable analogs have stemmed from attempts to mimic an
intermediate of the following form.
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This intermediate is derived by nucleophilic attack of a water molecule on the scissile-bond carbonyl.
Szelke pioneered the use of reduced-bond analog (-CH2-NH-), which were incorporated into the 613
peptide of angiotensinogen [12]. Cocrystallisation with endothiapepsin revealed that the reduced-bond
analog associates tightly with aspartate carboxyls (32 and 215) probably via a salt link. The naturally
occurring transition-state analogue statine (-CHOH-CH2-CO-NH-) [9] is a closer analogue of the
putative intermediate and has been incorporated into many inhibitors [13]. The scissile bond has also
been replaced by the ketone analogue (-CO-CH2-) [12] or by a C-terminal aldehyde group in a series of
tetrapeptides [14,15]. Although these appear to mimic the substrate more closely than the intermediate,
the carbonyl probably binds to the enzyme in the hydrated gem-diol form (-C(OH)2-CH2) [16]. Use of
the hydroxyethylene analog (-CHOH-CH2-) has led to exceptionally potent inhibitors [17] as has
substitution of fluorines into ketone analogs [16,18] giving, for example, -CO-CF2- which undergoes
hydration of the carbonyl to form -C(OH)2-CF2- and exhibits tight binding to renin. The hydrated gemdiol is thought to closely mimic the putative transition state -C(OH)2-NH-.
All inhibitors solved in complex with aspartic proteinases by x-ray diffraction are observed to adopt
similar main-chain conformations and form a conserved set of hydrogen bonds involving the inhibitor's
main-chain groups interacting with enzyme moieties. The inhibitors bind in extended conformations and
residues in the P3-P1 region form antiparallel -sheet-like interactions with residues 217219 on the
enzyme. A -hairpin turn formed by residues 7478 lies between the inhibitor and bulk solvent and
forms a number of generally conserved hydrogen bonds with the bound peptide. These interactions are
indicated in Figure 2. The binding pockets for the inhibitor's side chains are shallow and contiguous with
a greater hydrophobic character towards the central region of the active site cleft.
The elaboration of several classes of transition state analog are now considered in greater detail.
Statine Analogs
The natural transition analog statine possesses one less main chain atom than a dipeptide and has been
shown by x-ray analysis of inhibitor cocrystals to occupy the S1 and S1' sites of the enzyme [19]. The
hydroxyl group of statine binds symmetrically between the catalytic carboxyl groups displacing a
solvent molecule bound to the native enzyme. The carboxyl diad, therefore, provides a stereospecific
binding site for statine and hydroxyethylene analogues with preference for the S-enantiomer.
Replacement of the hydroxyl by an ammonium group might be expected to improve the potency of an
inhibitor by introduction of a salt link with the enzyme. The corresponding deoxy-aminostatine (ASTA)
analogs have been synthesised [20, 21] and were found to be nearly as potent as
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Figure 2
A schematic diagram of the putative hydrogen bonds formed between oligopeptide inhibitors and the fungal aspartic
proteinase endothiapepsin. The latter enzyme provided a useful model system for structural studies of interactions formed by renin
inhibitors with the active site cleft of aspartic proteinases prior to the determination of the human renin structure. The inhibitor is shown
horizontally with enzyme groups above and below. Intervening hydrogen bonds are indicated by dashed lines. Note the extensive
hydrogen-bond interactions made between the transition state analogs and the catalytic apparatus of the enzyme.
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the equivalent statine analogs with the benefit of improved solubility. Preference for the S- versus Renantiomer at the C3 amino position is observed in accordance with the statine inhibition data [22].
Oxahomostatine (-CHOH-CH2-O-CO-) and azahomostatine (-CHOH-CH2-NR-CO-) analogs eliminate
the main-chain frameshift that occurs with statine and, in addition, the azahomostatine conveniently
reduces the stereochemical complexity of the dipeptide surrogate by introducing a 7-membered urea-like
planar group into the S1' binding region. The crystal structure of such a compound complexed with
endothiapepsin has been solved at 1.8 resolution [23] and reveals that the large planar group is
accommodated by the active site and that hydrogen bonds to the P1' CO and P2' NH groups, observed in
other complexes, are retained.
One shortened analog of statine -CHOH-CO-O-R referred to as norstatine (where R is a C-terminal alkyl
group) has been found to be more potent than some equivalent statine analog [24]. The x-ray structure of
such an inhibitor complexed with endothiapepsin reveals that the carbonyl oxygen of this analog is held
by a hydrogen bond to the active site flap region of the enzyme involving the Gly76 >NH group (pepsin
numbering) in much the same way as the P1' >C=O group of other isosteres (Figure 2).
Aminoalcohols
In principle, a good analog of the putative intermediate would be the aminal CHOHNH group but
this would be in equilibrium with the aldehyde and amino fragments. Interposing a methylene group
between the hydroxymethyl and amino groups stabilizes the analog and may still allow tight binding to
the enzyme. Such aminoalcohols (-CHOH-CH2-NH-) have been synthesized [25] and were shown to be
potent inhibitors.
Cocrystallisation of two such compounds extending from P1 to P3' with endothiapepsin allowed their
bound structures to be solved at high resolution [26]. The bound structures revealed that despite the
insertion of a methylene group in the analog a frameshift in the binding mode does not occur since the
residue following the aminoalcohol occupies the S1' pocket. In contrast, the single amino acid, statine,
replaces two residues of the substrate. The hydroxyl of the aminoalcohol (S-enantiomer) is bound
symmetrically to both essential carboxyls as is the case for the hydroxyl of the statine and
hydroxyethylene analogs.
Glycols
Incorporation of glycol or vicinal diol analogs of the peptide bond (-CH(OH)-CH(OH)-) has led to
potent inhibitors and the x-ray structure for one such compound complexed with endothiapepsin is
available [27]. The first hydroxyl in
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this analog interacts with the catalytic aspartate carboxyls in the same manner as statine or
hydroxyethylene moieties; whereas, the second hydroxyl forms a hydrogen bond with the NH group of
Gly 76, thereby mimicing the carbonyl oxygen at P1' of other analogs (see Figure 2).
Phosphorus-Containing Analogs
Aspartic proteinase inhibitors in which the scissile bond is replaced by a phosphinic acid group (shown
below) have been reported [28].
These may mimic the tetrahedral intermediate more closely than statine or hydroxyethylene analogs.
One of the oxygens binds to the carboxyl diad and the other resides adjacent to Tyr75 (pepsin
numbering) forming a hydrogen bond with the outer oxygen of Asp32 [27]. This isostere is very
effective against pepsin. However, it ionizes at physiological pH and the resulting anion is ineffective as
an inhibitor of renin [29].
Fluoroketone Analogs and Implications for Catalysis
Fluoroketone analogs (-CO-CF2-) have been reported [16, 30] and found to be substantially more potent
than the unhalogenated statone molecules, presumably due to the ease of hydration and greater
complementarity of the resulting hydrated gem-diol with the catalytic site. The structure of a
difluorostatone inhibitor complexed with endothiapepsin [31] revealed interactions that indicate how the
catalytic intermediate is stabilized by the enzyme (Figure 3). One hydroxyl of the hydrated fluoroketone
associates tightly with the aspartate diad in the same position as the statine hydroxyl or the native
solvent molecule and the other hydroxyl is positioned such that it hydrogen bonds to the outer carboxyl
oxygen of Asp32. It has been suggested that the tetrahedral intermediate is uncharged, because if the
carboxyl of Asp32 carries a negative charge instead, the latter can be stabilized by a full complement of
hydrogen bonds donated by the gem-diol intermediate and surrounding protein atoms [31]. The current
mechanistic proposals are based on the key suggestion by Suguna et al. [32] that, although transition
state analogs appear to displace the active-site water molecule located between the two catalytic
aspartate carboxyls, the more weakly bound substrate may not. Instead as the substrate binds, the water
may be partly displaced to a position appropriate for nucleophilic attack on the scissile bond carbonyl.
Details of the proposed mechanism are given in Figure 3.
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Figure 3
The catalytic mechanism proposed by Veerapandian et al. [31] based on the x-ray
structure of a difluoroketone (geminal-diol) inhibitor bound to endothiapepsin. A water
molecule tightly bound to the aspartates in the native enzyme is proposed to
nucleophilically attack the scissile-bond carbonyl. The resulting geminal-diol
intermediate is stabilised by hydrogen bonds with the negatively charged carboxyl of aspartate 32.
Fission of the scissile C-N bond is accompanied by transfer of a proton from Asp215 to
the leaving amino group.
B. Complementarity of the Inhibitor

Optimizing the fit of a ligand to its binding site improves the potency by burying lipophilic residues and
by maximizing the number of van der Waals contacts, hydrogen bonds, and chargecharge interactions.
The principles that apply to ligand binding are similar to those involved in protein folding. Inhibitor
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binding involves displacement of hydrogen-bonded water molecules from both the ligand and the
binding cleft. This process is entropy favored since waters in the solvent lattice are more disordered than
those bound to protein. The change in enthalpy on forming NHCO bonds in the complex from
>COHOH and >NHOH2 is favorable but relatively small [33]. The hydrophobic effect is therefore
thought to play a dominant role in the energetics of binding with hydrogen bonds providing precise
alignment of the ligand with respect to the catalytic apparatus. The main chain >CO and >NH groups
from P3 to P3' of aspartic proteinase inhibitors are nearly always satisfied by hydrogen-bond interactions
on formation of the complex. Therefore, given that polypeptides can form the same hydrogen bonds to
the binding cleft regardless of amino acid sequence, differences in affinity for ligands of equal length
must be due to other interactions at the specificity pockets, presumably those between the ligand's side
chains and the enzyme.
One example of optimizing these interactions for renin is the use of the cyclohexylmethyl side chain at
P1, which has been shown to improve the potency by two orders of magnitude relative to the equivalent
leucine-containing inhibitor [13]. Structure/Activity Relationship (SAR) studies have shown that in
many inhibitor types, the cyclohexylmethyl group is optimal for the S1 pocket of human renin; whereas,
other analogs such as cyclohexyl, cyclohexylethyl, and the very bulky dicyclohexyl and adamantyl rings
generally have significantly reduced potency [10]. The use of a cyclohexylmethyl appears to introduce
selectivity for renin versus other human aspartic proteinases. This has been partly rationalized for
endothiapepsin where it was shown by x-ray analysis that the cyclohexylmethyl group at P1 can force
the Phe at P3 to adopt a less energetically favorable X2 angle. Hence, differences at the S3 pocket in renin
may allow the P3 Phe to adopt a more favorable X2 angle in the presence of a cyclohexyl at P1. In
contrast the S2 site is able to accommodate a wide variety of side chains depending on inhibitor type,
e.g., Phe and His are equipotent in some analogs [34]. The x-ray structures of a number of bound renin
inhibitors complexed with endothiapepsin have shown that His at P2 can adopt different X1 angles
separated by about 120 degrees [8]. In one conformation the imidazole is lying partly in the S1' pocket,
which has a definite hydrophobic character. In the other conformation, the His side chain is in a more
polar environment. The ability of aspartic proteinases to accept a variety of both polar and hydrophobic
groups at the P2 position may be due to this bifurcation. Many inhibitors possess naphthylalanine side
chains at P3 and P4 [14,24,35]. Compounds of this type are potent renin inhibitors with binding constants
in the nanomolar range. Cocrystallisation of such an inhibitor with endothiapepsin revealed that one
naphthalic ring is accommodated in the S3 pocket by significant conformational changes of local enzyme
side chains (Asp77 and Asp114). The other naphthalene lies in the S4 binding region [36].
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C. Rigidification
The number of conformations that a peptide can adopt in solution is reduced by cyclization. This can be
optimized, at least in theory, to lock the peptide in the conformation that has the highest affinity for the
receptor, resulting in a gain of affinity, primarily for entropic reasons. The structures of the fungal
aspartic proteinases reveal that the binding cleft is a wide channel with no obvious division between the
pockets, e.g., S1 and S3 are contiguous. The bound structures of numerous inhibitors have shown that
alternate side chains are in van der Waals contact due to the extended conformation that these ligands
adopt. In addition, at certain positions, e.g., P2, the side chains are allowed very different conformations
due to the permissiveness of the pocket. Hence, the cross-linking of certain side chains may, at least, not
be detrimental to inhibitory potency and may also reduce the susceptibility to degradation in the gut or
plasma. It might therefore be expected that oligopeptide renin inhibitors would be suitable' candidates
for cross-linking experiments. A similar philosophy of rigidification was pursued in the development of
the ACE inhibitor cilazapril [37].
A number of statine-containing inhibitors possessing disulphide links between P2 and P5, and P2 and P4'
have been synthesized [13] although the best potencies were slightly less than for the linear peptides. An
alkyl cycle of varying length was introduced between the hydroxyl of a serine residue at P1 and the main
chain nitrogen of P2 in a series of reduced-bond inhibitors [53]. Potencies similar to the uncrosslinked
molecule were achieved but none were greater. This was attributed to the cis isomerisation of the P3P2
peptide bond giving a conformation that cannot fit the active site of the enzyme. Difficulties in
achieving more potent cyclic inhibitors may be due to the tight binding environment provided by some
pockets (especially S1 and S3), and the possibility that other unproductive conformations of the inhibitor
become favorable. More recently similar findings have been reported for cyclic analogs of pepstatincontaining alkyl crosslinks of variable length between the P1 and P3 side chains [38].
D. In Vivo Stability
Peptides, when administered orally, are susceptible to degradation in the stomach by gastric enzymes
and the proteinases of the pancreas and brush border of the small intestine. Their lifetimes in the plasma
are often short due to rapid proteolysis and other metabolic processes. Early efforts were made to
improve the resistance of renin inhibitors to hydrolysis in vivo by the use of blocking groups at the Nand C-terminii [39] and replacement of susceptible peptide bonds other than the renin cleavage site.
Studies of SAR have shown that various N- and C-terminal groups, some based on the morpholine
nucleus and derivatives of it, have a favorable effect on the duration of inhibition in the
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plasma. This may arise from reduced nonspecific plasma binding due to the relatively polar nature of
these blocking groups [24].
Resistance of inhibitors to gut proteolysis has been improved by various methods such as replacement of
phenylalanine at P3 with O-methyl tyrosine (or naphthylalanine), which was shown to abolish
chymotrypsin cleavage and yet retain high inhibitory potency for renin [40].
III. Structural Studies of Rennin Complexed With Inhibitors
The three-dimensional structures of renin-inhibitor complexes had long been sought as an aid to the
discovery of clinically effective antihypertensives [41]. X-ray analyses of recombinant human renin [42]
and mouse submandibulary renin [43] have given an accurate picture of active-site interactions and
largely confirm the predictions of models based on homologous aspartic proteinases [4]. A large number
of questions concerning the specificities of renins have been answered by these x-ray analyses. The
renin-inhibitor structures also make an important contribution towards the rational design of effective
antihypertensive agents.
A. X-Ray Analysis of Mouse and Human Renin Complexes
For both of these renins multiple copies of the molecules have been independently defined in the x-ray
analysis and shown to have very similar structures. These x-ray structures were refined to final
agreement factors and correlation coefficients of 0.19 and 0.91 for human renin at 2.8 resolution and
0.18 and 0.95 for mouse renin at 1.9 resolution. As expected from the high degree of sequence
identity of human and mouse renins (approximately 70%), they have very similar three-dimensional
structures as shown in Figure 1.
The active-site cleft has a less open arrangement in renins than in the other aspartic proteinases. Many
loops as well as the helix hc (residues 224236) belonging to the C-domain (residues 190302) are
significantly closer to the active site in the renin structures compared to those of endothiapepsininhibitor complexes. This is partly due to a difference in relative position of the rigid body comprising
the C-domain. For instance, there is a domain rotation of ~4 and translation of ~0.1 in the human
renin complex with respect to the endothiapepsin-difluorostatone complex.
The entrance to the active-site cleft is made even narrower in renins as a consequence of differences in
the positions and composition of several well-defined loops and secondary structure elements. Unique to
the renins is a cis proline, Pro111, which caps a helix (hN2) and contributes to the subsites S3 and
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S5. This helix is nearer to the active site in renins than in other aspartic proteinases. On an equivalent
loop in the C-lobe (related by the intramolecular pseudo 2-fold axis), there is a sequence of three
prolinesthe Pro292Pro293 Pro 294 segment. This structure is also unique to the renins among the
aspartic proteinases with Pro294 and Pro297 in a cis configuration. Such a proline-rich structure
provides an effective means of constructing well-defined pockets from loops that would otherwise be
more flexible.
This rather rigid poly-proline loop, together with the loop comprised of residues 241250, lies on either
side of the active site flap formed by residues 7281. Hence, in the renins, the cleft is covered by the
flaps from both lobes rather than from the N-lobe alone as in other pepsin-like aspartic proteinases. This
gives renin a superficial similarity to the dimeric, retroviral proteinases where each subunit provides an
equivalent flap that closes down on top of the inhibitor [44,45].
B. The Role of Hydrogen Bonds in Inhibitor Recognition
Whereas the mouse renin inhibitor extends from P6 to P4', the human renin inhibitor extends only from
P4 to P1'. The cyclohexyl norstatine residue at P1 in the human renin inhibitor mimics a dipeptide analog
with its isopropyloxy group occupying the subsite for the side chain of P1'. The mouse renin inhibitor
(CH-66) possesses a Leu-Leu hydroxyethylene transition state analog [12]. Both inhibitors are bound in
the extended conformation that is found in other aspartic proteinase-inhibitor complexes. Both inhibitors
make extensive hydrogen bonds with the enzymes as shown in Figure 4. In general the two renininhibitor complexes described here demonstrate that a similar pattern of hydrogen bonding is probably
used in the substrate recognition of all aspartic proteinases although their specificities differ
substantially.
There is also great similarity between aspartic proteinases in terms of interactions with the transitionstate analog inhibitors at the catalytic center. The catalytic aspartyl side chains and the inhibitor
hydroxyl group are essentially superimposable in both renin complexes. The isostere C-OH bonds lie at
identical positions when the structures of inhibitor complexes of several aspartic proteinases are
superposed, in spite of the differences in the sequence and secondary structure. Most of the complex
array of hydrogen bonds found in endothiapepsin complexes are formed in renin with the exception of
that to the threonine or serine at 218, which is replaced by alanine in human renin. The similarity can be
extended to all other pepsin-like aspartic proteinases and even to the retroviral proteinases [44,45]. This
implies that the recognition of the transition state is conserved in evolution, and the mechanisms of this
divergent group of proteinases must be very similar.
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Figure 4
The inhibitors complexed with human (top) and mouse (bottom) renin
showing the putative hydrogen-bond interactions made with the enzyme moieties.
C. Specificity
If the main-chain hydrogen bonding of substrates is conserved among aspartic proteinases, how are the
differences in specificities achieved? Table 1 defines the enzyme residues that line the specificity
pockets for both mouse and human renin. In modeling exercises (e.g., Reference 4) it was assumed that
specificities derive from differences in the sizes of the residues in the specificity pockets (Sn) and their
ability to complement the corresponding side chains at positions Pn in the substrate/inhibitor. A detailed
analysis now shows that this simple assumption only partly accounts for the steric basis of specificity.
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For example, in the specificity subsite S3 the phenyl rings of Phe P3 occupy almost identical positions in
both renin inhibitor complexes. Modeling studies have predicted the specificity subsite S3 to be larger in
renins than in other aspartic proteinases [4] due to substitution of smaller residues, Pro 111, Leu114, and
Ala115, in place of larger ones in mammalian and fungal proteinases. However, a compensatory
movement of a helix (hN2) makes the pocket quite compact and complementary to the aromatic ring as
shown in Figure 5. Thus, the positions of an element of secondary structure differ between renin and
other aspartic proteinases with a consequent important difference in the specificity pocket.
The differing positions of secondary structural elements may also account for the specificities at P2'.
Mouse submaxillary and other nonprimate renins do not appreciably cleave human angiotensinogen or
its analogs [46], which have an isoleucine at P2', although they do cleave substrates with a valine at this
position. In contrast, human renin not only cleaves the human and nonprimate substrates but also the rat
angiotensinogen with tyrosine at P2', albeit rather slowly [47]. This can be explained in terms of the threedimensional structures. In the mouse renin complex, the P2' tyrosyl ring is packed parallel to an
adjoining helix (h3) in a narrow pocket and there is only limited space available beyond the C
methylene group. This appears to be able to accommodate a valine, but not the larger isoleucine at P2',
which will suffer greater steric interference from several residues that are conserved in identity and
position in the two renins. On the other hand, in human renin differences in the orientation and position
of
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Figure 5
The S3 specificity pocket of human renin occupied by phenylalanine in the
cyclohexylnorstatine inhibitor.
helix h3 bring it closer by (by 0.5) to the substrate-binding site than in mouse renin. It is orientated in
such a fashion in human renin that, although it can accommodate the isobutyl side chain of isoleucine at
P2', aromatic rings on substituents such as phenylalanine and tyrosine will have severe short contacts
with the side chain of Ile130 (valine in mouse renin). Thus the reorientation of a helix, coupled with
subtle differences in the shapes of the side chains, makes significant changes in the substrate specificity
at this subsite. It is interesting to note that in pepsin this helix is in a similar position with respect to the
active site as in human renin. This provides a structural rationale for the negative influence of peptides
containing phenylalanine [48], tyrosine, or histidine [49] at this subsite (S2') on the rate of proteolytic
pepsin cleavage, while isoleucine and valine enhance catalysis.
Differences in the specificity subsites at S1' in the human and mouse renins have a more complicated
explanation. At first sight the situation appears to be explained by complementarity of the subsites to the
valine and leucine at
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P1' in human and mouse angiotensinogens. Accordingly residue 213 is leucine in human renin and valine
in mouse renin. The S1' pockets of chymosin, pepsin, and endothiapepsin have an aromatic side chain at
residue 189 while the renins have amino acids with smaller side chains (valine in human and serine in
mouse renins). This would be expected to make the pocket larger in renins. However the structure of the
mouse renin complex shows that the substrate moves closer to the enzyme in renins as a result of the
smaller residue at 189 and the pocket is made even more compact due to a compensatory change in the
position and composition of the polyproline loop (residues 290297). Thus, the specificity difference at
this site arises not only from a compensatory movement of a secondary structure, in this case a loop
region, but also from the substitution of an enzyme residue that allows the substrate to come closer to
the body of the enzyme.
Elaboration of loops on the periphery of the binding cleft in renins also influences the specificity. This is
most marked at P3' and P4', for which it has been particularly difficult to obtain complexes with welldefined conformations for other aspartic proteinases. In endothiapepsin, which has been the subject of
the greatest number of studies, different conformations are adopted at P3' and the residue at P4' is
generally disordered. In contrast these residues are clearly defined in mouse renin. This is mainly a
consequence of the polyproline loop, illustrated in Figure 6, which occurs uniquely in renins. The x-ray
analysis of the mouse renin complex shows that the S3' and S4' subsites are formed by the polyproline
loop together with residues of the flap, and a similar situation is likely to occur in human renin. The welldefined interactions of P3' described in the mouse renin complex explains the significant affinity when
inhibitors have phenylalanine or tyrosine at P3' as well as the importance of a P3' residue for catalytic
cleavage of a substrate by renin [50].
Hydrogen bonds between the side chains of the inhibitor and the enzyme do not play a major role in
most specificity pockets. However, S2 is an exception. This subsite is large and contiguous with S1', so
that in human renin the S-methyl cysteine (SMC) side chain of P2 is oriented towards the S1' pocket,
which is only partly filled by the isopropyloxy group of the putative P1' residue. The carbonyl oxygen of
P2 accepts a hydrogen from the O of Ser76, which is unique to human renin; residue 76 is a highly
conserved glycine in all the other aspartic proteinases, including mouse renin. In mouse renin the P2
histidyl group has a different orientation and forms a hydrogen bond with the O of Ser222. If such a
conformation were adopted by the human angiotensinogen in complex with human renin, the two
imidazole nitrogens would be hydrogen bonded to the O of both Ser76 and Ser222. The observed
reduction in the rate of cleavage of a human angiotensinogen analog containing a 3-methyl histidine
substituent at P2 [51] could be explained on the basis of the hydrogen bonding scheme proposed above.
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Figure 6
The P3' tyrosine residue of the mouse renin inhibitor complex showing
the unique polyproline loop on the right. Specificity of this and
neighboring subsites in renins must derive partly from this rigid loop
region.
IV. Rational Drug Design

The pioneering work of Burton, Szelke, and others in developing peptide-based renin inhibitors has been
followed by a worldwide commercial effort to elaborate such compounds into therapeutically active
antihypertensives. The twin problems of insufficient oral bioavailability and rapid clearance has
seemingly presented major obstacles to success. In addition, the possible advantages of renin inhibitors
compared with ACE inhibitors remain questionable. Never-theless information from human-renin
crystallographic studiessuch as the more recent high resolution analyses [52] and algorithms for
analysing voids in the complexes as potential sites for elaborating the drug molecule (e.g. Figure
7)may yet provide leads for compounds with suitable therapeutic characteristics.
The detailed analyses of renin-inhibitor complexes reported here confirm the general structural features
thought to contribute to renin's specificity but
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Figure 7
Schematic illustration of the voids between enzyme and
inhibitor in the crystal structure of human renin complexed with a norstatine
inhibitor. The figure was produced using the GapE software
(Dr. Roman Laskowski). The inhibitor (dark bonds) is
enclosed by a net surface and the gaps (where the
enzyme and inhibitor are not in
contact) are represented by solid surfaces [42].
demonstrate the need for careful, high-resolution x-ray analyses for more confidence in drug design. In
particular, they show that even minor alternations in the positions of secondary structural elements can
lead to major changes in the disposition of the subsites and thus the recognition of substrates. Since such
molecular recognition defines the species specificity and determines the catalytic efficiency of the
enzymes, a through understanding is indispensable for the synthesis of suitable inhibitors. The
specificity pocketsthe molecular recognition sitesare modified by elaboration, particularly of
surface loops, which can be disordered in the uncomplexed enzymes and difficult to model with
precision from homologous structures. These data establish a new foundation for the rational design of
renin inhibitors and have provided a rational base for development of clinically successful HIV
proteinase inhibitors.
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14
Structural Aspects in the Inhibitor Design of Catechol O-Methyltransferase
Jukka Vidgren and Martti Ovaska
Orion Corporation Orion Pharma, Espoo, Finland
I. Introduction
Catechol O-methyltransferase (COMT) plays an important role in the catabolic inactivation of
catecholamines. It is present both in extracerebral tissues and in the central nervous system. During the
last few years there has been a remarkable interest in COMT. Basic biochemical and molecular biology
research has given detailed insights into the function and nature of the enzyme. The knowledge of the
crystallographic structure has allowed researchers to analyze the molecular mechanism of the catalytic
reaction and to accomplish the structure-based design of inhibitors. The development of potent and
selective inhibitors has provided effective pharmacological tools to investigate the physiological role of
the enzyme. The main clinical interest has been the possible application of COMT inhibitors as adjuncts
in the L-dopa therapy of Parkinson's disease. Parkinson's disease is a dopamine deficiency disorder. The
dopamine-producing neurons in striatum are destroyed. The medication strategy is to replenish the
missing dopamine. L-Dopa, given together with a peripheral inhibitor of dopa decarboxylase (DDC), for
example, carbidopa, is a standard therapy in Parkinson's disease. While dopamine does not penetrate
into the brain, L-dopa penetrates the blood-brain barrier and is decarboxylated into dopamine in the
brain. The half-life of L-dopa is short and in the presence of DDC inhibitor a large amount of the drug is
eliminated by COMT. The COMT enzyme produces the metabolite 3-methoxytyrosine (3-OMD), which
has no benefit in the treatment of Parkinson's disease, but has a long elimination half-life and may be
harmful during chronical treatment. Also a gradual loss of the efficacy of L-dopa occurs during longterm medication. Since the early 1980s active
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Figure 1
The rationale of COMT inhibition as adjunct in the L-dopa therapy of
Parkinson's disease (reproduced by permission from COMT News, Issue 1, Orion
Corporation, Orion Pharma, 1994).
research in pharmaceutical companies has been carried out to develop new potent, selective, and orally
active COMT inhibitors. Some of them (e.g., entacapone), are now in final clinical trials and the results
have been promising. The rationale of COMT inhibition can be seen in Figure 1. It can be concluded
that COMT inhibition in peripheral tissues improves the brain entry of L-dopa and decreases the
formation of 3-OMD. The dose of L-dopa can be lowered and the dose interval prolonged. Also a
decrease of the fluctuations of dopamine formation has been observed. The inhibition of COMT seems
to be the next step in improving the L-dopa therapy of Parkinson's disease. This paper discusses the
structure-based approach for the understanding of the enzyme function and inhibitor design.
II. The Enzyme
A. Physiological Role of COMT
Catechol O-methyltransferase (COMT, EC 2.1.1.6) was originally detected in rat liver extracts [1]. Since
then, COMT has been found in many species:
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Figure 2
The reaction catalyzed by catechol O-methyltransferase. Dopamine:
R=CH2-CH2-NH2; L-dopa: R=CH2-CH(NH2)-COOH.
animals, plants, and procaryotes [2]. In mammals the highest COMT activities have been found in the
liver and kidney, but COMT is common in almost all mammalian tissues [24].
The COMT enzyme catalyzes the transfer of the methyl group from the coenzyme S-adenosyl-Lmethionine (AdoMet) to one of the phenolic hydroxyl groups of a catechol or substituted catechol [1]
(Figure 2). The presence of magnesium ions is required for the catalysis. The reaction products are Omethylated catechol and S-adenosyl-L-homocysteine (AdoHcy). Physiological substrates of COMT are
catecholamine neurotransmitters, dopamine, noradrenaline, and adrenaline, and some of their
metabolites. The COMT enzyme inactivates catecholic steroids such as 2-hydroxyestradiol, drugs with a
catechol structure such as L-dopa, and a large number of other catechol compounds [1,2,57]. The
general physiological function of COMT is the inactivation of biologically active or toxic catechols. A
schematic view of the major catecholamine pathways in the brain is shown in Figure 3. L-Dopa is the
dopamine precursor used in the treatment of Parkinson's disease [8].
B. Primary Structures
There are no isoenzymes of COMT known in different mammalian tissues. Two distinct forms of
COMT have been found: one is soluble (S-COMT) and the other membrane bound (MB-COMT) [9,10].
Both soluble and membrane-bound COMT have been cloned and characterized [1116]. The soluble and
membrane-bound COMT are coded by one gene using two separate promoters [17]. The soluble COMT
contains 221 amino acids, whereas the membrane-bound form has a 50-(human) or 43-(rat) residueslong amino-terminal extension containing the hydrophobic membrane anchor region. The sequences of
COMT enzymes from different species are highly similar (see Figure 4). The soluble human protein is
81% identical with the rat enzyme. The 165-amino-acids-long fragment of porcine COMT has 82%
homology with the human
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Figure 3
The main metabolic routes of dopamine and noradrenaline in
the brain. COMT, Catechol O-methyltransferase; MAO, monoamino
oxidase; DDC, dopa decarboxylase; DBH, dopamine
-hydroxylase; 3-OMD, 3-methoxytyrosine; Dopac,
dihydroxyphenyl acetic acid.
enzyme [13]. The existence of a thermolabile low-activity and a thermostable high-activity COMT in
human population has been reported [18]. Interestingly, the two published sequences of human soluble
COMT differ in only one amino acid. Recent kinetic studies have shown that this difference affects
unambiguously the thermostability of the enzyme [19].
C. Kinetics of Human COMT
The kinetic mechanism of the methylation reaction of human COMT has been studied exhaustively
using recombinant enzymes [19]. The mechanism is sequential ordered: AdoMet binding first, then
Mg2+ and the catechol substrate as the last ligand. Human S-COMT and MB-COMT have similar kinetic
properties. The main difference is the one-order lower Km value of MB-COMT for dopamine as
substrate (S-COMT 207 M and MB-COMT 15 M). The COMT enzyme is a rather slow enzyme with
a low catalytic number. At saturating substrate levels S-COMT has a double efficiency compared with
MB-COMT (kcat=37 and kcat =17, respectively). At low substrate concentrations (<10 M) the MBCOMT seems to methylate catecholamines more rapidly than S-COMT.
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Figure 4
Amino acid sequence comparison of known COMT sequences (rat [11], human
[14], pig [13]). Secondary structure elements in the sequence of rat soluble COMT
are indicated as well as important active-site residues involved in binding of ligands
(a, AdoMet; m, magnesium; s, substrate/inhibitor). The numbering of the residues
corresponds to the soluble enzyme. The extension of the MB-COMT consists of the first
50 amino terminal residues. In the human sequence of COMT determined by Bertocci
[13], Val108 is replaced by Met108.
Under physiological concentrations of catecholamines in the brain, MB-COMT may play a more
important role than S-COMT [19,20].
D. Three-Dimensional Structure of COMT
Backbone
The crystal structure of rat soluble COMT has been solved at 2.0 resolution [21]. The COMT enzyme
has a single domain /-folded structure, in which eight -helices are arranged around the the central
mixed sheet. The sheet contains five parallel strands and one antiparallel hairpin. An overview of
COMT is illustrated in Figure 5.
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The AdoMet binding motif is similar to the Rossmann fold, which is well known from the nucleotide
binding proteins [22]. It has been shown that the known crystal structures of methyltransferases are
strikingly similar in the AdoMet-binding regions [23], which indicates that all AdoMet-utilizing
enzymes may share a common divergent evolution.
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Figure 5
Schematic stereo view of the three-dimensional structure of COMT. The ligands
bound to COMT are the methyl-donating coenzyme AdoMet and the magnesium ion.
Figures 57 and 13 were produced using the program MOLSCRIPT [50].
Figure 6
Stereo view of the AdoMet binding to COMT. The most important amino acid
residues are shown as well as the magnesium ion and the inhibitor
3, 5-dinitrocate-chol (DNC).
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Active Site
The active site of COMT consists of the AdoMet binding domain and the catalytic site. The structural
elements and individual interactions between COMT residues and the enzymatic-action-participating
ligands are demonstrated in detail in Figures 5 to 8.
AdoMet Binding by COMT. The active-site residues, which have significant interactions with the
coenzyme, are shown in Figure 6. The loop region between strand 1 and helix 4 forms the AdoMetbinding consensus sequence (in COMT GAxxG) that is conserved in methyltransferases [23]. In this
region the terminal amino and carboxyl groups of AdoMet are bound. The last residue of the strand 2,
Glu90, forms a hydrogen bond to the ribose hydroxyls. The residue Met91 has face-to-face van der
Waals contacts on one side, and His142 has edge-to-face contacts on the opposite side of the adenine
ring. The residue Trp143 closes the adenine of AdoMet into the protein with face-to-edge contact.
Furthermore, the N-6 atom of the adenine hydrogen binds to Ser119. The Met40 residue holds the
sulphur of AdoMet with the methyl group in right position towards the hydroxyl group of the catechol
substrate. As a result of the various hydrogen bonds and van der Waals contacts, AdoMet has a high
affinity to COMT with a dissociation constant of 23 M [19].
Catalytic Site. The catalytic site of COMT is a rather simple environment formed by the metal ion and
by the amino acids important for substrate binding and catalysis of the methylation reaction.
The magnesium ion plays a crucial role for the catalytic activity of COMT. Figure 7 shows the binding
of magnesium to COMT as derived from the
Figure 7
Magnesium binding in COMT. The magnesium ligands are Asp141, Asp169, Asn170,
both hydroxyls of 3, 5-dinitrocatechol (DNC) and a water molecule (W).
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Figure 8
The catalytic machinery of COMT. Shown are the COMT residues
important for catalysis, Mg2+ binding, the catechol as substrate, and the
methyl-donating coenzyme AdoMet. The hydrophobic walls are defined
by two tryptophane residues and a proline residue.
crystallographic studies. Magnesium has an octahedral coordination to two aspartic acid residues
(Asp141 and Asp169), to an asparagine residue (Asn170), to both catechol hydroxyls of the substrate,
and to a water molecule. In addition to the Mg2+ ion, Lys144 and Glu199 participate directly in the
methylation reaction as shown in Figure 8. The gate keeper residues Trp38 and Pro174 form the
hydrophobic walls and define the selectivity of the enzyme to different side chains of the substrate.
They play a significant role in the binding of the substrates and inhibitors of COMT [19, 21].
III. Mechanism of the Catalytic Action of COMT
The catalytic site is a shallow groove with the catalytic machinery at the bottom as illustrated in Figure
8. The two hydroxyl oxygens of a catechol substrate bind directly to the Mg2+ ion. The active methyl
group of AdoMet is near one of the hydroxyl groups, on one side of the catechol ring. The amino group
of Lys144 is also located near this hydroxyl group, on the other side of the catechol ring from AdoMet.
The Glu199 residue is near the other hydroxyl group.
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Figure 9
The energy profile (as calculated at the 3-21G/PM3 level [24]) for the proton
transfer step A rarrow.gif B and the methyl transfer step B rarrow.gif C for catechol as a
substrate.
The pKa of the catecholic hydroxyl is about 9.8. The role of the Mg2+ ion bound to the enzyme is to
make the hydroxyl groups more easily ionizable. It has been shown by quantum mechanical calculations
that the hydroxyl protons can be transferred to Lys144 and Glu199 [24]. The proton transfer OH
rarrow.gif Lys144 activates the hydroxyl group for the methyl transfer AdoMet rarrow.gif O-. Thus
the reaction coordinate for methylation of catechols by COMT consists of a proton transfer from a
hydroxyl group to Lys144 and a subsequent methyl transfer from AdoMet to the hydroxyl group (Figure
9). The Lys144 residue acts as a typical catalytic base in a general base-catalysed SN2-like nucleophilic
substitution reaction [24].
IV. Inhibitors of COMT
A. First-Generation Inhibitors
First generation COMT inhibitors such as pyrogallol, U-0521 (3, 4-dihydroxy-2-methylpropiophenone),
tropolone, and 8-hydroxyquinoline (Figure 10) were
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Figure 10
Structures of some first generation COMT inhibitors: pyrogallol,
U-0521 (3, 4-dihydroxy-2-methylpropiophenone), tropolone,
and 8-hydroxyquinoline.
Figure 11
Structures of second-generation COMT inhibitors discussed in this
paper: nitecapone (OR-462), entacapone (OR-611), tolcapone
(RO-40-7592), and 2-((3, 4-dihy-droxy-2-nitrophenyl) vinyl) phenylketone.
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Table 1 Inhibitor Constants of Selected COMT Inhibitors
Ki (nM) Humanb
Ki (nM)Pigc
Nitecapone
1.0
700
Entacapone
0.3
N.D.
Tolcapone
0.3
N.D.
Vinylphenylketone
4.0a
200
aT.
Lotta, unpublished results
bReference
19.
cReference
41.
N.D.: not determined
used as in vitro tools to investigate COMT inhibition, but because of the lack of potency and selectivity
and because they were toxic, they were not clinically useful [2]. These inhibitors have inhibition
constants (Ki values) in the micromolar range. Many of them contain the catechol structure and are also
substrates of COMT.
B. Second-Generation Inhibitors
The invention of a new structural family of COMT inhibitors in the late 1980s lead the COMT research
into a new active epoch [25,26]. The most potent second-generation inhibitors are nitrocatechol
derivatives; some examples are shown in Figure 11. Entacapone (OR-611) and tolcapone (RO-40-7592)
are now in clinical trials for the treatment of Parkinson's disease, and have been extensively studied
[2740]. Both compounds are very potent and selective tightbinding inhibitors of human COMT with Ki
values of 0.3 nM [19]. They differ mainly in their pharmacokinetic properties. Entacapone acts
peripherally while tolcapone inhibits COMT both peripherally and centrally.
Recently it was reported that 2-((3,4-dihydroxy-2-nitrophenyl)vinyl) phenylketone is a tight-binding
inhibitor of pig COMT [41], and it is also a potent inhibitor of human COMT (Table 1). This inhibitor
has a vinylphenylketone group at position 4 of the catechol ring, i.e., in the position ortho to the nitro
group.
V. Enzyme Inhibitor Interactions
A. Background
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From quantitative structure activity relationship studies (QSAR) of COMT inhibitors it became evident
that the acidity of the catechol hydroxyl group is the most important factor that influences the inhibitory
activity of catechol
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derivatives [26,42]. Structures containing a catechol ring optimally substituted with a nitro group in
position 3 and other electron-withdrawing substituents in position 5 showed high-potency inhibition
[25,26,42,43]. It was also detected that the side-chain hydrophobicity at position 5 correlates
significantly with the inhibitor activity [42].
B. X-Ray Structures of COMT-Drug Complexes
The structure of 3,5-dinitrocatechol complexed with COMT has been solved [21]. This inhibitor is a
typical nitrocatechol derivative with a high affinity for COMT. The excellent electron density of the
inhibitor in the active site of COMT is represented in Figure 12. The planar structure of this compound
fits well into the active-site cavity of the enzyme and forms nearly ideal contacts
Figure 12
A portion of the structure model and the 2F0-Fc electron
density map contoured at 1.0 standard deviation. The region
containing the inhibitor, magnesium, parts of AdoMet, and
the residue Glu199 is shown. The sphere marked with W
represents a water molecule.
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with the tryptophane residues 38 and 143. The hydroxyl groups of the inhibitor are coordinated to the
Mg2+ ion. The hydroxyl group in position 1 has an important hydrogen bond to the carboxyl group of
Glu199. The other hydroxyl is near the methyl group donated by AdoMet. The pKa of this hydroxyl
group is low (about 3.4) [26]. The 3-nitro group of the inhibitor has favourable van der Waals
interactions with Trp143. The Trp38 residue is located edge-to-face with the catechol plane, which
allows an ideal aromatic hydrophobic contact. Such aromatic hydrophobic interactions have been
described to be important in proteins and for the binding of ligands [44,45]. The binding mode of the
catechol ring of other crystallographically determined potential nitrocatecholtype inhibitors complexed
with COMT is essentially the same as with 3,5- dinitrocatechol (J.Vidgren, unpublished results).
C. Differences in the Active Site of Human, Rat, and Pig COMT
Models for human and pig COMT are easy to build using the experimental structure of the rat COMT,
due to the high degree of homology between the rat, human, and pig COMT enzymes (Figure 4). The
active sites are especially well conservedthe few differences in the active-site residues are collected in
Table 2. The kinetic data show that the Km values of common substrates for rat and human COMT are
very similar. Pig COMT shows, however, a considerably higher Km value for catechol [46]. The same
difference is apparent for inhibitors represented by the Ki values in Table 1.
The model for the binding of vinylphenylketone to pig and rat COMT is shown in Figure 13. Assuming
that the catechol part of the inhibitor adopts the same position as found in the crystal structure with
dinitrocatechol, the vinylphenylketone substituent has enough room to bind to both enzymes. The most
significant difference between these enzymes lies in residue 38, the hydrophobic tryptophan in rat (and
human) COMT and the polar arginine in pig COMT. If Arg38 is directed towards the hydrophobic core
of the enzyme in a similar conformation as Trp38 (shown in Figure 13), it causes repulsion with the
catechol ring of the inhibitor. However, it is probable that the polar Arg38 is directed towards the
solvent. In this case the substrates and inhibitors will lack the favorable contacts that exist with Trp38 in
human and rat enzymes. ObviTable 2 Differences in the Active Sites Between Rat, Human, and Pig COMT
Position
Rat
Human
Pig
38
Trp
Trp
Arg
173
Val
Cys
Cys
201
Met
Arg
Ser
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Figure 13
The inhibitor 2-((3,4-dihydroxy-2-nitrophenyl)vinyl)phenylketone modeled
into the active site of rat (a) and pig (b) COMT.
ously this one amino acid differences in the active site of the isoenzymes can explain the significant
differences in the inhibitory potency of vinylphenylketone against these enzymes. From the structural
point of view it seems that the position of the side-chain substitution (for example at C5 in entacapone
and C4 in vinylphenylketone) is not critical for the inhibitor binding. In both cases the substituent has
sufficient space to adapt to the protein structure, and in fact, large substituents reach from the active site
cavity to the solvent (Figure 13).
The tenfold higher inhibitory activity of entacapone compared with vinylphenylketone against human
COMT can be accounted for by the electron- withdrawing effect of the side-chain substitution. In the
case of entacapone, the side chain at position C5 has a more beneficial electronic influence on the 2hydroxyl of the inhibitor producing a better inhibition (see Section VI).
VI. Mechanism of the COMT Inhibition By Nitrocatechols
As described above, catechols with strong electronegative groups are potent inhibitors of COMT. These
compounds seem to bind well to the active site, but in spite of that they are very poor substrates. It has
been shown, with nitecapone
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Figure 14
The energy profile (as calculated at the 3-21G//PM3 level [24]) for the proton
transfer step A rarrow.gif B and the methyl transfer step B rarrow.gif C for 3,4-dinitrocatechol as a
substrate.
in a rat COMT assay, that the rate of nitecapone methylation is equivalent to about 1% of the rate of
dopamine methylation [47].
The energy profile for the hypothetical methylation of 3,5-dinitrocatechol is shown in Figure 14 [24].
The electronegative nitro groups strongly stabilize the ionized catecholCOMT complex, and the energy
barrier for the methylation step is high (see Figure 9 for comparison). This can be understood as
decreased nucleophilicity of the hydroxyl oxygen, due to the electron-withdrawing properties of the
nitro groups.
The electronic effect of the substituents of the catechol ring to the nucleophilicity of the hydroxyl group
at the active site can be readily seen from the molecular electrostatic potential (MEP) surfaces of the
system. The MEP surfaces were calculated at the PM3 level and plotted at 20 kcal/mol for catechol and
3,5-dinitrocatechol at the active site of COMT [24]. The results are summarized in Figure 15. In the case
of catechol the effect of the proton transfer form OH to Lys144 is seen as a remarkable increase in the
negative potential between AdoMet and the substrate. 3,5-Dinitro substitution of the catechol ring
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Figure 15
The energy profiles and MEP surfaces (20 kcal/mol) for catechol (black) and
3,5-dinitrocatechol (grey).
decreases the activating negative potential substantially. As a consequence, the OH group is weakly
nucleophilic and 3,5-dinitrocatechol is not a substrate of COMT but a potent inhibitor.
VII. Inhibitor Design
The drug-design process of COMT inhibitors started long before the structure of the target molecule was
available. The most important results were extracted from the QSAR studies of substituted catechols
[26,42]. Those investigations clearly indicated the importance of the acidity of one of the two hydroxyl
groups in the catechol ring. The ionization of the hydroxyl was greatly influenced by electronwithdrawing substituents in the positions ortho and para to the hydroxyl. The lipophilicity of the side
chain was predicted, but after the determination of the enzyme structure it became clear that the active
site of COMT is a relatively shallow groove where ligands with longer side chains
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easily reach the surface of the enzyme. The pharmacophore model constructed with biochemical and
QSAR knowledge [42] was surprisingly good and realistic in comparison to the experimental structure.
The crucial role of the magnesium ion for the binding of substrates and catalysis was not given enough
attention. Even without the experimental knowledge of the three-dimensional structure of COMT, the
correct decision for the direction of the drug-design process was possible. Many open questions were
still waiting for the determination of the molecular structure. The most important drug-design aspects
under consideration included the optimization of the pharmacokinetic properties of the molecules,
especially the penetration across the blood brain barrier. The structure of COMT clearly showed that
with catechol-type inhibitors, one of the positions ortho to the hydroxyls is optimally substituted by a
nitro group, while the other ortho position has to be unsubstituted. Sterically, the two remaining sites can
be substituted quite freely, so that these substitution positions can be used to modify the
physicochemical properties of the inhibitors. The question of the possibility of designing an inhibitor
without the catechol structure is important, because the potent inhibitors which rely on the ionization of
a catechol hydroxyl, penetrate very poorly into the brain. In clinical trials of the therapeutic use of
COMT inhibitors it has become evident that the beneficial effect to L- dopa metabolism is fully reached
with peripheral inhibitors such as entacapone [2729]. As demonstrated above, based on the threedimensional structure of COMT, the design of potent noncatechol type inhibitors may be very tedious.
The active site of COMT is a rather simple environment with a few catalytic residues and a magnesium
ion defining the structural limits of the catechol ligands.
VIII. Clinical Possibilities of Inhibitors of COMT
A. Parkinson's Disease
Parkinson's disease is a neurological disorder that affects voluntary movement. The symptoms are
slowness of movement, rigidity, and tremor. The reason for the disease is unknown. Parkinson's disease
is a progressive disorder and involves the deterioration of dopaminergic nerve fibers in substantia nigra,
which leads to a striatal deficiency of dopamine. The symptoms of Parkinson's disease are detected only
after about 80% of the dopamine-producing neurons are degenerated. There is no known cure but the
symptoms are treated with a combination of different drugs.
Current and Future Therapy of Parkinson's Disease
Dopamine does not penetrate the blood-brain barrier. L-Dopa is actively transported into the brain and
then converted to dopamine. L-Dopa is rapidly metabolized and only about 1% of an oral dose reaches
the brain. In the current
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therapy of Parkinson's disease L-dopa (precursor of dopamine) is administered together with a

peripheral dopadecarboxylase (DDC) inhibitor (such as carbidopa or benserazide). The major peripheral
L-dopa metabolizing enzymes are DDC and COMT. After inhibition of DDC, COMT is responsible for
the main catabolism of L-dopa. In the central nervous system COMT together with monoamino
oxidase (MAO) participitates in the metabolism of L-dopa and dopamine. Large amounts of orally
administered L-dopa are converted by COMT to 3-O-methyldopa (3-OMD). Having a long plasma halflife (approximately 15 h compared with the dopamine 1-h half-life), 3-OMD accumulates in the plasma
during the L-dopa treatment. L-Dopa and 3-OMD also compete for the same active transport system into
the brain. It has been proposed that 3-OMD could cause some side effects of the L-dopa treatment
(dyskenisia, on-off phenomenon). Inhibition of COMT enzyme decreases the 3-OMD formation and
improves the brain entry and bioavailability of L-dopa. The use of COMT inhibition should prolong the
L-dopa effects and permit a decreased does [27 29].
Preclinical and clinical results indicate that both entacapone and tolcapone are orally active, nontoxic
and well-tolerated drugs. The adjuvant L- dopa therapy with DDC inhibitor + COMT-inhibitor (+
possible MAO inhibitor) may substitute for the present double therapy in the treatment of Parkinson's
disease [2740]. Together with the development of dopamine agonists and MAO inhibitors, the
inhibition of COMT will constitute major progress in the treatment of Parkinson's disease in the near
future.
B. Other Possible Indications of the COMT Inhibition
It has been proposed that COMT inhibitors co-administered with L-dopa could have beneficial effects in
the treatment of depressive illness [40]. This can be caused by either the better availability of dopamine
or by the elevated noradrenaline levels in the brain. Another hypothesis suggests that the increasing level
of AdoMet caused by COMT inhibition may cause an antidepressive effect [33].
Dopamine has also natriuretic and diuretic effects in kidney. There has been evidence that abnormalities
of the renal dopamine system can lead to salt- sensitive hypertension [48]. In rat kidney, deamination
represents the major pathway in the metabolism of dopamine, but when MAO is inhibited, methylation
appears to offer an alternative metabolic pathway [49]. Thus COMT inhibition may be important in the
regulation of renal sodium excretion.
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39. Mnnist PT. Clinical potential of catechol-O-methyltransferase (COMT) inhibitors as adjuvants in
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15
Antitrypanosomiasis Drug Development Based on Structures of Glycolytic
Enzymes
Christophe L. M. J. Verlinde, Hidong Kim, Bradley E. Bernstein, Shekhar C. Mande, * and Wim G.J.
Hol
University of Washington, Seattle Washington
I. Trypanosomiasis
A. Disease and Treatment
Human African trypanosomiasis, also called sleeping sickness, is caused by the parasites Trypanosoma
brucei gambiense and Trypanosoma brucei rhodesiense. These unicellular organisms live freely in the
bloodstream of the human host and invade the brain during the later stage of the disease. Without
treatment the disease is always fatal [1]. The course of the gambiense form may last from months to
years, while T. brucei rhodesiense usually kills within weeks. Sleeping sickness occurs in thirty-six subSaharan African countries, putting fifty million people at risk. Each year 25,000 new cases are reported,
but the actual number of cases is more likely to be about 250,000 [2]. The current state of
antitrypanosomal chemotherapy is dismal; many parasitologists do not want to take the risk of being
infected with T. brucei rhodesiense and prefer to study the parasite T. brucei brucei, which is harmless
to humans [3].
Not more than four drugs are available to treat the disease: pentamidine, suramin, melarsoprolall from
the first half of this centuryand eflornithine, introduced in 1990 (Figure 1). Except for eflornithine,
which is an irreversible ornithine decarboxylase inhibitor [4], the mechanisms of the drugs are poorly
understood [5]. All four drugs require administration by injection in a hospital setting, which is a major
drawback in rural Africa [6]. Pentamidine is useful for treating early stage T. brucei gambiense
infection, suramin for both early stage gambiense and rhodesiense infection. The permanent charges on
pentamidine
*
Current affiliation: Institute of Microbial Technology, Chandigarh, India
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Figure 1
Available drugs for the treatment of human African trypanosomiasis.
and suramin explain why they show poor oral absorption and do not cross the bloodbrain barrier,
making them unsuitable for the treatment of late-stage trypanosomiasis. Melarsoprol, an organoarsenical
compound, was until 1990 the only drug effective in the late stage of both forms of trypanosomiasis.
Unfortunately, it is also highly toxic, causing reactive encephalopathy in up to 10% of the patients, of
which about one half die. This deadly complication is well known to villagers of areas where the disease
is endemic and, ironically, discourages people from participating in diagnostic surveys. Eflornithine,
heralded as the resurrection drug upon its introduction [7], cures patients infected with late-stage T.
brucei gambiense but is ineffective against the more virulent rhodesiense form. Additionally, it causes
bone marrow suppression in half of the patients and occasionally convulsions [6]. A serious concern is
that resistance has been reported against each of the four antitrypanosomal drugs [1].
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Sleeping sickness has been largely ignored by the pharmaceutical industry because the poor
socioeconomic situation in the part of the African continent afflicted by this debilitating disease offers
little prospect of reasonable financial returns [8]. It is revealing that eflornithine was originally
developed as an anticancer drug. It was screened for antitrypanosomal properties only when the
biochemistry of the trypanosomal polyamine metabolism was understood [9]. Fortunately, the cell
biology of trypanosomes is so extraordinary that they have been the subject of more fundamental
research than most other protozoan parasites [5]. Each of these unique features of T. brucei may become
a target for new drugs, provided they prove to be essential for the survival of the parasite in the human
host. With such a wealth of biochemical information, structure-based drug design provides a tremendous
opportunity to arrive at new drugs to cure sleeping sickness.
B. Targets for Future Drugs
Preventing trypanosomiasis would be a nobler goal than curing it. Unfortunately, trypanosomes are
experts in evading our immune system. They achieve this by varying their dense surface coat. It is
composed of ten million copies of a single protein, the variant surface glycoprotein (VSG), for which
they have no less than a thousand different genes. In this way trypanosomes can change surface antigens
more rapidly than the host can produce new antibodies [10]. Clearly, such a mechanism leaves little
hope for preventing sleeping sickness by vaccination.
In contrast to the small number of drugs available to treat trypanosomiasis, the opportunities for
developing new drugs are ample, as can be seen from Table 1. They range from unique RNA processing
to reduced metabolism, salvage systems, and different rates of protein turnover. All of these features
were the subject of an outstanding review by C.C. Wang [5].
An inventory of the structural information waiting to be exploited by structure-based drug design reveals
eight potential target enzymes (Table 2). Since for most trypanosomatid proteins there is a human
counterpart it is mandatory that designed inhibitors be selective, i.e., have very little affinity for the
equivalent enzymes of the human host. As a consequence, selective design requires pairs of equivalent
structures from the parasite and from the host. A complication for selective design is that for three of the
mammalian enzymes only the structure of one isoenzyme is known. For example, the structure of human
aldolase A has been determined [23] but not the structures of isoenzymes B and C, which share only 69
and 82% sequence identity to isoenzyme A [4042]. Of course, homology modeling might be a way to
overcome this problem.
From Table 2 it is evident that only for three proteins, TIM, GAPDH, and trypanothione reductase the
structures of the parasite and host enzymes are
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Table 1 Trypanosomal Targets, Their Human Equivalents, and Current Leads for Drug Design
Human host
T. brucei
Lead
RNA editing by trans-splicing [11]
cis-splicing
none
All energy from fast glycolysis [12]
glycolysis and oxidative

phosphorylation
MMBAa[13]
Glucose transporter [14]
human erythrocyte glucose

transporter
none
Purine P2 transporter [15]
none
Purine salvage enzymes, e.g., HGPRTb[16]
HGPRT
Slow rate of enzyme turnover [17], e.g., ornithine

decarboxylase [18]
fast rate of enzyme turnover
Polyamine metabolism, e.g., S-adenosyl

methionine decarboxylase [19]
S-adenosyl methionine
decarboxylase
MDL 73811c
Trypanothione reductase [20]
glutathione reductase
mepacrine
VSG anchor: a myristate-containing GPI [21]

aMMBA
= 2' -deoxy-2' -(m-methoxybenzamido)-adenosine.
bHGPRT
= hypoxanthine guanine phosphoribosyltransferase.
cMDL
none
eflornithine (= drug) [9]
10-(propoxy)-decanoated
73811 = 5' -{[(Z)-4-amino-2-butenyl]methylamino}-5' -deoxyadenosine.
dIt
has not been established whether the trypanocidal effect of this compound is due to its incorporation in the GPI anchor
[5].
known. For five more targets the crystal structure of only the mammalian enzyme is available. Efforts to
solve the structures of trypanosmatid aldolase, PGK, and PK counterparts are underway in our lab. This
review explains how we attempted to arrive at selective inhibitors of three trypanosomal glycolytic
enzymes.
C. Trypanosomal Glycolysis: Enzyme Inhibition as a Target
In the bloodstream of the human host, trypanosomes are metabolically
Document
lazy. Since there is plenty of glucose and oxygen available, they rely solely on glycolysis to the stage of
pyruvate for their energy supply [12]. Their glycolysis proceeds at an amazing rate, which is about fifty
times faster than in the cells of the human host [43]. This fast rate is a necessity because only two
molecules of ATP are generated per molecule of glucose instead of the thirty-six produced by complete
oxidation. These findings led to the proposal that inhibitors of trypanosomal glycolysis might be turned
into drugs. Support for this idea comes from in vitro experiments where salicylhydroxamic acid (SHAM)
was used to bring the parasite under anaerobic conditions, after which glycolysis was
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Table 2 Three-Dimensional Structures Available for Trypanosomal Drug Design
Trypanosomatid
PDB code
Mammalian
PDB code
(a) Glycolytic:
PGIa
Porcine
1PGI[22]
Aldolase
Humanb
1ALD[23]
TIM
T. brucei
5TIM[24]
Human
1HTI[25]
GAPDH
T. brucei
L.mexicana
1GGA[26,27]
[30]
Humanc
3GPD[28,29]
PGK
Horse
Pig
2PGK[31][32]
PK
Catd
1PYK[33]
HumanGR
3GRS[35]
Human
1HMP[38]
(b) Other:
TR
T. cruzi
1NDA[34]
C. fasciculata
1TYT[36]
1PPR[37]
HGPRT
VSG
T. brucei
1VSG[39]
No equivalent
aAbbreviations:
PGI = phosphoglucose isomerase, PGK = phosphoglycerate kinase, PK = pyruvate kinase, TR =

trypanothione reductase; GR = glutathione reductase, HGPRT = hypoxanthine-guanine phosphoribosyl
transferase, VSG = variable surface glycoprotein.
bA
isoenzyme, from muscle; other mammalian isoenzymes are B in liver and C in brain.
cMuscle
isoenzyme; a liver isoenzyme exists.
dM1
isoenzyme, from muscle; mammals also have M2 in kidney, adipose tissue and lung, L in liver, and R in
rood blood cells.
Document
blocked by mass action through the addition of glycerol (Figure 2). As a result trypanosomes were lysed
within five minutes [44,45]. Treatment of infected rodents with the SHAM/glycerol mixture proved to
be effective to clear the blood of the animals from T. brucei [46], although only with sublethal doses was
permanent aparasitemia obtained [47]. If glycolysis could be blocked selectively, i.e., without affecting
the equivalent enzymes of the host, one might have a promising therapy against trypanosomiasis.
D. Beyond Enzyme Inhibition: Protein Routing as a Target
In trypanosomes, seven enzymes involved in glycolysis, from hexokinase to phosphoglycerate kinase,
are sequestered in specialized organelles, called glycosomes [48]. These microbodies are probably
evolutionary relics of an endosymbiont [49] but are devoid of genetic material encoding for the
glycosomal enzymes. Instead, these enzymes are encoded in the nucleus and are post-translationally
imported into the glycosome. Since the import process likely involves unfolding one might envision
blocking import by stabilizing the folded
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Figure 2
Glycolysis in bloodstream-form trypanosomes. All net energy production takes place
in the cytosol as the result of ATP formation by pyruvate kinase. However, the
majority of glycolytic enzymes are sequestered into a specialized organelle, the
glycosome. There the net ATP synthesis is zero irrespective of the presence of oxygen.
Under aerobic conditions the NADH produced by glyceraldehyde-3-phosphate dehydrogenase is reoxidized via the G-3-P/DHAP shuttle, which couples glycolysis to a
mitochondrial glycerophosphate oxidase. Under anaerobic conditions or when the
oxidase is blocked by SHAM, equimolar amounts of glycerol and 3-phosphoglycerate
are formed. However, the addition of an excess of glycerol to the cytosol prevents the
reoxidation of NADH. As a result trypanosomes treated with a mixture of SHAM and
glycerol die in a matter of minutes.
Document
state through the tight binding of ligands. In this way inhibitors might act at two levels, directly by
blocking catalysis and indirectly by preventing proper enzyme routing in the parasite. Thus far, we have
engaged in a collaborative effort to inhibit three of the glycosomal enzymes. (From Ref. 95. Copyright
1988 by Elsevier.)
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II. Three Glycolytic Enzymes Of T. Brucei: Molecular Biology, Biochemistry, and X-Ray
Crystallography
A. Triosephosphate Isomerase (TIM)
TIM is a homodimeric enzyme that interconverts dihydroxyacetone phosphate and glyceraldehyde-3phosphate. It ensures that both trioses derived from glucose can be used for ATP production in the
glycolytic pathway. Triosephosphate isomerase does not require any cofactor. Both the T. brucei and
human enzymes have been overexpressed in Escherichia coli [50,25] and their crystal structures were
solved in our group [24,25]. In addition, the structures of T. brucei TIM in complex with seven
nonselective competitive inhibitors, with inhibition constants of 300 M or higher were determined:
monohydrogen phosphate [51], 2-phosphoglycerate [52], 3-phosphoglycerate [53], 3phosphonopropionate [53], glycerol-3-phosphate [53], 2-(N-formyl-N-hydroxyamino)-ethyl phosphonic
acid [54], and N-hydroxy-4-phosphonobutanamide [55]. These studies gave an excellent picture of
different ligand binding modes and of the conformational flexibility of the enzyme.
All ligands interact with the main features of the catalytic machinery of the enzyme (Figure 3): (1) the
phosphate is sequestered by the positive end of a 310-helix and Lys13; (2) polar groups on the carbon
framework interact with His95 and Glu167, the catalytic electrophile and the catalytic base of the
enzyme, respectively; (3) the entire inhibitor is shielded from the bulk solvent by a flexible loop, which
normally closes over the substrate during catalysis to prevent phosphate elimination [56] (Figure 4). The
only exception to flexible loop closure is N-hydroxy-4-phosphono-butanamide. It binds to the enzyme
with the flexible loop in the open conformation because its size precludes loop closure. Thus, the
crystallographic binding studies point out that it should be possible to design two very different classes
of selective inhibitors: a class that binds to the enzyme in the closed loop conformation and one that
binds to the open loop conformation.
Selective inhibitor design in the case of TIM appears to be a formidable task. All residues within 10
of the active site are conserved [25]. This is also reflected in the similarity of the kinetic characteristics
between trypanosomal and human TIM: for T. brucei TIM, Km (glyceraldehyde-3-phosphate) = 0.25
mM, kcat = 3.7 105min-1 [57]; for human TIM, Km = 0.49 mM, kcat = 2.7 105 min-1 [25]. There are
significant differences in the surface protein of the two enzymes about 15 away from the substrate
phosphorus atom [58]. In a shallow cleft, T. bruceiTIM has Ala100-Tyr101, while the human
counterpart of these residues is His-Val (Figure 5). The cleft is formed by the flexible loop of one
subunit of the enzyme and a different loop originating from another subunit. When the flexible loop
changes its conformation from the closed to the open form the cleft widens substantially. Moreover, the
Ala-Tyr dipeptide becomes then directly accessible from the active site, the distance being about
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Figure 3
Schematic representation of the structure of a TIM monomer. Helices and strands
are labeled as H and B, respectively. The view is along the axis of the barrel, into
the active site. Key catalytic residues Lys13, His95, and Glu167 are shown along with
the helix that binds the substrate phosphate and the flexible loop that covers the
substrate during catalysis. Black dots indicate residues in contact with the second
monomer of the enzyme. (From Ref.24. Copyright 1991 by Harcourt Brace.)
10 instead of the 15 in the closed loop conformation of the enzyme. In any case, selective inhibitor
design for TIM appears to require de novo design as there are no leads known that interact with the AlaTyr region of the enzyme.
B. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)
Glyceraldehyde-3-phosphate dehydrogenase is a homotetramer that carries out the oxidative
phosphorylation of glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate. During this reaction
NADH is formed. Each subunit of the enzyme consists of two domains and has an NAD+ binding site.
The N-terminal domain anchors the adenosine portion of the cofactor while the nicotinamide portion is
involved in the catalytic reaction at the C-terminal domain. T. brucei
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Figure 4
Stereoview of superimposed TIM monomers, one with an open flexible loop (full black)
and another one with a closed flexible loop (open gray). The loop location is marked
by an asterisk. Note the proximity of the active site, indicated by the catalytic residues
Lys13, His95, and Glu167 (all sterofigures in this paper were drawn with
MOLSCRIPT [93]).
Document
Figure 5
Exploitable structural differences between T.brucei(full) and human (dashed)
TIM. The inhibitor 2-phosphoglycolate as observed in the structure of the human
enzyme indicates the location of the active site. Drug design targets are the T.brucei
Ala100-Tyr101, which are considerably different from their human counterpart
His-Val. (From Ref.25. Copyright 1994 by Cambridge University Press.)
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Figure 6
Stereoview of NAD binding by trypanosomal GAPDH (full black). For clarity
only 2 of the 4 subunits are shown. A substantial deviation of the protein backbone
occurs in human GAPDH (open gray, only one subunit shown) near the adenosine
part of the cofactor. The nearby cleft (marked by an asterisk) is important for
introducing selectivity in inhibitor binding and has therefore been termed selectivity cleft.
glycosomal GAPDH has been overexpressed in E. coli [59] while human erythrocyte GAPDH is
available from commercial suppliers. The sequences of the two enzymes are only 55% identical [60].
The crystal structure of the parasite enzyme was solved from Laue data at 3.2 resolution in our group
(Figure 6) and in a second crystal form at 2.8 [26]. The structure of the human muscle enzyme was
solved at 3.5 resolution in the group of the late Herman Watson [28]. Its resolution was improved to
2.3 in our group [26,29]. Both structures were of the holo-enzyme, i.e., the enzyme in presence of the
cofactor.
The active site and the nicotinamide-binding site of the two enzymes are very well conserved. This is
reflected in similar Km (glyceraldehyde-3-phos-phate) values of 0.15 and 0.17 mM for the trypanosomal
and human enzyme, respectively [61]. Surprisingly, the Km (NAD+) values differ by a factor ten: 0.45
mM for T. brucei and 0.04 mM for human GAPDH [61]. In view of the conservation of the
nicotinamide and pyrophosphate binding sites, the substantial difference in Km (NAD+) has to be
ascribed to the adenosine binding environment. Indeed, some of the residues embracing the adenine ring
of the cofactor are not identical. In the trypanosomal enzyme the adenine is sandwiched in between a
Thr in the back and a Met in the front. This Met is replaced by Pro and Phe in the human enzyme
(Figure 7). A second difference involves the residue flanking the C2 atom of the purine ring. It is a Val
in the trypanosomal enzyme, and Asn in the human enzyme. These differences apparently account for a
ten-fold lower affinity for the cofactor.
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Figure 7
Comparison of the binding modes of the adenosine moiety of NAD to GAPDH:
(left) in T.brucei,(right) in the human enzyme. Note the identical hydrogen bonds to
the purine N6 and the ribosyl hydroxyls. The purine ring is embraced by
hydrophobic residues that are not conserved. Also, a unique cleft near O2', which we
called the selectivity cleft, is present in the T.brucei enzyme. (From Ref. 13.Copyright
1994 by the American Chemical Society.)
Other differences in the vicinity of the adenosine portion of the NAD+ cofactor are prime targets for
selective inhibitor design. Close to C8 of the adenine ring, the trypanosomal GAPDH exhibits a Leu
while its human counterpart has a smaller residue, namely Val (Figure 7). Also, the parasite enzyme
possesses a hydrophobic cleft near the 2' -hydroxyl of the adenosine ribose. This cleft, termed the
selectivity cleft is almost absent in the human enzyme due to a different local backbone conformation
and the presence of the Ile37 side chain. In conclusion, the adenosine binding region looks like an
excellent target for selective inhibitor design.
It is exciting that the residues responsible for binding adenosine in T. brucei GAPDH are identical to
their counterparts in glycosomal GAPDH of Leishmania mexicana, another trypanosomatid [31]. L.
mexicana is one of the most common species of Leishmania throughout Central and South America and
the southern United States. In humans it hides as amastigotes in the macrophages and causes hideous
skin lesions. Together with about twenty other species of Leishmania these parasites infect about twelve
million people annually [63]. Though they are less dependent on glycolysis than T. brucei there is
evidence that stibogluconate, a well-known drug for treating leishmaniasis, specifically inhibits
glycolysis in these parasites [64]. Therefore, we decided to study GAPDH of L. mexicana in parallel
with the T. brucei enzyme. Its structure
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was recently solved at 2.8 resolution in our lab [30]. The kinetic parameters of the two enzymes are
virtually identical: Km (glyceraldehyde-3-phosphate) = 0.13 mM and Km (NAD+) = 0.41 mM for the L.
mexicana enzyme [62]. As expected, the structures of the two parasite enzymes are very similar: the rms
deviation for all backbone atoms is 0.7 and the adenosine binding environment is perfectly
superimposable except for Asn39 of T. brucei GAPDH, which is a Ser in the L. mexicana enzyme. In
this way drug design for one disease may have implications for another one.
C. Phosphoglycerate Kinase (PGK)
A monomeric enzyme, PGK transfers the acylated phosphoryl group from 1,3- bisphosphoglycerate to
ADP, thus forming 3-phosphoglycerate and ATP. The enzyme uses a metal ion as a cofactor, namely
Mg2+ [65]. The PGK enzyme from T. brucei has been overexpressed in E. coli [66]. Human PGK is not
Figure 8
Steroview of B.stearothermophilus PGK [69] in complex with ADP bound to the
C-terminal domain. To illustrate the sugar binding site, the 3-phosphoglycerate has
been added to the figure based on the crystal structure of pig muscle PGK [32].From
the distance between the two substrates it is obvious that during catalysis a
hinge-bending motion between the domains of the protein has to occur to bring the
substrates together.
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Table 3 Residues Involved in ADP Binding in Glycosomal and Human PGK
T.brucei
Human_1a
Human_2b
In contact with ADP moiety
Ala 242
Gly 238
Gly 238
Adenine
Tyr 245
Phe 241
Tyr 241
Adenine
Lys 259
Leu 256
Leu 256
Adenine
Ala 314
Gly 309
Gly 309
Adenine
Ser 378
Thr 375
Thr 375
-phosphate
aSomatic
bPGK
PGK [67].
in spermatogenic cells [68].
commercially available. However, its sequence has been determined [67] and appears to be 97%
identical to horse and pig PGK. For completeness it should be mentioned that there is a second human
PGK in testis tissue that is 87% identical to the somatic enzyme [68].
The crystal structures of the apo-enzyme from horse [31] and of the binary complex between pig PGK
and its substrate [32] (Figure 8) are available from the Protein Databank. The substrate was found to
bind to the N-terminal domain of the enzyme. The binding site for ADP is known from the structure of
its binary complex with PGK from B. stearothermophilus [69]. It resides in the Cterminal domain. Since
the substrate and ADP binding sites are 10 apart, a hinge-bending motion between the two domains
has been postulated to occur during catalysis [70].
Kinetically glycosomal PGK from T. brucei and mammalian PGK are very similar: the Km values for
ATP are 0.29 and 0.46 and mM, respectively; the Km values for 3-phosphoglycerate are 1.62 and 0.62
mM, respectively (due to the unavailability of the human enzyme the rabbit muscle enzyme was used as
a substitute) [71]. The residues responsible for binding the substrate [32] are identical between human
and glycosomal PGK [72]. However, five of the residues involved in the binding of ADP differ between
the two enzymes (Table 3). Apparently, the biggest difference between human PGK and the parasite
enzyme is the charged residue Lys259, which has the apolar Leu256 as a human counterpart. Molecules
that bind at the ADP binding site and specifically recognize Lys259 might therefore be good starting
points for drug design.
III. Search For New Leads
A. Triosephosphate Isomerase: The Crystallographic Cocktail Soak Approach
Document
Because there are no known leads that bind to the selectivity region of TIM, the design of selective
inhibitors is an exercise in de novo ligand design. We tried to
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design such molecules on the basis of the trypanosomal TIM structure by a linked-fragment approach
[57]. In that strategy small building blocks are designed to be complementary to the targeted surface of a
protein. Such fragments can then be synthesized or purchased, tested for their effect on enzyme kinetics
and for their binding mode by crystallography. Promising fragments are then linked together into larger
molecules. The idea behind this stepwise approach is to obtain early experimental feedback in the drugdesign cycle.
The crystallographic follow-up of our linked-fragment approach design for trypanosomal TIM was
disappointing. Two designed fragments were soaked into a crystal of trypanosomal TIM, namely 4hydroxy-2-butanone and D-asparagine. Despite high concentrations of these molecules in the mother
liquor, 220mM and 30 mM respectively, no convincing electron density could be seen in difference
Fourier maps calculated between 10.0-2.8 [72]. Common to both molecules is that they are fairly
polar, rather flexible, and were expected to displace crystallographically observed water molecules.
Apparently, de novo design of tightly binding small ligands is far from trivial.
We also tried to find new leads by a completely experimental approach. For that purpose the
crystallographic cocktail soak (CCS) approach was developed. In this method cocktails of fine
chemicals are soaked into a crystal in the hope of finding crystallographic evidence of binding for one of
the molecules from the cocktail. The identification of such a molecule might not be clear immediately
because several molecules in the cocktail might be compatible
with the shape of the electron density, especially if the resolution is not very high. An outcome would be
provided by a dichotomic approach (Figure 9), in which the crystallographic soaking experiment is
repeated with ever smaller subcocktails of the original one. For example, if a ligand shows up from a
cocktail soak of 32 compounds, a second experiment should be done with only half of the compounds. If
the ligand fails to show up, one knows that it is one of the alternative 16 compounds. After at most six
experiments the identity of the ligand is known. One might like to think of the CCS approach as the
experimental analog of the computational methods in programs like GRID [73] or MCSS [74], but with
thirty-two compounds at a time.
Figure 9
Dichotomic search for unknown ligand from
cocktail 1. The number of compounds in the sub
cocktail is indicated by n, and the
interpretation about the presence of compound
X in the subcocktail is given as Y/?/N.
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For trypanosomal TIM we experimented with three different cocktails of 32 compounds (Table 4).
Molecules were chosen in such a way that they would be compatible, soluble, cheap, and as varied as
possible. Each compound was present at a concentration of 1 mM. The final cocktail solutions were
clear and devoid of precipitate. Since this was a pilot experiment both subcocktails were checked at each
stage of the dichotomic strategy. Only the soak with cocktail 1 revealed electron density that could not
be accounted for by water molecules, hereafter called peak X. The soaks with cocktails 2 and 3 led to
featureless difference Fourier maps. The quality of the data and refinement can be inspected from Table
5, while Figure 9 illustrates the dichotomic search to identify peak X. An oxidized molecule of DTT,
identified in the high-resolution structure of the native TIM crystals [24], served as an internal reference
to judge the quality of the data and the noise level in the final difference Fourier maps.
Peak X was found near His95 of the second subunit of the enzyme, i.e., the subunit where the flexible
loop adopts the closed conformation in this crystal form. Its signal was somewhat weaker than that of
DTT. The same density showed up when crystals were soaked with subcocktail 1B but not with 1A,
narrowing down the list of potential ligands to sixteen compounds. However, the next round of the
dichotomic search led to a problem that has not been solved thus far. Peaks of roughly the same shape as
the original peak X appeared with both subcocktails 1BA and 1BB. Several strategies were followed to
improved the quality of the maps. First, the model was further refined with all data while a bulk solvent
scattering correction [75] was incorporated. Second, a variety of maps were calculated: (|Fo|-|Fc|) eic,
(2|Fo|-|Fc|) eic, (3|Fo|-2|Fc|) eic and (|Fo|-|Fo,native|)eic. Third, all maps were SIGMAA-weighted [76].
Since the shape of peak X varied substantially between the different maps it can be tentatively
concluded that peak X did not originate from the presence of a compound but was noise. The lesson of
this experiment seems to be that the crystallographic cocktail soaking approach should only be tried
when high- resolution data can be obtained, probably better than 1.8 resolution.
B. Glyceraldehyde-3-Phosphate Dehydrogenase: Docking
In order to discover new ligands that would block GAPDH of T. brucei by occupying the adenosine
binding region we used the program DOCK [77], version 3.5. This program characterizes a binding site
by filling it with a set of overlapping spheres. The centers of these generated spheres constitute an
irregular grid, called a graph by mathematicians. Docking of a ligand then consists of matching
subsets of ligand interatomic distances onto subsets of the receptor graph. Finally, the quality of the fit
between a docked ligand and the receptor is evaluated. Within DOCK 3.5 three methods are available
for this evaluation: contact scoring, which measures shape complementarity; force-field scoring, which
is an estimate of the enthalpy of the intermolecular interaction; and elec-
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Table 5 Crystallographic Cocktail Soaking Experiments of Trypanosomal TIM Crystalsa
res
DTT
a()
b()
c()
m()
()
R-sym
Compl
()
()
112.7(1)
97.7(1)
46.7(1)
0.7
2.85
0.044
0.97
0.136
5.0
3.5
1A
112.6(1)
97.6(1)
46.8(1)
0.9
2.50
0.099
0.96
0.173
7.0
2.0
1B
112.7(2)
97.5(3)
46.8(2)
0.7
2.30
0.044
0.88
0.172
5.0
4.0
1BA
112.6(2)
97.9(2)
46.7(2)
0.7
2.40
0.051
0.96
0.202
7.8
3.0
1BB
112.6(1)
97.8(1)
46.7(1)
0.7
2.30
0.034
0.90
0.203
5.0
3.0
112.9(1)
97.8(1)
46.7(1)
0.9
2.40
0.060
0.92
0.172
5.0
112.9(1)
97.7(3)
46.7(2)
0.7
2.40
0.057
0.93
0.170
4.0
aThe
following data are tabulated column-wise: C = cocktail; a, b, c = cell parameters of the P212121 crystals; m =
mosaicity; res = resolution; R-sym = agreement between symmetry-related reflections; Compl = completeness; R =
agreement between data and model; DTT = signal of oxidized DTT in the final difference Fourier map. DTT is
present in the mother liquor but not incorporated in the model. X = signal of peak X in the final difference Fourier
map. Maps were calculated with data between 10.0 and the high resolution limit.
trostatic scoring, where the linearized Poisson-Boltzmann equation is solved [78]. We report here on
docking experiments to identify GAPDH inhibitors from the Available Chemicals Directory-3D 93
(ACD) [80].
Force-field scoring and electrostatic scoring require the assignment of partial atomic charges.
Unfortunately, such charges are not available in the ACD, mainly because there is no consensus on a
method to calculate them. Since the number of molecules in the ACD is very large, about 73,000 in
1993, we opted for the charge-equilibration algorithm developed by Rapp and Goddard [81] as
implemented in the BIOGRAF program [82]. This method leads to charges that are in excellent
agreement with experimental dipole moments and with atomic charges obtained from electrostatic
potentials of accurate ab initio calculations. A script was written that processed the entire ACD
automatically on an R4000 processor in about two days. Charges of the protein atoms were assigned
from the table of AMBER-derived charges provided with DOCK 3.5.
Document
Because there are tens of parameters that can be varied in the program there is no such a thing as the
DOCK run for a given protein target. We chose to perform two parallel runs that differ in receptor
description. In run 1 the DOCK sphere center description was used while in run 2 the atomic coordinates
of 2'- deoxy-2'-(3-methoxybenzamido)adenosine, a designed selective inhibitor of T. brucei GAPDH
(see Section IV), were picked to describe the receptor site. For each run the same program parameters
were chosen (Table 6) and the ACD database was split up in batches of 10,000 molecules. The
computation was done on an Indigo2 workstation with an R4400 processor operating at 175 MHz. It
took 3 h 19 min of CPU time for run 1 and 34 h 48 min for run 2. The ten-fold time difference between
the two runs originates from the different number of
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Table 6 Parameters Chosen for the DOCK Runs with T. brucei GAPDHa
Program
Variable
SPHGEN
DISTMAP
aVariables
Value
Program
dentag
DOCK
dotlim
Variable
Value
distance_tolerance
1.0
0.0
nodes_maximum
radmax
5.0
nodes_minimum
radmin
1.0
ligand_binsize
0.4
polcon
2.3
ligand_overlap
0.1
ccon
2.8
receptor_binsize
0.8
discut
4.5
receptor_overlap
0.2
perang
atom_minimum
as defined in the DOCK 3.5 manual and discussed in References 79 and 83.
centers to describe the receptor, namely 20 for run 1 and 34 for run 2. According to theory [83], the
difference should scale as 344/204 = 8.3, which fits our observation. Since it is well known that scores
exhibit poor correlation with real affinities [84] we decided to subject the 200 best-scoring ligands of
each batch to inspection on the graphics. Scanning through the 3200 compounds required about ten
days. Eventually, sixteen compounds were selected for purchase on
Table 7 Parasite GAPDH Inhibitors Discovered with DOCK
Contact
Inhibitor
IC50 (mM)
Delphi
Score
Rank
Score
Rank
Run 1
2-Guanidinobezimidazole
1.2
97
1432
1.01
1427
2-Benzimidazoylurea
1.8
102
958
0.27
1241
4-Nitrophenyl sulfone
2.8
106
608
-0.46
370
Tryptophan
6.0
128
32
-0.48
357
Document
5-Methoxytryptamine
19.0
92
1495
-0.32
488
Ephedrine
25.0
99
1315
-0.22
590
Epinephrine
>7a
102
989
-3.09
27
Aspartic acid dimethyl ester
>10
87
1551
-1.66
146
3-Amino-L-tyrosine
>11a
102
969
-0.14
703
1,3-Diphenylguanidine
>11a
112
301
-1.28
209
Octopamine
>50a
106
660
-2.03
83
4.4
132
900
0.11
1112
12.0
143
241
-2.02
156
Norepinephrine
>5.4a
151
84
-2.09
137
4'-Amino-N-methyl acetanilide
>7.8a
131
1027
0.04
1021
>9.3
142
300
0.13
1156
Run 2
2-Nitrobenzoic acid hydrazide
Dopamine
L-histidinol
aCould
not be tested at higher concentrations due to solubility problems.
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Figure 10
Binding mode of 2-guanidinobenzimidazole to T.brucei GAPDH as predicted by
DOCK. The benzimidazole moiety occupies roughly the position occupied by adenine
in the holo-enzyme, whereas the guanidino group a salt bridge to Asp37.
the basis of structural rigidity, chemical stability, solubility, and electrostatic complementarity. The
latter property was evaluated with the program DELPHI [85].
Each of the sixteen compounds was tested for GAPDH inhibition (Table 7). Half of them were inactive
while the other ones showed inhibition in a range between 1.2 and 25 mM. Unfortunately, there appears
to be no correlation between the DOCK scores and the IC50 values. For examples, norepinephrine and
1,3-diphenylguanidine are inactive while they have a better score than 2- guanidinobenzimidazole
(Figure 10), the compound with the best IC50. Also, it appeared that the two different receptor
descriptions used led to almost completely different lists of compounds. Only 156 molecules occurred in
both lists of top-scoring molecules. In the modeled binding mode all of the inhibitors occupy roughly the
same position as the purine ring of NAD in the crystal structure of GAPDH. While the values obtained
for IC50 are indicative of poor inhibition, one has to keep in mind that adenosine exhibits an IC50 of 50
mM [13]. By using the program DOCK we were able to discover ligands that have a substantially higher
affinity for GAPDH than the natural ligand.
C. Phosphoglycerate Kinase: Leads from the Past
The starting point for drug design in the case of PGK is quite different from TIM or GAPDH because a
number of nonsubstrate-like inhibitors have been
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Figure 11
Two-dimensional structure of SPADNS, a
micromolar inhibitor of T.brucei PGK.
discussed in the literature. Suramin inhibits glycosomal PGK of T. brucei with a Ki of 8.0 M [69]. In
addition a number of yeast PGK inhibitors are known: gallic acid with a Ki = 0.4 mM [86],
hydroxyethylidene biphosphate with a Ki = 24 mM [87]. 1,3,6-naphthalenetrisulphonic acid with a Ki =
5.5 mM, and 2-(p- sulphophenylazo)-1,8-dihydroxy-3,6-disulphonic acid, also known as SPADNS
(Figure 11), with a Ki = 126 M [87]. None of the four yeast PGK inhibitors are potent, but, for
SPADNS, the binding mode has been further characterized. Studies by Williams et al. [87] have
demonstrated that SPADNS is directly competitive with both enzyme substrates, 3-phosphoglycerate
and ATP. Moreover, by 600 MHz 1H-NMR it was shown that SPADNS interacts with the nucleotide
binding site while the conformation of the enzyme changes substantially [87].
Since the four yeast PGK inhibitors are commercially available it was logical to test them for T. brucei
PGK inhibition. The first three compounds were active in the millimolar range. However, SPADNS
exhibited a Ki of 10.0 M in these in preliminary tests [88]. Moreover, when assayed against a
commercially available rabbit muscle PGK, SPADNS had no influence on the enzyme kinetics up to a
concentration of 250 M [88]. In conclusion, SPADNS appears to be an excellent lead because of its
potency and selectivity. Crystallographic experiments to determine its binding mode to T. brucei PGK
are underway.
IV. Lead Optimization: Glycosomal Gapdh
From the selectivity point of view the adenosine binding site of GAPDH is attractive for drug design, as
we explained in Section II.B. Unfortunately, inhibition studies on T. brucei and L. mexicana GAPDH
revealed the poor affinity of our natural lead adenosine with IC50 values of 100 mM and 50 mM,
respectively. Moreover, adenosine is an antiselective lead because the IC50
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for human GAPDH is better than for the parasite enzymes, namely 35 mM [13]. Despite the fact that
these IC50 values are about ten thousand times higher than what would be considered a lead in the
pharmaceutical industry we decided to optimize the affinity and selectivity of adenosine.
Each of the three areas where differences occur between the parasite and the human enzyme are
hydrophobic. Therefore, we modeled hydrophobic substituents at positions C2, C8, and O2' of adenosine
under the constraint that they were conformationally compatible with the C2'-endo pucker of the ribose
sugar. Designing derivatives at O2' was a problem, however. Each of the two ribosyl hydroxyls forms a
hydrogen bond with the carboxylate of Asp37. Since making direct derivatives of the hydroxyl, such as
ethers or esters, would deprive the Asp of a hydrogen-bond partner while burying the carboxylate,
resulting molecules would have a dramatically reduced affinity. Moreover, an alignment of 47 GAPDH
sequences made it clear that the Asp is highly conserved [89]. An elegant way to overcome this problem
was to replace the 2' -hydroxyl by a 2'- amino function. Moreover, coupling with carboxylic acids was
appealing from a synthetic point of view while the conformational properties of the amido-substituted
system would ensure the correct orientation of substituents into the selectivity cleft. The modeled
inhibitors were evaluated for the quality of their fit to the protein surface and subsequently synthesized.
From Table 8 it can be seen that our predictions were successful. The addition of a methyl group at C2
of the adenine ring, which is close to Val36, increased the affinity for parasite GAPDH by an order of
magnitude. The effect of a thienyl substitution on C8, targeted to Leu112, was even bigger, namely two
orders of magnitude. However, both substitutions are only mildly selective (Table 8). As expected, the
greatest gain in selectivity was obtained by modifying the 2'-position of the ribose, so that the selectivity
cleft is filled up (Figure 12). The 2'-deoxy-2'-(3-methoxybenzamido) adenosine compound (Figure 13)
bound at least 48 times better to L. mexicana GAPDH than to the human enzyme. The selectivity versus
T. brucei GAPDH appeared to be smaller. This has to be ascribed to a difference in residues contacting
the 3-methoxy moeity. The residue Asn39 of T. brucei GAPDH has a Ser equivalent in the L. mexicana
Table 8 Inhibition Gains of Designed GAPDH Inhibitors with Respect to Adenosine
C2-subst
C8-subst
CH3
T. brucei
L. mexicana
human
OH
12.5
6.25
<3.5a
thien-2-yl
OH
167
100
27
NHCO-phenyl
16.7
NDb
3.5
NHCO-(3-OCH3-phenyl)
45
167
<3.5a
aUpper
bNot
C2'-subst
limit because solubility problems did not allow for an IC 50 determination.
determined
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Figure 12
Predicted binding mode of 2' -deoxy-2'-(3-methoxybenzamido)adenosine to T.brucei
GAPDH. (From Ref. 13. Copyright 1994 by the American Chemical Society.)
enzyme. In conclusion, the strategy of burying hydrophobic residues with lipophilic substituents paid
off.
Despite the rather poor IC50 values of our optimized inhibitors, an evaluation of their effect on live
trypanosomes appeared to be useful. Enzyme inhibitors that are not taken up by the parasites would be
of no use as a drug. Therefore, the effect of 2-methyl-adenosine, 8-(thien-2-yl)-adenosine and 2'- deoxy2'-(3-methoxybenzamido)adenosine on the growth of T. brucei in cultures, as described by Baltz et al.
[90], was monitored. At 0.1 mM all compounds inhibited the growth completely, unlike adenosine
derivatives that were without inhibitory effect against T. brucei GAPDH [91]. Experiments are
underway to confirm that the growth inhibition is due to blockage of the glycolytic pathway. Also, te
mechanism of uptake of te inhibitors will be examined because it is now well established that
trypanosomes possess a unique P2 purine transporter that they use for uptake of purines from the host
[15]. The experimental antitrypanosomal drug 5'-{[(Z)-4-amino-2-butenyl}methylamino}-5'deoxyadenosine(MDL 73811) Figure 13), which is an irreversible S- adenosyl-L-methionine
decarboxylase inhibitor, is actively taken up through the P2 transporter. Moreover, MDL 73811 is not
actively transported in the human host, which presumably contributes to the drug's selectivity [19]. It is
not unthinkable that our inhibitors might use the same transporter because of the nature of their scaffold,
adenosine.
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Figure 13
Two-dimensional structures of 2'-deoxy-2'-(3-methoxybenzamido)
adenosine (MMBA), a selective T.brucei GAPDH inhibitor and
MDL 73811, an irreversible
inhibitor of trypanosomal S-adenosyl
methionine decarboxylase.
Finally, we want to point out that pessimism about adenosine derivatives as drugs is not necessarily
warranted. This doubt stems from the argument that many proteins recognize NAD(P), adenosine, or
ATP. Cross-reactivity of adenosine with these different proteins and, therefore, toxicity may be
expected. That this is not necessarily so is evident from the use of adenosine derivatives as antileukemia
agents. For example, fludarabine, a C2'-epimer of adenosine, exhibits relatively low toxicity [92]. The
much bigger changes to the adenosine scaffold in our inhibitors may hence lead to a surprisingly high
overall selectivity.
V. Conclusions
Our goal is to discover and design selective inhibitors of trypanosomal glycolsysis. Thusfar, three
enzymes have been targeted. Whereas little success was obtained with TIM, substantial progress is being
made with GAPDH and PGK.
Obstacles encountered during this project were the need for selective inhibitors and the absence of
potent inhibitors as lead compounds. From our studies to inhibit TIM it appears that it is difficult to
come up with inhibitors for areas on the protein surface for which no known inhibitors exist. On the
other hand, lead optimization for GAPDH by over two orders of magnitude in just one cycle of drug
design was straightforward. Selectivity was obtained by using part of the cofactor as a lead and
exploiting the hydrophobic patches at the surface of the parasite enzyme. In particular, 2'-deoxy-2' (3methoxy-benzamido) adehttp://legacy.netlibrary.com/nlreader/nlReader.dll?bookid=12640&filename=Page_387.html (1 of 2) [4/5/2004 5:42:15 PM]
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nosine proved to inhibit the parasite more than fifty times better than the human enzyme. Additionally,
by means of the program DOCK, eight new leads for GAPDH inhibition were found. None of them
were micromolar inhibitors but all of them were more potent than the natural lead, adenosine. For
trypanosomal PGK a potent lead compound, SPADNS, was discovered by testing inhibitors that had
been described as weak inhibitors of yeast PGK. Moreover, this lead had no effect on mammalian PGK
at concentrations up to twenty-five times higher than that needed for T. brucei inhibition. At present,
none of our inhibitors is potent enough to consider clinical tests. There is hope, however, since our
GAPDH inhibitors inhibit the growth of trypanosomes in cultures.
Acknowledgments
It is a pleasure to thank the many colleagues and collaborators who have contributed to this project: Paul
Michels, Veronique Hannaert, Sylvie Allert, and Linda Kohl (Institute for Cellular Pathology in
Brussels) for cloning and overexpressing trypanosomatid enzymes; Phil Petra (University of
Washington, Seattle) for helping us out with protein purification protocols; Mia Callens and Fred
Opperdoes (Institute for Cellular Pathology in Brussels) for enzymology and parasitology; Rik
Wierenga, Martin Noble, Fred Vellieux, Randy Read, Risto Lapatto, Hillie Groendijk, Tjaard Pijning,
and Kor Kalk for laying the structural foundation of the project in Groningen and Heidelberg; Cees
Witmans and the late Alan Horn (University of Groningen), Michle Willson and Jacques Peri
(University of Toulouse), Serge Van Calenbergh, Arthur Van Aerschot, and Piet Herdewijn (University
of Leuven) for synthesizing TIM and GAPDH inhibitors; Kim Simons (University of Washington) for
going after completely new GAPDH inhibitors; Vronique Mainfroid and Joseph Martial (University of
Lige) for providing human TIM; Klaus Muml;uller and Klaus Gubernator (Hoffmann-La Roche, Basel)
for valued modeling advice; and Mike Gelb (University of Washington) for valued discussions.
Financial support for these investigations has been provided by the World Health Organization,
Hoffmann-LaRoche in Basel, the Dutch Organization for the Advancement of Science (NWO), the STD
program of the European Community, the School of Medicine of the University of Washington, and the
Murdock Charitable Trust.
Note Added in Proof
We just solved the ternary structure of PGK from Trypanosoma brucei in complex with ADP and 3phosphoglycerate. The enzyme is in the closed conformation that has eluded crystallographers for 20
years.
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16
Progress in the Design of Immunomodulators Based on the Structure of
Interleukin-1
Glen Spraggon
Pandi Veerapandian
Axiom Biotechnologies, Inc., San Diego, California, and
La Jolla Institute for Experimental Medicine, La Jolla, California
I. Introduction
The interleukin-1 (IL-1) family of cytokines exhibit both normal and pathological effects in almost
every tissue and organ system and, as such, have been associated with cells engaged in the immune
response, inflammatory cells, and cells engaged in development, differentiation, and repair processes
[14]. Interleukin-1 can produce either a direct response on one specific target cell or act as an indirect
effector molecule, inducing the expression of a variety of genes and synthesis of several proteins such as
IL-2IL-8, tumor necrosis factor (TNF), colony-stimulating factors (CSF), platelet-derived factors
(PDFs), and other cytokines. Pathologically uncontrolled, IL-1 activity can induce disease states and has
been linked to septic shock, the growth of acute and chronic myelogenous leukemia cells, inflammation
associated with arthritis and colitis, development of atherosclerotic plaques, insulin-dependent diabetes,
osteoporosis, parsitemia, and cancer [1,5,6]. Strategies to treat such diseases are being developed and
usually involve the inhibition of IL-1 synthesis or the blocking of its activity [7]. The therapeutic
advantage of reducing the activity of IL-1 resides in preventing its deleterious biological effects without
interfering with homeostasis. In order to achieve such a goal, an understanding of the structure-function
relationship of the IL-1 family of proteins at the molecular level is important. Such a knowledge could
lead to the design and development of
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synthetic agents with the ability to modulate IL-1 responses. Here we review the present knowledge of
the IL-1 system and the attempts toward the development of its modulators.
A. Cytokines and Physiological Responses
Cytokines are multifunctional hormones produced by a variety of cells to carry out a wide range of
overlapping biological actions including communication within the immune system and between other
cell types. They are produced by macrophages, endothelial cells, fibroblasts, keratinocytes, T cells, B
cells, and natural killer cells in response to injury and infection [7]. Upon production they act either
locally or as systemic intercellular signaling factors and induce the production of other cytokines for
continuing their communications via cell surface receptors. The cytokines network also has associated
with it a complementary set of soluble or membrane-bound antagonist or mediator molecules that are
capable of shutting down these effects [8]. During infection and injury, the leukocytes adhere
themselves to the endothelium and migrate into tissues. Cells like T-lymphocytes, monocytes,
macrophages, and neutrophils collect themselves as inflammatory infiltrate. Once within the tissues,
these cells neutralize the microorganisms or infected cells, secreting other cytokines that can modulate
the adhesion molecule expression on the endothelium. In addition, chemotactic signals are delivered to
cells passing in the circulation. Cytokines are also involved in tissue repair, i.e., remodeling of
connective tissues and revascularization of damaged areas. Soluble mediators are released from the site
of damage into the circulation to act in an endocrine fashion. The cytokines then control hepatic
responses to tissue damage. During the regulation of hematopoiesis, cytokines induce the production and
release of a number of colony-stimulating factors and other interleukins. Thus induced factors are
responsible for the replacement of leukocytes and erythrocytes that were lost after trauma.
A large number of cytokine molecules have been characterized [7]. Structurally it appears that these
cytokines can be classified into the following main groups based on their folding pattern: 4-helix
bundles, beta-trefoil, beta sandwich, EGF-like, beta cysteine knot, and alpha/beta. Thus far the structures
of many representatives of each family have been solved by both x-ray crystallography and NMR
(Table 1). Extensive experimentation has indicated that despite a large degree of structural diversity,
there is a large degeneracy in the cytokine network and the molecules have a wide range of overlapping
biological functions. For example, IL-1, TNF, PDGF, and TGFB all exhibit similar biological behavior.
The structures of the receptors for these molecules have not been forthcoming and only three examples
of the extracellular domains of the receptors have been reported. They are growth hormone receptor in
complex
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Table 1 Known Three-Dimensional Structures of Cytokines
Molecule
Brookhaven
Structure
Code
Determined by
Type
Interleukin-1 alpha (IL-1)
Beta trefoil
2ILA
X-ray crystallography
Interleukin-1 beta (IL-1)
Beta trefoil
1ILB, 2ILB, 4ILB,

5ILB, 6ILB
X-ray crystallography and

NMR
Interleukin-1 receptor antagonist

(IL-1ra)
Beta trefoil
1ILR, 1IRP
X-ray crystallography and

NMR
Fibroblast growth factor (acidic)

(aFGF)
Beta trefoil
1AFC
Fibroblast growth factor (basic)

(bFGF)
Beta trefoil
1BFG
Granulocyte-colony stimulating
factor (G-CSF)
Long-Chain 4 helix
bundle
1RHG, 1GNC
Growth hormone
Long-Chain 4 helix
bundle
1HGU
Leukemia inhibitory factor (LIF)
Long-Chain 4 helix
bundle
1LKI
Granulocyte-macrophage colony
stimulating factor (GM-CSF)
Short-Chain 4 helix
bundle
1CSG, 1GMF
Interleukin-2 (IL-2)
Short-Chain 4 helix
bundle
3INK
Interleukin 4 (IL-4)
Short-Chain 4 helix
bundle
1BBN, 1ITM,
2CYK
NMR
Interferon gamma (IFN-)
Dimeric 4 helix bundle
1IKI
Dimeric 4 helix bundle
1ILK
Nerve growth factor (NGF)
Beta cysteine knot
1BET, 1BTG
Platelet derived growth factor

(PDGF)
Beta cysteine knot
1PDG
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Beta cysteine knot
1TFG, 2TGI
Transforming
growth factor
alpha
(TGFA)
Beta EGF-like
2TGF
NMR
Tumour
necrosis
factor
(TNF)
Beta sandwich
1TNF
Macrophage inflammatory protein

1-beta
Alpha/Beta
1HUM
NMR
Alpha/Beta
1IL8
NMR
Melanoma growth stimulating

activator (MGSA)
Alpha/Beta
1MGG
NMR
Platelet factor 4
Alpha/Beta
1RHP
Transforming growth factor beta2

(TGFB2)
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with growth hormone, prolactin receptor (PRLR), and tumor necrosis factor with a ligand. In the case of
the human growth hormone-receptor complex and tumor necrosis factor-receptor complex the
interaction between the ligand and the receptor is intricate and takes place over a large area. It is likely
that this will be a characteristic feature of all cytokine-receptor complexes.
B. Cytokine-Based Therapy
Certain disease states can occur due to a disregulation of the cytokine network. This can lead to chronic
stimulation of the immune and inflammatory response and ultimately to disease. Many cytokines have
been implicated in autoimmune diseases like myasthenia gravis, insulin-dependent diabetes,
atherosclerosis, systemic lupus erythematosus, and rheumatoid arthritis [1,7,9]. The intimate relationship
between cytokines and the pathology of disease development and progress can be exploited to provide
therapeutic benefit. By subtly manipulating the cytokine communication network one can modulate the
disease process. Understanding of the functional roles of cytokines that mediate the communication
between them and the factors involved in the immune system's cell-cell interactions forms the
foundation for cytokine therapies. Such therapeutic agents are now a possibility due to modern
techniques such as molecular biology, structural biology, high-power computation, combinatorial
chemistry, and functional screening. The findings from biological techniques in conjunction with the
structural insights as obtained through protein crystallography and nuclear magnetic resonance form the
foundation for rational drug design [1013]. Discovery of IL-1-based therapeutic agents have led to
promising applications in the treatment of the above mentioned diseases. Design of synthetic adjuvants,
for exogenous immunomodulation, is also an important factor to be considered in the field of vaccine
development. In this review we will discuss the available literature on interleukin-1 and the recent
attempts toward the design of immunomodulators.
II. The Interleukin-1 Family
The three molecules of the IL-1 family, interleukin-1 (IL-1), interleukin-1 (IL-1), and interleukin-1
receptor antagonist (IL-1Ra) map to the long arm of chromosome two in the human genome. It appears
that the family arose via a gene duplication event some 350 million years ago, and the molecules possess
between 27.5 and 36% sequence identity with each other (Table 2) [1,14,15]. In addition, the genes for
the two IL-1 receptors IL-1R1 and IL-1RII [16,17], and an IL-1R accessory protein (IL-1RacP), which
binds to the IL-1, IL-1 receptor complex [18], have been identified. Together, these molecules via their
differential activity serve primarily to modulate the host defense mechanism.
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Table 2 RMS Distance of Aligned C Moieties of the Three Interleukin-1 Structures
IL-1
IL-1Ra
IL-1
IL-1
0.0
1.8 (36.3%)
1.42 (30.7%)
IL-1
1.8 (36.3%)
0.0
1.8 (27.5%)
IL-1Ra
1.42 (30.7%)
1.8 (27.5%)
0.0
Number in parentheses referred to sequence identities between the aligned structures.
A. Expression and Processing of IL-1

All three IL-1 molecules, IL-1, IL-1, and IL-1Ra are synthesized as 31 kDa precursor molecules
produced primarily by mononuclear phagocytes. These precursor proteins can be subsequently
processed to mature 17 kDa molecules. The means whereby this is achieved appears to be different for
each type of molecule and may help to explain the roles of the three. Both agonist molecules IL-1 and
IL-1 lack a classical hydrophobic leader sequence and thus must be processed in an alternative way.
The two can be separated by their biological activity in the precursor form, IL-1 being active in both
precursor and mature forms while IL-1 produces a biological response only in its processed form. In
addition, a specific enzyme, Interleukin-1 beta converting enzyme (ICE), cleaves IL-1 to its mature
form. This enzyme is a cysteine protease whose only known substrate is proIL-1, which it cleaves at
Ala 117 [19,20]. The means whereby pro IL-1 is processed is still largely a mystery although it has
been postulated that a calpain or related protein may perform the task.
Naturally occurring IL-1Ra is a 22 kDa glycosylated protein [14,15,21 24] that possesses a signal
sequence. It is likely that it is processed in the conventional manner as can be concluded by the
glycosylation states of the molecule not present in the agonists [25]. The LPS-stimulated human blood
monocytes initially express the gene for IL-1Ra [25]. An alternatively spliced form of IL-1Ra also exists
(intracellular IL-1Ra), which remains inside the cell presumably to block intracellular IL-1 action [26].
Both soluble and intracellular forms of IL-1Ra block IL-1R but do not trigger any biological response.
B. Receptors and Responses
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All effects of the IL-1 family are exerted via their receptors, which are present on a variety of different
cell types. There are two known IL-1 receptors. The first, type I (IL-1RI) is an 80-kDa formation found
mainly on T cells and fibroblasts; the second, type II (IL-1RII) is present on B cells, monocytes,
neutrophils, and heptoma cells and is approximately 60 kDa in size. From sequence analysis these
molecules are believed to belong to the Ig-superfamily, the extracellular portion of the receptors
consisting of three immunoglobulin-like domains of approximately 100 residues each [27,28]. The IL1RI receptor
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Figure 1
Schematic representations of the members of the IL-1 family.
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has a single membrane-spanning region and a cytoplasmic region of 213 amino acids. The IL-1RII
receptor has a single membrane-spanning region and a cytoplasmic region of 29 amino acids.
All three IL-1 molecules bind with a high affinity to the receptor IL-1RI. However only IL-1 and IL1 produce a detectable response [29]. The IL-1Ra molecule completely blocks the binding of the
former two molecules without inducing any signal transduction events. The second IL-1 receptor, IL1RII, similarly binds IL-1 tightly but does not produce any signal [30]. The soluble portion of IL-1RI
binds to IL-1Ra, IL-1, and IL-1 in decreasing order of affinity whereas the soluble portion of IL-1RII
binds IL-1, proIL-1, IL-1, and IL-1Ra, in that order. It therefore appears that both IL-1Ra and IL1RII take part in modulation of the agonist molecules, an argument supported by the finding that soluble
IL-1RII binds IL-1 with a similar affinity to the cell-associated receptor; whereas, the affinity of IL1Ra to such a soluble receptor is some 2000 times less [31,32,33]. Greenfeder, et al., [18] have
identified a new molecule, the IL-1 accessory protein IL-1RacP. The complex of IL-1 and IL-1 receptor
binds to IL-1RacP and together they induce the signal [18]. Greenfeder also observed that antibodies to
IL-1RI and to IL-1RacP block IL-1 binding and activity. From sequence analysis it appears that the
cytoplasmic domains of IL-1R and IL-1RacP contain the same amino acid domains commonly found in
the members of the GTPase family of proteins [34]. It has been proposed that such a complexation may
lead to a closer proximity of these cytoplasmic domains and thus facilitate signal transduction. A scheme
showing functional relationships between molecules that make up the IL-1 systemIL-1, IL-1, IL1Ra, IL-1RI, IL-1RII, and related receptorsis shown in Figure 1.
C. Autoantibodies of IL-1
In addition to these molecules, naturally occurring neutralizing autoantibodies of IgG type to IL-1 have
been identified. These have been detected in serum isolated from human donors. [35,36]. These
antibodies bind to both proIL-1 and 17-kDa IL-1 [37] and completely prevent the binding of IL-1 to
type-I cell surface receptors [38]. Patients with autoimmune diseases have higher populations of these
antibodies [39].
III. Three-Dimensional Structural Information
In an effort to understand the natural and interactions of the IL-1 family, various laboratories have
undertaken the task of elucidating the three-dimensional structure of the molecules. To the present this
has resulted in the structures of all three members of the IL-1 family being solved independently by both
x-ray
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crystallography and heteronuclear NMR spectroscopy [4049]. In addition to these, the structure of IL1 converting enzyme has been determined [50]. The molecule is an oligomeric cysteine protease
consisting of 10- and 20-kDa subunits. To date proIL-1 is its only physiological substrate. As
processing is essential for IL-1 extracellular transport and activity, inhibitors of ICE could provide a
method to block the production of the active form of IL-1. Although the elucidation of these structures
have been important in determining various differences among the individual proteins, perhaps a more
important result would be the elucidation of the structures of the two interleukin-1 receptor molecules,
either individually or in complex with their substrates. This information could provide a means to draw
together the present structural and biochemical knowledge into a coherent picture of the agonistic
activity of IL-1 and IL-1 as opposed to the antagonist effect of IL-1Ra. The structures should also
provide a foundation upon which structure-based drug design could proceed. To date no crystals or
structural reports for the receptors have been published.
A. The -Trefoil Fold
Structurally IL-1 exhibits a unique fold, known as a -trefoil fold. Each IL-1 molecule show the
characteristic -trefoil fold and small deviations of the back-bone despite the relatively low sequence
identity (Table 2). Figure 2 shows the structural alignment of the IL-1 molecules. The -trefoil fold was
first observed in Kunitz-type soybean trypsin inhibitor [51]. The fold has since been identified numerous
times in the Kunitz family of protease inhibitors, the interleukin-1 system molecules, and the acidic and
basic fibroblast growth factors [52]. Although these proteins have diverse biological function and low
sequence identity to each other, all appear to have a common structural core. Each one, however, binds
to its specific receptor with high affinity. The present known examples of proteins with such a fold are
composed of between 125 and 170 amino acids. The overall fold itself is an antiparallel barrel
consisting of six two-stranded hairpins. Three of these form a barrel structure (strands denoted 1, 4,
5, 8, 9, and 12) while the other three are in a triangular array that caps the barrel. The arrangement
of these moieties is such as to give the molecule a pseudo-three-fold axis [52]. Each fragment
contributes one pair of antiparallel beta strands to the barrel and one pair to the cap of the barrel, thus
forming a so called open and closed end to the structure [42].
B. Three-Dimensional Structure of IL-1
The structure of IL-1 was the first of the IL-1 family to be solved and has been solved independently
five times: four times by x-ray crystallography [40
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Figure 2
Structural alignment of IL-1, IL-1, and IL-1Ra. Residue numbering is taken from IL-1. Residues bordered in black are conserved
over the three molecules while those in gray constitute a conservative substitution. Arrows indicate sheet region, and the cylinders
indicate helix. Figure produced by Stamp [109] and Alscript [110].
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Figure3
(a) Stereo diagram of the secondary structural elements of IL-1. Produced by Molscript [106].
(b) Stereo plot of all the atoms of IL-1 viewed parallel to the axis of the barrel.
42,44], and once by NMR [43]. The four crystal structures, each to 2.0 , were all solved in the same
space group, P43, each structure being in relatively good agreement with the others [53]. As pointed out
previously, the molecule adopts a -trefoil fold with about 65% of the molecule in beta sheet and 35% in
random coil/turn (Figure 3a). The IL-1 molecule resembles a conical barrel with a shallow open face
on one end and a closed face on the other. The length of the long tubular core of the molecule is about
23 . Twelve antiparallel
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strands constitute the secondary structural elements of IL-1. Three pairs of strands (one pair from
each of the fragments) form the six-stranded barrel and the other three pairs cover one end of the barrel,
referred to as the closed end. The amino and carboxy termini are close to each other at the open end
of the barrel (Figure 3b). The overall structure of the molecule consists of three similar fragments (F1,
F2, F3) related by a pseudo-3-fold symmetry, with each fragment having a L motif. Residues 1 to
52 (F1), 53 to 107 (F2), and 108 to 153 (F3) form these fragments. These L motifs can be
superposed to display structural similarities.
The core of the molecule consists entirely of hydrophobic residues, two-thirds of which are leucines and
phenylalaninesa feature that seems to be essential to maintain the structural integrity of the molecule.
Most of the aromatic groups have their planes aligned along the barrel axis and both ends of the barrel
have concentrations of exposed polar residues that may be involved in binding interactions with the
receptor (see below). No alpha-helical structure is observed in IL-1 although one short region of 310
helix is observed between residues Gln34 and Gln38. Two of the hairpins are in the open end and
three are at the closed end. Residues 18 to 28, 69 to 82, and 122 to 135 form the three hairpins at the
closed end where three strands (residues 2428, 7882, and 129135) are in close proximity and form
three sides of a triangle, covering this end of the barrel.
C. Open End of the Barrel
Analysis of the surface of IL-1 reveals an epitope consisting of many polar residues widely spaced,
approximately in an annular fashion, around the open end of the barrel (Figure 3b). It has a broad
surface area containing many polar residues [42]. Residues from six -turns [type I, turn 1 (T1=1117); type III, turn 3 (T3=33-36); type I', turn 6 (T6=52-55); type I, turn 7 (T7=62-65); type I,
turn 9 (T9=86-89); type I', turn 10 (T10=106-109)] are present in this open end. There is a bulge
between strands 4 and 5 in one of the hairpins; this bulge may play a key role in the formation of the
putative binding surface. The bulge in IL-1 is of the wide type stabilized by multiple interactions,
whose conformation is such as to place the side chains of polar residues (Glu51, Asn53, Asp54) in the
proposed binding surface, fanning out in the direction of the open end of the barrel. Multiple interactions
serve to stabilize the strained conformation of the loop, between residue 86 and 94, presenting the side
chains of Asp86, Lys88, Asn89, and Lys93 at the open end of the barrel. In well-defined residues having
abnormal , values, strained conformations often may be related to functional properties and are
observed either in the active site or in the region responsible for its activity. The reason for having such
strains in the loop at this open end may be to place these charged side chains in the putative receptor
binding surface. There is a short 310-helix in the region between 3 and
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4 (residue Gly33 and Gln39). By such a conformation, residues Gln32, Gln34, and Glu37 have their
side chains in the proposed binding surface. A turn between 62 and 65 is folded in such a way that the
charged groups of the residues Lys63, Glu64, and Lys65 present themselves on the proposed binding
surface. Conformational arrangement via saltbridge scaffolds and other multiple hydrogen-bonding
interactions stabilize the residue 102 to 113 loop, and orient the side chains of Lys103, Glu105, Asn107,
and Asn108 in the proposed surface at the open end of the barrel. Similar arrangement places the side
chains Arg11 and Gln15 in the proposed epitope. The amino and carboxy termini form a part of the
proposed epitope, contributing the polar side chains of Arg4, Ser5, Asn7, Gln149, Ser152, and Ser153.
Based on extensive analysis, we found that the structural elements like electrostatic and hydrogenbonding interactions in the loops between strands allow the polypeptide to adopt a conformation that
enables an unusual concentration of polar and charged groups to be presented at the open end of the
barrel. Such a cluster of charged residues around an area that is almost perpendicular to the barrel axis
forms a hydrophilic patch with which IL-1 might bind to the receptor [42]. The core of the barrel,
though probably not involved in binding, must nevertheless be important to the function of IL-1 because
mutations within it reduce activity but not binding. We hypothesized that binding and cell proliferation
through signal transduction involve separate regions of the IL-1 molecule; the surface polar loops are
required for the binding and the core of the barrel is required for the physiological response.
D. Three-Dimensional Structure of IL-1
The structure of IL-1 has been determined by x-ray crystallography [45] to a resolution of 2.7 in
space group P21. Its general fold is very similar to that of IL-1, having the same central barrel along
with the adjoining loops (Figure 4a, b). The overall rms distance between 133 aligned C positions of IL1 and IL-1 is 1.8 (Figure 5). The major difference between the two molecules is an N-terminal
extension of 8 residues beyond the N-terminus of IL-1. This projection forms a short strand (residues
6-10), the presence of which positions the N-terminus of IL-1 in an alternative conformation. It has
been postulated that this conformation is responsible for the differences in binding of precursor IL-1
and : while precursor and mature IL-1 bind to IL-1RI and elicit response, only processed IL-1 binds
with the receptor. This suggests that the N-terminal region of IL-1 probably plays a role in receptor
binding. Extra residues in the alternate conformation of immature IL-1 serve to inhibit the receptor
binding and thereby the biological activity. In contrast to IL-1, IL-1 incorporates two other secondary
structural elements: a short strand (residues 9799) and about two turns of a 310 helix (residues
101105), neither have been shown to be important in the function of the molecule.
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Figure 4
(a) Stereo diagram of the secondary structural elements of IL-1. Produced by Molscript [106].
(b) Stereo plot of all the atoms of IL-1 viewed parallel to the axis of the barrel.
E. Three-Dimensional Structure of IL-1Ra

The x-ray structure of IL-1Ra was first reported in 1994 [47] and has since been solved by x-ray
crystallography and NMR by several different laboratories [4649]. Again, this molecule possesses the
characteristic trefoil fold (Figure 6a) and similar tertiary structures (Figure 6b). The structure is similar
to that of IL-1 and IL-1 with an rms deviation of 1.8 and 1.42 , respectively, when considering their
C positions (Figure 7a). Structural superposition of these three molecules shows a common trefoil core
(Figure 7b). In the absence of receptor substrate complexes, the comparison of all three structures
combined with mutational studies (see below) is invaluable in obtaining a model of the regions
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Figure5
Superposition of the C atoms of IL-1 and . Thinner line represents IL-1
and the thicker line is that of IL-1.
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Figure 6
(a) Stereo diagram of the secondary structural elements of IL-1Ra. Produced
by Molscript [106]. (b) Stereo plot of all the atoms of IL-1Ra viewed parallel to the axis
of the barrel.
involved in binding and exertion of biological effect in the IL-1 system. Similarity between the three
molecules has been observed mainly in the strands. One notable region of
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difference between IL-1Ra and IL-1 agonists has been observed on the side of the beta barrel on site B
of the receptor binding site (see below). The C positions in this region (residues 8494 in loop 78)
differ by 9.1 [26] in comparison with IL-1. This region also contains Asn84, which is the site for at
least two forms of N-linked glycosylation that occur in IL-1Ra in addition to the nonglycosylated form.
Mutagenesis experiments have also
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Figure 7
(a) Superposition of the C atoms of IL-1, IL-1, and IL-1Ra. (b) A
common trefoil core based on the structural superposition of all the three IL-1
molecules.
identified this region and the surrounding area in IL-1 with a large receptor binding epitope
encompassing the N and C termini of the protein as well as loops 45 and 910. The other
postulated binding epitope (epitope A) is structurally conserved in IL-1Ra. Other differences in structure
not related to any implicated biological structure are an extended 310 helix in residues 9299 in IL-1Ra
as opposed to a type-1 turn found in IL-1.
F. Three-Dimensional Structure of Interleukin-1 Beta Converting Enzyme (ICE)
As mentioned above, the processing of the different members of the IL-1 family is important to
discovering their function and may also provide clues in the
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design of inhibitors. The structure of ICE has been solved by x-ray crystallography to 2.6 in complex
with an acetyl-Tyr-Val-Asp-H tetra-peptide [50]. Figure 8a illustrates the tertiary structure of the
heterodimer with a dimension of about 45 35 25 . The molecule itself is oligomeric and
contains two subunits, p20 (residues 120297) and p10 (317404) of relative molecular weight 20 kDa
and 10 kDa, respectively. The two subunits form an intimately connected heterodimer. The core of ICE
is a six-stranded sheet containing 5 parallel strands and one antiparallel strand. The core is bounded by
six alpha helices that lie parallel to the beta sheets. Physiologically ICE occurs as a (p20)2(p10)2
tetramer. The molecule is a cysteine protease, which has a unique preference among the mammalian
proteases for cleaving bonds. It has only one known with aspartic acid adjacent and N-terminal to the P1
scissile bond. It has only one known physiological substrate, proIL-1, which it cleaves to active IL-1.
The struc-
Figure 8
(a) Stereo diagrams of the heterodimer structure of Interleukin-1 Converting
Enzyme (ICE). (b) The tetrapeptide inhibitor (Asp-Ala-Val-Tyr) covalently
bound to Cys285 in the active site. Tetrapeptied shown in black, p20 subunit in
dark gray, and p10 subunit in light gray. Produced by Molscript [106].
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ture is unlike other cysteine proteases such as papain and appears to have only one known possible
homologue, CED-3 protein [54]. The crystal structure with the tetrapeptide (acetyl-Tyr-Val-Ala-Asp-H)
substrate in the binding pocket (Figure 8b), coupled with the results obtained by the mutational
experiments, has provided a detailed understanding of the enzyme mechanism and substrate bonding of
the molecule. Structural analysis has pinpointed a catalytic diad of residues Cys285 and His237
responsible for the catalytic activity of the molecule: Cys285 acts as an active-site nucleophile while the
imidazole ring of His237 participates in destabilizing the cysteine O hydrogen. Although both of above
these residues are contained in the p20 subunit, the p10 subunit is essential for the maintenance of a
binding pocket (and thus the specificity of the molecule), providing binding sites S2 to S4 (residues
338341) and jointly contributing, with subunit p20, the S1 (Arg179Arg341) site. The uniqueness and
substrate specificity of ICE make it an ideal target for small molecules to block the production of
mature, active IL-1. Since the substrate peptide sequence is known (Tyr-Val-Ala-Asp), it has been a
starting point to develop peptidomimetics to inhibit ICE.
IV. Therapeutic Strategies
A. Manipulating the IL-1 System
Since IL-1 is an important mediator of human disease processes, modulating its activity or completely
inhibiting its synthesis may be of therapeutic benefit. Blake and Henderson [7] reviewed and portrayed a
general strategy to interfere with the cytokine at any one of the following stages: induction or initiation
of gene expressiontranscription, RNA processing and translation in IL-1 production, folding, release
and secretion of extracellular protein, IL-1 in circulation, IL-1 binding to its cell surface receptors, signal
transduction and resulting activities (Figure 9). All of the above approaches hold promise for the future.
The wealth of structural information on IL-1 combined with extensive site-directed mutagenesis studies
on the molecules have helped to build up a picture of the regions involved in biological function. A
schematic representation of the present-day efforts to design efficient antagonists of IL-1 its shown in
Figure 10.
B. Site-Directed Mutants
Extensive mutagenesis studies have provided information related to the structural integrity, receptor
binding region, and residues that are important for IL-1 function. Site-directed mutagenesis (SDM) can
create a single-site mutant and its receptor binding and bioactivity values can be calculated. The results
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Figure 9
Points of intervention for IL-1-based immunomodulation.
obtained from such studies can be imported into the available 3-dimensional structures. A combination
of such structural insights coupled with the bioassay results provide clues leading to IL-1 functional
information. In a previous structural report on IL-1, we summarized the SDM results and identified a
plausible receptor-binding epitope of interleukin-1 [42]. As mentioned before, electrostatic and
hydrogen-bonding interactions in the loops between strands allow the polypeptide to adopt a
conformation that enables an unusual concentration of polar and charged groups to be presented at the
open end of the barrel. This cluster of charged residues forms an epitope with which IL-1 might bind to
the receptor. Following our proposal, many groups have supported this hypothesis by employing
mutagenesis studies. For a list of mutants and their activity results refer to References 42, 56, 59, and 61.
Ju and coworkers are employing SDM to characterize interleukin-1 [55,56,58,61]. Substitution of Lys
for the Asp145 of IL- (D145K) greatly reduced agonist activity, while retaining 100% binding to the IL1RI [56]. Based on the sequence alignment of IL-1 with IL-1Ra, they selected Lys145 of IL-1Ra for
mutagenesis and converted it to an aspartic acid. This mutant analog (IL-1Ra K145D) maintained
receptor binding and gained partial agonist activity [56]. Following this study, Ju and coworkers selected
five other amino acid residues in IL-1Ra for further analysis because the side chains of these residues
appear to be in close proximity to Lys145 in IL-1Ra [61]. Mutations were made at Val18, Thr108,
Cys116, Cys122, and Tyr147, usually by a replacement with the corresponding amino acid of IL-1 at
each position. None of these muta-
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Figure 10
Schematic diagram showing the IL-1-based therapeutic molecules.
tions provided enough information related to the structure-activity relationship. The mutant IL-1Ra
K145D was mutated further to V18S, T108K, C116F, C122S, C122A, Y147T, Y147G, H54P, and a
H54I. Of these, K145D + T108K showed a 2-fold decrease in IL-1RI binding and a 3-fold decrease in
bioactivity compared to the IL-1Ra K145D analog. The K145D + C116F combination resulted in the
complete loss of bioactivity, whereas full receptor-binding activity was maintained. The observation that
receptor-binding activity is preserved indicates that the binding site is not altered that much. Structural
alignment
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indicates that Cys116 in IL-1Ra is in a homologous position with Phe117 in IL- 1. The K145D +
Y147T analog lost all detectabled activity (both binding and bioactivity), whereas the Y147G analog
lost all bioactivity but retained 100% binding. These data suggest that Tyr147 is important for
bioactivity of IL-1Ra K145D.
C. Insertion of Bulge
A region of charged amino acids (Gln48 to Asn53, a bulge) positioned between strands 4 and 5 has
been implicated in IL-1 binding to its receptor and its immunostimulatory properties [42, 6467]. The
-bulge residues form a protrusion on the edge of the open end of the barrel. Evidence for this patch of
amino acids involved in the function of this molecule comes from mutagenesis studies in which
deletions or substitutions of residues in this region reduced IL- 1 agonist activity without affecting
receptor binding [62,63]. Simoncsits et al. have shown that deletion of amino acids 5254 (SND) in IL1 reduces IL-1RI binding by 10 fold and biological activity by 1000 fold [63]. Also, studies indicate
that a synthetic peptide derived from IL-1 (VQGEESNDK), which contains these six bulge amino
acids, has immunostimulatory but no inflammatory effects normally associated with IL-1 [6466]. The
insertion of VQGEESNDK into recombinant human ferritin H chain and recombinant flagellin from
Salmonella muenchen increased the immunogenicity of these antigens in mice [67].
Greenfeder et al. inserted this -bulge region into IL-1Ra K145D either after Ile51 of after Pro53 [61].
The insertion of the bulge (QGEESN) after either position 51 or 53 of IL-1Ra K145D resulted in
analogs that retained full IL-1RI binding and increased bioactivity by 34 fold. Aslo they tried to obtain
the analogs of the QGEESN insertion in the absence of the K145D mutation either after amino acid 51
or 53 of IL-1Ra. None of the plasmid clones with the insertion at position 51 of IL-1Ra produced the
appropriate protein, whereas they were able to isolate clones with the insertion at position 53. Based on
these results, Greenfeder et al. suggest that in the first case the insertion interfered in the proper folding
of the protein, whereas the second mutant folded properly and exhibited only 10 to 20% of the IL-1RI
binding activity. The mutants with K145D + QGEESN insertion after Ile51 of IL-1Ra showed an
increase in bioactivity in the range of 38 fold. The triple mutant IL-1Ra K145D/H54P/QGEESN
showed a higher bioactivity, and based on their mutagenesis study, Greenfeder et al. suggest that this
increase in activity may be due to the introduction of Pro and not the removal of His at position 54 in the
K145D mutant of IL-1Ra. The cumulative effects of these three mutations are also interesting since their
positions on the IL-1Ra protein appear to be spatially separated. The residues Ile51 and His54 are
located on the open face of the barrel of IL-1Ra, whereas Lys145 is located away from the open barrel
end. In IL-1, the same relative
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positions of the bulge and Asp145 are observed [42] with the two regions separated by the known IL1RI binding site.
We have compiled the available site-directed mutants of IL-1 and sorted them according to the
following four categories: (1) mutants that shows significantly higher agonistic activity, A1T, P2M,
S5R, N89G, K92R, and K93R; (2) mutants that show significantly higher antagonistic activity, R4E,
C8S, T9G, T9Q, T9E, L10T,C,S,A, C71X, R11G, K93M, M95R, E96Q, K103Q, and D145K;, (3)
mutants that show significantly higher binding, A1T + P2M, S5R, T9L, T9W, P87S, P87H, K88V,G,L,
N89R, E96Q, K103Q, G, C and M148A; and (4) mutants that show significantly lower binding,
R4A,K,D, L6A, T9E, L10N,T,C,S,A, H30R, M44S, F46D, A, 156A, V58A, K92E, K93L,A,F,S,E,Q,L,
K103S, and E105S,K. These four types of mutants are shown in Figure 11ad.
Taken together, this information supports our earlier proposal of the receptor-binding epitope. This was
further characterized as functional sites A and B of interleukin-1. The first site, Area A is structurally
conserved in all three molecules and contains residues Arg11, His30, and Asp145 in IL-1 Asn17,
Ala36, and Asp147 in IL-1; and Trp16, Tyr34, and Lys145 in IL-1Ra. Present in both active IL-1
molecules, Asp145 has been recognized as an important residue in IL-1 binding. Area B has also been
identified in both IL-1 and IL-1 it contains a large hydrophilic ridge around solvent-accessible
hydrophobic residues. In-IL-1, this region contains the 7-residue hydrophilic ridge (Arg4, Gln48,
Glu51, Asn53, Lys93, Glu105, Asn108) around 5 hydrophobic solvent-accessible residues (Leu6,
Val47, Ile56, Leu110, Val151). Figure 12 shows a surface presentation of the proposed receptor-binding
epitope. In IL-1, the 7-residue hydrophilic ridge has been identified with residues (Arg12, Ile14,
Asp60, Asp61, Ile64, Lys96, and Trp109, which when mutated resulted in significant loss of binding to
the receptor. Area B is structurally conserved in IL-1 and in IL-1, but is lacking in IL-1Ra. For this
reason Area A has been proposed as the binding region while Area B has been proposed as the
triggering region.
D. Peptide Fragments
Peptide-based IL-1 antagonists have been derived from the primary sequence of IL-1 or IL-1.
Stepwise synthesis of a series of peptides from amino-terminal to carboxy-terminal regions did not
provide any satisfactory results [68]. Polypeptide fragments of IL-1, termed somnogeneic peptides,
induced sleep in mammals [69], 61.5% NREMS at a dose of 20 ng. The numbering of these peptides in
proIL-1 are 178207, 199225, 208240 and that of mature IL-1 are 6291, 83109, and 92124 (seq
92124=KKKMEKRFVFNKIENNKLEFESAQFPNWYIST). Monsanto company has identified a
peptide fragment of IL-1 (4170) exhibiting inhibitory effects for IL-1 and IL-1, but not TNF-, at 5
nM (5 ng/mL). Peptide 5670 is a
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Figure 11
Functional residues of IL-1, identified by the
site-directed mutagenesis results. Produced by
Molscript [106].
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Figure 12
The open end of IL-1, shown as a surface. The positions of some of the
residues that have been subjected to site-directed mutagenesis studies have
been marked. Produced using the program GRASP [108].
weak agonist. Peptide 4755 (VQGEESNDK) has been identified as an activator of T cells. It also
stimulates glycosaminoglycan synthesis, excites antitumor activity in vivo, and lacks proinflammatory
and pyrogenic activities [70]. Later a shorter segment (4953 = GEESN) with higher activity than 4755
has been identified. Peptides based on IL-1 and IL-1 sequences were claimed to induce production of
prostaglandin E2 but actually maintain other biological activities. Few other peptides or peptidecontaining epitopes were identified by using neutralizing antibodies. Labriola-Tompkins and his
colleagues have reported obtaining epitopes for neutralizing antibodies by fractionation of a goat
polyclonal antiserum over columns containing individual immobilized synthetic
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Figure 13
The identified peptide fragments. Peptide fragments are shown as striped
coil, with residues at the start and end of the peptides are numbered. Produced by
Molscript [106], modified by R. Esneuf.
peptides derived from an IL-1 sequence [58]. Their work resulted in 4 peptide regions with residue
numbers 412, 4463, 6488, and 89105. Peptides 4755, 8199, 92124, 121153 in the 3dimensional structure of IL-1 are shown in Figure 13.
E. Other Strategies
Biochemical and structural knowledge has opened many pathways for the development of novel
therapeutics. Such strategies include inducer blockers, nucleotide intercalators, antisense RNAs, and
other novel molecular mimics. It is known that potent inducers such as lipopolysaccharides, c5a, and
integrins bind to IL-1-producing cells and induce the over expression of IL-1. Such an induction can be
interrupted by raising the level of neutralizing monoclonal antibodies against the inducers. Alternatively,
one can design ligands that can bind to the inducer receptors, which leads to the inhibition of the IL-1
synthesis process. Transcription factors bind to specific DNA sequences and stimulate gene
transcription. Controlling such a specific gene transcription can be achieved by a number of means.
Intercalators or fragments of the same DNA sequences as those bound by the transcription factors can
selectively inhibit transcription. Double-stranded oligonucleotides, having the same consensus sequence,
could complete with the transcription factor for binding to the promoter region. Antisense RNA, which
when introduced into eukaryotic cells induces sequence-specific inhibition of target gene expression,
can be used. The
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antisense strand hybridizes the complementary mRNA to form a double-stranded helix thereby
achieving the inhibition of the gene products. Antisense oligo-deoxynucleotide derivatives have been
shown to inhibit species-specific fibroblast PGE2 synthesis stimulated by IL-1. These approaches
generated much interest and many laboratories are pursuing the design of compounds based on antisense
nucleotides, triple-helical transcription inhibitors, aptamers, and other novel nucleic acid derivatives.
V. Neutralizing Soluble IL-1 in Circulation
Once IL-1 is released into the extracellular fluid, the following molecules can be employed to
manipulate its activity: soluble receptors, IL-1Ra, IL-1 neutralizing antibodies, IL-1-specific binding
proteins, high-affinity small molecules (Figure 10).
A. Soluble IL-1 Receptors
The most effective neutralizer of a cytokine is likely to be its receptor. Some viruses have been reported
to use an IL-1 receptor mimic to evade the immune system [72]. Natural shedding of cytokine receptors
is a common occurrence and may form part of a normal homeostatic regulatory system, and there is a
high potential for the use of such soluble forms as therapeutic agents. The binding affinity of the mature
forms of the interleukin-1 molecules and their receptors have been reported [8,3133,71]. These studies
show that the affinity of both the membrane-bound and soluble forms of human IL-1RI for the mature
forms of human IL-1, IL-1, and IL-1Ra are approximately the same. In contrast to IL-1RI, IL-1RII
binds IL-1preferentially. Based on the binding-affinity studies one can infer that soluble IL-1RI is a
better inhibitor of IL-1 than IL-1 and soluble IL-1RII is a better inhibitor of IL-1. Both soluble
receptors, at sufficiently high concentrations, will completely block the binding of both IL-1 forms to
cells. Dower and coworkers [8] have studied the real-time binding of human IL-1, IL-1, and IL-1Ra
to human soluble IL-1RI and IL- 1RII. It seems that the binding of IL-1Ra to IL-1RI is essentially
irreversible, whereas its binding to IL-1RII is rapidly reversible. In contrast, IL-1RII binds IL-1
irreversibly [8,1]. This indicates that the IL-1RII can be used as a high-affinity antagonist of IL-1.
Dower further suggest in his studies that the soluble receptors have several potential advantages over
anticytokine antibodies due to the fact that they have much higher affinities (100 to 1000 fold) and
should not be recognized by the immune system. Even though soluble receptors have high-binding
affinity they are difficult to synthesize in large quantities and they have a low half-life.
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B. Interleukin-1 Receptor Antagonist

Over the past 510 years a number of inhibitors of IL-1 and TNF have been found in biological fluids
and cell-culture supernatants. Interleukin-1 receptor antagonist was the first protein receptor antagonist
to be described. Recombinant IL-1Ra has been shown to block the activity of IL-1 and IL-1 both in
vitro and in vivo in animal models by binding to both type I and type II IL-1 receptors without
demonstrable agonist activity. These effects have been extensively studied. However clinical trails
carries out by Synergen on human subjects provided a negative support for the antagonist behavior of IL1Ra for sepsis. It may be that the system took an alternative route by inducing TNF in excessively high
levels thus achieving signal transduction. Therefore neutralizing TNF in combination with IL-1
inhibition could be an alternate procedure. Even though IL-1Ra exhibits very high binding affinity it is
rather a poor inhibitor of IL-1 action in vivo. It must be present at greater than a 100-fold molar excess
over either agonist form to block action, and large doses are required to block IL-1-mediated effects in
vivo [1]. Burger and Dayer suggest that the simultaneous use of IL-1Ra and IL-1RII might be beneficial,
since this mixturecontrary to the use of IL-1Ra alonecompletely abolished the production of
interstitial collagenase in the inflammatory pathway [73].
C. Monoclonal Antibodies
Chimarized or humanized neutralizing monoclonal antibodies for IL-1 can be used as IL-1 can be used
as IL-1 antagonists. Otsuka Pharmaceuticals has developed a monoclonal antibody (IgG1 kappa) against
IL-1. It can be used in the immunoassay method for the selective detection of human IL-1 and also to
determine the biological activity of IL-1. Their patent (EP 0-364-778) also covers the use of such an
antibody against IL-1. when it is abnormally produced in disease states. Another patent of Otsuka
Pharmaceuticals (EP 0- 408-859-A2) relates to an antibody and its application to inflammatory
processes. This antibody is directed to a specific antigen on activated human endothelial cells (1E7/2G7)
and blocks the binding of white blood cells causing inflammatory responses. Based on the antibody
complementarity-determining region low-molecular-weight nonpeptide mimetics can be developed. This
attempt might result in low-molecular-weight, orally active compounds.
D. Low-Molecular-Weight Antagonists
Low-molecular-weight antagonists are attractive due to their low cost and bioavailability. Available
literature indicates that many laboratories are attempting to create immunomodulators of small synthetic
molecules, peptidomimetics, bacterial cell-wall components, macrocycles, corticosteriods, and others
[74].
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Figure 14
2-Dimensional structures of small molecules that exhibit IL-1 modulating
activities. They are tenidap, ciprofloxacin, 3-Deazaadenosine, (SK&F 86002),
E5110, DMARDs (Chloroquine, Auranofin, Sodium aurothiomalate and
Dexamethasone) tiaprofenic acid, dexamethasone, tricyclic-ylidene-acetic
acid and its derivative, Probucol, eicosapentenoic acid + docosahexenoic,
pentoxifylline, Denbufylline, and Romazarit (Ro-31-3948).
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American Home Products has patented molecules ranging from substituted quinoline, piperidine, and
naphthpyridine compounds as immunomodulators. Ciprofloxacin, a quinolone antibiotic, reduced the
extracellular IL-1 activity in human monocytes and delayed the peak production of IL-1 IL- by 24 h
and decreased total IL-1 production, but did not change total IL-1 production [75,76].
Tenidap, an antiarthritic drug, has shown efficacy both in rheumatoid arthritis and osteoarthritis [77,78].
It is a known inhibitor of 5-lipoxygenase and cyclooxygenase (5-LO/CO) and in vitro studies indicate
that it also inhibits the synthesis of mature IL-1 and pro IL-1 [77,79]. Kadin reports analogs of Tenidap
as antiinflammatory agents and analgesics [80]. An antiarthritic molecule, 3- Deazaadenosine, has been
demonstrated to inhibit IL-1 production by LPS- stimulated human PBMCs acting at the level of RNA
synthesis and also by blocking the effects of IL-1 on EL4 cells and induction of PGE2 release by
human fibroblast [81,82]. Another 5-LO/CO inhibitor (SK&F 86002) has been reported to inhibit the
synthesis of IL-1 in human monocytes and human synovial cells in a dose-dependent manner [83,84].
Analogs of this compound
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Figure 14
(Continued)
have been patented as IL-1 inhibitors [8587]. In human monocytes, E-5110 is a dual 5-LO/CO
inhibitor found to reduce extra- and intracellular IL-1 activity induced by LPS in a dose-dependent
manner. This compound also inhibits the IL-1 generation induced by antigen-antibody complexes,
zymosan, and silica particles [88].
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Antinflammatory DMARDs such as chloroquine, auranofin, sodium aurothiomalate, and dexamethasone

have been shown to inhibit IL-1 synthesis [89]. Analogs of these compounds have exhibited potent
inhibition of IL-1- induced cartilage resorption [90]. Elevated collagenase and proteoglycanase levels
caused by IL-1 in human cartilage were found to be reduced by tiapro-
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Figure 14
(Continued)
fenic acid and dexamethasone [91]. A patent report from the National Institutes of Health describes a
method of treating diseases associated with elevated levels of interleukin-1. Rosenthal, as the inventor of
this patent, describes a method for inhibiting the release of IL-1 from IL-1-producing cells by
administering a therapeutically effective amount of an aromatic diamidine (WO9115201-A).
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These aromatic diamidines inhibit IL-1 production and also block IL-6 and TNF. Imidazoline blocks IL1 and TNF and is less toxic to the cell with an in vitro LD50 >>10-4. Examples of these compounds are
1,5-bis(4-amidophenoxy) pentane (pentamidine), in the form of pentamidine isothionate, and an
imidazoline in the form of 1,5-di(4-imidazolinophenoxy)pentane. Tricycli- cylidene-acetic acid [92] and
its 2-chloro derivative [93] were found to be inhibitors of IL-1 release, claiming clinical improvement in
patients with psoriasis, periodontal disease, and Alzheimer's disease. In vitro this compound blocks the
synthesis of prostaglandins and inhibits the release of IL-1 and IL-1 from human monocytes and
murine macrophages.
Probucol, a hypocholesterolemic drug that possesses antioxidant activity, inhibits the ex vivo release of
IL-1 from LPS-stimulated macrophages of mice pretreated orally with 100 mg/kg/day of this compound
[94,95]. This compound has been shown to inhibit LPS-induced zinc-lowering effect, is cited as direct
evidence for the inhibition of IL-1 release, and may be useful candidate for the treatment of
atherosclerosis [95,96]. An amino-dithiol-one derivative (RP 54745) blocked the proliferative action of
IL-1 on murine thymocytes in vitro and also inhibited the production of IL-1 in mouse peritoneal
macrophages in vitro and in vivo. The compound RP 54745 selectively inhibited the expression of IL1 and IL-1 mRNA while TNF mRNA was unaffected [97, 98].
Administration of a cocktail containing eicosapentenoic acid and docosahexenoic acid to volunteers for
up to 6 weeks, resulted in a significant depression in IL-1 (61%), IL-1 (39%), and TNF (40%)
synthesis. These levels returned to normal after a few weeks [99]. In vitro studies indicate that
Pentoxifylline can block the effects of IL-1 and TNF on neutrophils [100]. It is a phosphodiesterase
(PDE) inhibitor that causes increased capillary blood flow by decreasing blood viscocity and is used
clinically in chronic occlusive arterial disease of the limbs with intermittent claudication. Denbufylline,
a closely related xanthine, has been patented as a functional inhibitor of cytokines and exhibits a similar
profile to Pentoxifylline [101]. Romazarit (Ro-31-3948) derived from oxazole and isoxazole propionic
acids has been shown to block IL- 1-induced activation of human fibroblasts in vitro and in animal
models reduces inflammation [102,103,104]. By using a spontaneous autoimmune MRL/lpr mouse
model, a significant efficacy was shown [105]. Two-dimensional structures of some of these molecules
are shown in Figure 14.
Even though the above mentioned small molecules exhibit IL-1 inhibition none of them were discovered
based on defined functional or structural aspects. An understanding of the three-dimensional structure of
IL-1s and their receptors, by themselves or in complexes, will form a very strong foundation for
structure-based design of more specific and potent IL-1-based immunomodulators.
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Page 428
VI. Conclusion
The design of novel compounds to inhibit or manipulate the IL-1 system remains a daunting task. At this
time, the design of immunomodulators for the IL-1 system is still in its infancy and has largely been
confined to the use of whole or fragmented proteins or the identification of nonspecific small molecules.
In addition, newer approaches have also been initiated and these include the use of antisense
oligonucleotides, small molecules designed to compete with IL-1's binding to its receptor, ICE
inhibitors, and intracellular signaling inhibitors. All such strategies show promise.
Structure-based design has not been explicitly used in the design of agonists and antagonists of IL-1. But
as of now we have the structures of IL-1, IL-1, and IL-1Ra. A new insight may be forthcoming once
the complex crystallographic structure of one of the interleukin-1 molecules and its corresponding
receptor molecule is available. This structural information, coupled with the anticipated IL-1 + IL-1R
complex structure, will form the foundation for rational design of inhibitors with improved selectivity
for the treatment of various IL-1-mediated diseases.
Acknowledgements
Our special thanks to Professor Russell Doolittle for his encouragement and support, and also to Dr.
Mitch Lewis for providing us with the very high-resolution coordinates of IL-1. We gratefully
acknowledge San Diego Super Computing Center for their assistance and support in providing valuable
software and high-power computing time. We thank Dr. Donald Kyle for his valuable comments. We
also thank Dr. Per Kraulis for providing us the latest version of MOLSCRIPT, Dr. Anthony Nicholls for
the program GRASP, Dr. Rob Russell for the structural alignment, and Professor Lynn Ten Eyck and
Dr. Jerry Greenberg for their help.
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17
Structure and Functional Studies of Interferon: A Solid Foundation for
Rational Drug Design
Michael A. Jarpe
Cambridge NeuroScience, Inc., Cambridge, Massachusetts
Carol H. Pontzer
University of Maryland, College Park, Maryland
Brian E. Szente* and Howard M. Johnson
University of Florida, Gainesville, Florida
I. Introduction
The interferons (IFNs) were discovered in 1957 by Isaacs and Lindenman when they observed that a
substance secreted by virally infected cells could protect other cells from viral infection [1a]. They
called this substance interferon and found that it was a protein that caused uninfected cells to produce
other proteins that made them resistant.
Researchers since then have been finding a growing family of structurally related molecules: the
interferons. Through the years, the interferons have been given many different names including immune,
fibroblast, leukocyte, Type I, and Type II interferons. The recognized nomenclature includes alpha, beta,
omega, tau, and gamma (, , , , and ) interferons. Alpha, beta, omega, and tau all belong to the
similar Type I subclass. Gamma is the sole member of the Type II or immune interferon class. The Type
I interferons all share a greater sequence homology to each other than they do to IFN- (for a recent
general review of the IFNs, see Reference 1b).
The IFNs exert their actions on cells via cell surface receptors. Type I IFNs share the IFN Type I
receptor (IFN-R1) while IFN- has its own unique Type II receptor. The signal transduction pathways of
Type I and Type II
*
Current affiliation: Brigham and Women's Hospital, Boston, Massachusetts.
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Figure 1
Interferon activity.
receptor activation are similar. These pathways involve ligand and receptor binding followed by the
activation of tyrosine kinases and the phosphorylation of various proteins and their subsequent
interaction with transcription elements on DNA. The two receptor activation pathways differ at the level
of ligand binding. Type I IFNs bind their receptor as a complex of a single ligand, ligand binding
element, and an accessory molecule. The Type II binding event occurs as a dimer of IFN- binding to
two identical receptor molecules leading to receptor dimerization and activation.
The IFNs of all subclasses posses antiviral activity. Additionally, they produce cellular responses that
are distinct from antiviral activity, including antiproliferative and immunomodulatory activities (Figure
1). These activities have led to an interest in their use as potential therapeutics to combat viral disease,
cancer, and autoimmune disease. Currently, the IFNs have a worldwide market in excess of 2 billion
dollars annually. There are six FDA-approved indications in the United States with several more in
clinical trials (Table 1). In fact, two of the top-ten grossing biotechnology-based drugs on the U.S.
market are IFNs. Intron A is an IFN- used for immune protection and has an annual U.S. market of
$570 million. Roferon-A is another IFN- used for hairy-cell leukemia and Kaposi's sarcoma with an
annual market of $170 million. These sales are despite profound negative side effects associated with
IFN treatment. High doses are required to achieve positive clinical results and can lead to severe flu-like
symptoms including nausea, vomiting, and fever. These side effects can cause patients to drop out of
treatment before beneficial effects are seen. Another drawback of IFNs as drugs is that they require
parenteral delivery. The IFNs are protein drugs that must be administered by injection and
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Table 1 Approval Indications for IFNs
FDA Approved
IFN-
Clinical Trials
Chronic hepatitis
HIV infection
Kaposi's sarcoma
Colon tumors
Genital warts (papillomavirus)
Kidney tumors
Bladder cancer
Hairy cell leukemia
Malignant melanoma
Non-Hodgkin's lymphoma
Chronic myelogenous leukemia
Throat wartz (papillomavirus)
IFN-
Relapsing remitting multiple sclerosis
Basal cell carcinoma
IFN-
Chronic granulomatous disease
Kidney tumors
Leishmaniasis
cannot be given orally. For many of the clinical indications, treatments of many months are needed
requiring repeat injections.
These drawbacks, coupled with the market value of IFN-related treatments, now and in the future, have
created an interest in producing second-generation molecules that can mimic IFN activity. These
mimetics could potentially have greater specificity with fewer side effects. They may also have the
advantages of reduced manufacturing costs and more versatile delivery. The design of mimetics can be
achieved through structure-based drug design methodologies that are currently being developed.
However, in order to apply structure-based drug design to a protein, a solid understanding of the
structure/function relationship is needed. A three-dimensional structure, taken alone, gives little insight
into the activity of a protein. Structure/function studies must be done for the full potential of structurebased drug design to be realized.
Document
Structure/function studies can take a variety of forms and use a number of techniques including the use
of molecular biology, synthetic peptides, and antibodies, or combinations of these methods. Molecular
biology is a powerful tool for structure/function analysis. Mutagenesis of cDNAs to produce mutant
proteins with point mutations, truncations, or deletions can identify functional sites. One drawback to
this approach, especially with large proteins, is the proverbial needle in a haystack problem. One has
difficulty determining where to begin placing mutations. The synthetic peptide approach can be equally
as powerful. One can synthesize individual domains or segments of proteins and test them for agonist or
antagonist activities thereby identifying functional domains. Synthetic peptides can be used to map the
epitope specificity of antibodies that block the activity of a protein. Peptides can also be used to produce
monospecific antisera to a defined region of a protein. The antibody approach has also proved quite
useful in determining functional sites of
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proteins. One potential disadvantage to using antibodies is the possibility of over interpreting the
blocking results because of steric hindrance. A large antibody molecule may inhibit function through
binding to a distant site and covering up a functional site. These approaches have all been used with
success on a variety of proteins, but are best used in combination. For example, the information obtained
from synthetic peptides and antibodies can significantly narrow down the region for site-directed
mutagenesis studies. The entire sequence is narrowed to a segment, which reduces the size of the
haystack in which the needle is hidden.
Even though these approaches are powerful methods for determining functional sites on proteins, they
are limited if not coupled with some form of structural determination. As Figure 2 illustrates, molecular
biology and synthetic peptide/antibody approaches are not only interdependent, they are tied in with
structural determination. Structural determination methods can take many forms, from the classic x-ray
crystallography and NMR for three-dimensional determination, to two-dimensional methods such as
circular dichroism and Fourier Transformed Infrared Spectroscopy, to predictive methods and modeling.
A structural analysis is crucial to the interpretation of experimental results obtained from mutational and
synthetic peptide/antibody techniques.
Figure 2
Flow diagram of structure/function studies.
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Table 2 Three-Dimensional Structure Studies of the IFNs
Type of Study
Reference
X-ray studies
IFN-
IFN- human
IFN- bovine
C.T. Samudzi, J.R. Rubin, unpublished data
IFN- rabbit
IFN- human + receptor
NMR studies
IFN- human
IFN- mouse N-terminal peptide (139)
Models
IFN-2a
IFN-8
IFN- sheep
10
T. Senda, S.I. Saitoh, Y. Mitsui, J.Li, and R.M. Roberts,
unpublished data
Note: This is not meant to be an exhaustive list of all structural studies of the IFNs. It only highlights some of the
three-dimensional studies that have been conducted.
While there are no hard-and-fast rules for conducting structure/function studies, the approaches taken for
studying the IFNs can be used to illustrate some of the methods that have been successful. Over the
years, a large body of work has accumulated on the IFNs, including a number of structural studies. Table
2 summarizes some of the studies exploring the three-dimensional structure of the IFNs. The following
sections review some of the structure/function studies that have begun to elucidate important features of
IFN activity and form a basis for future rational drug design.
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II. Type IFNs

A great deal of structure/function analysis has been done on the Type I IFNs. One Type I IFN in
particular, IFN- has received attention recently because of its lower cytotoxicity compared to the IFNs. Structure/function studies have concentrated on comparing IFN- with IFN-. Therefore, IFN-
provides an excellent example of structure/function studies of the Type I IFNs.
First isolated from the conceptuses of sheep, IFN- is the major conceptus secretory protein responsible
for signaling maternal recognition of pregnancy in ruminants [11]. it is produced in large quantities (200
g in 30 h from a day 16 conceptus culture). The protein was purified using a combination of anion
exchange and molecular sieve chromatography.
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A. IFN- Synthetic Peptide Studies

A sheep blastocyst library was screened with a probe based on the N-terminal sequence of the IFN-
protein and the cDNA obtained (Table 3). Surprisingly, it exhibited 4555% homology with various
IFNs from human, mouse, rat, and pig and 70% homology with bovine IFN- [12]. It shared both
molecular weight (19 kDa) and pI (5.45.6) with IFN-s, while its length, 172 amino acids, was
equivalent to the IFN-s. In competition studies, IFN- was found to compete with IFNs , , and for
binding to the Type I IFN receptor [13]. In contrast, IFN- exhibited several unique properties such as
its reproductive function, its poor inducibility by virus, and its apparent reduced cytotoxicity. Thus, IFN conceptus protein appears to be a novel IFN.
Structural studies began with production of overlapping synthetic peptides, each 3035 amino acids in
length, corresponding to the entire
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sequence of the molecule [14]. The peptides were used in competition assays with the native molecule.
Peptide inhibition of a particular function would implicate the region of the molecule that it represented
in the elicitation of that function. The effect of the IFN- peptides on the antiviral activity of ovine IFN was examined in a dose/response assay using Madin Darby bovine kidney (MDBK) cells challenged
with vesicular stomatitis virus. The carboxy-terminal peptide oIFN-(139172) was found to be the most
effective inhibitor of antiviral activity. Three additional peptides, oIFN-(137), (6292), and
(119150), also reduced IFN- antiviral activity. This suggested that multiple regions of the IFN-
molecule interact with the Type I IFN receptor and elicit antiviral activity. These regions are underlined
in Table 3. The data were consistent with studies of antiviral activity and receptor binding with IFN-
analogs demonstrating that 3 distinct sites, located in the amino-terminal, internal, and carboxy-terminal
regions of the molecule, influenced human IFN- activity [15].
To verify functional results using synthetic peptides, antipeptide antisera were produced [14]. All
antipeptide antisera were reactive with the native molecule. Interestingly, antisera titers correlated with
the hydropathic index of the peptide, rather than with the predicted surface accessibility of the specific
region in the 3-D configuration. Consistent with the peptide studies, antisera against the same four
regions of the molecule inhibited IFN- activity while antisera to other regions did not.
Since IFN- and IFN- bind to the same receptor, the ability of the IFN- synthetic peptides to block
both bovine and human IFN- was examined. Interestingly, only three of the four inhibitory peptides
were effective competitors of IFN-. Cross-inhibition of IFN- by the internal and carboxy-terminal
peptides was observed and suggested that these residues may adopt a similar conformation in both
molecules and bind to a common site on the receptor. The aminoterminal peptide failed to reduce IFN-
function entirely. Thus, either the IFN- amino-terminus has a much higher affinity for receptor or the
IFN- aminoterminus binds a unique site on the receptor complex that may be associated with its unique
properties. As expected, none of the peptides blocked the antiviral activity of IFN-, which interacts
with a different receptor.
Next, it was determined whether the same active regions of IFN- were involved in additional systems.
The Type I IFN receptor on cells has been reported to be somewhat more promiscuous than on other
cell types [16]; therefore, vesicular stomatitis challenge of Fc-9 cells was performed. Only the carboxyterminal peptide inhibited IFN- activity in this system [17]. This suggested that it was the carboxyterminus that was crucial to receptor interaction. In studies examining IFN--treated feline
immunodeficiency virus infected FeT-1 cells and human immunodeficiency virus-infected peripheral
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blood lymphocytes, peptide inhibition of IFN- antiretroviral activity implicated both the amino- and
carboxy-termini as functionally important [17].
The structural basis of the antiproliferative activity of IFN- was also investigated. While multiple
regions were again involved in IFN- antiproliferative activity, it was the area adjacent to the carboxy
terminus, rather than the carboxy-terminus itself, which was the most crucial for antiproliferative
activity, inhibiting cell division by blocking entry into the S phase of the cell cycle [18]. Since, for a
particular IFN- subtype, antiviral potency does not necessarily correlate with antiproliferative potency,
localization of these functions in different domains of the molecule is not unexpected [19]. Within all
known IFN-s, the 8 amino acids from 139 to 147 are highly conserved. These residues are contained in
both carboxy-terminal peptides, but while they may be involved in antiviral activity, they do not appear
to be solely responsible for antiproliferative activity since the two peptides are not equivalent inhibitors
of IFN- antiproliferative activity. This observation is consistent with inhibition of antiviral activity but
not antiproliferative activity by a monoclonal antibody in this conserved region in human IFN- and
with the requirement for tyrosine at position 123 for human IFN-1 antiproliferative activity [20,21]. It
has also been reported that mutations around Arg33 affected both antiviral and antiproliferative activity
of human IFN-4 on human cells [22], while the amino-terminus did not appear to be as important in
IFN- antiproliferative activity on bovine cells.
B. IFN- Monoclonal Antibodies
Another approach to structure/function analysis of IFN- involved generation of anti-IFN- monoclonal
antibodies. Four monoclonal antibodies were produced that reacted with the native IFN- protein. They
were epitope mapped using the available IFN- peptides. Two of the antibodies were directed against the
carboxy-terminus of the molecule, one against a region adjacent to the aminoterminus, and the final one
appeared to react with a conformational, rather than a linear determinant (C. Pontzer, unpublished data).
When these antibodies were used as competitors in binding assays, all four inhibited IFN- binding to
the Type I IFN receptor on MDBK cells. That anti-IFN- carboxy-terminal antibodies would inhibit
binding is not unexpected, but the inhibitory activity of the monoclonal antibodies directed against the
more amino-terminal region was not anticipated. There is the caveat that warns that results using
monoclonal antibodies to delineate function sites must be interpreted with caution since their size may
cause significant steric hindrance.
To proceed further with structural studies of IFN-, access to larger quantities of pure protein was
required. The obvious route to this end entailed production of recombinant protein. A synthetic gene for
IFN- was designed
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that allowed for optimal expression in both bacterial and yeast systems [23]. In addition, restriction sites
were incorporated at intervals throughout the length of the sequence to allow for cassette mutagenesis.
Using the Pichia pastoris expression system, 50 mg of purified IFN- were obtained from a one-liter
culture.
C. IFN- Binding and Signal Transduction
Detailed receptor-binding studies were performed comparing recombinant human IFN- and IFN-
[24]. The Kd of 125I-IFN- and 125I-IFN-A for receptor on MDBK cells was 3.9 10-10 M and 4.45
10-11 M, respectively. Consistent with the higher binding affinity, IFN-A was several fold more
effective than IFN- as a competitive inhibitor. Functionally, the two IFNs had similar specific antiviral
activities, but IFN- was 30 fold less toxic to MDBK cells at high concentrations. Phosphorylation of the
signal transduction proteins, Tyk2, Stat1a, and Stat2 did not appear to be involved in the cellular toxicity
associated with IFN- relative to IFN-. Excess IFN- did not block the cytotoxicity of IFN-A,
suggesting that they recognize the receptor differently. While maximal IFN antiviral activity required
only fractional receptor occupancy, toxicity was associated with maximal occupancy. Thus, spare
receptors may exist with respect to certain biological properties, and IFNs may induce a concentrationdependent selective association of receptor subunits.
D. Structural Biology of IFN-
In order to better interpret the information derived from the above studies, an understanding of the 3-D
structure of IFN- is required. Prior to resolution of the crystal structure, modeling techniques were
employed for structural predictions [10]. For IFN-, the homology it shares with the other IFNs can be
exploited. Since the x-ray coordinates for IFN- are known [2] (Figure 3), it was used as a template for
predicting the topology of IFN-. When the sequences of IFN- and IFN- are aligned, the overall
homology is approximately 30%. When residues are compared on the basis of conservative
substitutions, the similarity rises to about 50%, and if only the location of hydrophobic residues is
compared, the similarity is approximately 75%. This is important because hydrophobicity is thought to
be a critical factor in driving protein folding. The interferons IFN-, IFN--2, and several other
cytokines including IL-2, IL-4, growth hormone, and GM-CSF belong to a family in which all share a
four-helix bundle structural motif. Four-helix bundles exhibit a characteristic apolar periodicity in the
helices where every third or fourth residue is apolar, forming a hydrophobic strip down one side of the
helix, which facilitates packing. The aligned helical regions of IFN- show the same apolar periodicity,
suggesting a four-helix bundle motif.
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Figure 3
Stereo view of IFN- crystal structure [2].
The structure of IFN- was also examined by CD [10]. Analysis of the IFN- spectra predicts that the
secondary structural elements derived from CD spectra indicate approximately 70% -helix. The
remainder of the molecule is either predicted to be random or a combination of sheet and turn. Since it
is known that algorithms that predict secondary structures from CD spectra are most accurate at
identifying helices, we are confident that IFN- is mainly helical. The CD spectra for the synthetic
peptides of IFN- were also obtained. The peptides IFN-(137), IFN-(6292), IFN-(119150), and
IFN-(139172) all show the presence of helix, while IFN-(3464) and IFN-(90122) are mainly
random. The presence of an helix in the peptides supports the CD analysis of the intact protein and
also roughly indicates the location of helical segments.
The secondary structure of IFN-, including the location of the helices and loop region, was then
predicted using a neural network-based computer program called PHD that relies on sequence
alignments of all proteins related to the target sequence [25,26]. When this prediction is correlated with
the CD data, peptides that possess considerable helicity are predicted to contain entire helical
segments, and conversely, peptides with little helicity are predicted to be within loop regions.
A model of the 3-D structure of IFN- was constructed using a distance geometry-based homology
modeling method with mouse IFN- acting as a template. The distance constraints were generated
between residues within IFN- that are homologous to residues of IFN-. Dihedral-angle restraints of
helices were generated from the secondary-structure prediction of IFN-. No constraints were applied to
the 13-residue carboxy tail of IFN-, which is absent in IFN-, since it is likely to be flexible in a
manner similar to other proteins such as IFN-. Additional distance constraints were added from putative
disul-
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Figure 4
Stereo view of IFN- model. Highlighted sequences are from 137, 6292,
and 139172.
fide bridges between residues 1 and 99 and residues 29 and 139. Several structures were generated using
distance geometry routines, and the energy was minimized and averaged to yield a final model [16]. A
similar model was built by Senda et al. (unpublished results) using a homology modeling method. This
model was also built using the x-ray coordinates of IFN- and shows a similar topology to the IFN-
three-dimensional structure (Figure 4). The most striking feature of both models is that those
discontinuous regions, previously determined to be functionally important, are localized to one side of
the molecule and found to be spatially contiguous (Figure 4). This observation is consistent with
multiple binding sites on IFN- interacting simultaneously with the Type I IFN receptor and emphasizes
the importance of structural modeling in the understanding and interpretation of functional data.
III. Type II IFN
A. Functional Sites on the IFN- Molecule
The production of IFN--neutralizing antibodies specific for an N-terminal peptide of human IFN-
provided the first evidence that the N-terminus of IFN- contained an important functional site [27]. A
similar approach was used to produce N-terminus-specific neutralizing antisera against murine IFN-
[28]. Subsequent studies using IFN- synthetic peptides to map the epitope specificity of monoclonal
antibodies to murine IFN- showed that N-terminal specific monoclonal antibodies neutralize IFN-
antiviral activity [29]. In receptor-
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competition studies, murine IFN- N-terminal peptide consisting of residues 139 [IFN- (139)]
blocked both binding to receptor and antiviral activity of IFN- [30]. Overlapping peptides of other
regions of the IFN- molecule failed to block binding and function of IFN- [31]. Thus the combination
of peptide mapping of epitope specificities and receptor competition using peptides has identified the Nterminus as a structurally and functionally important region of the IFN- molecule. This region is
highlighted in the sequence of human IFN- found in Table 3.
Interestingly, site-specific antibodies to the C-terminus of murine IFN-, which were induced using the
peptide consisting of residues 95133 [IFN- (95133)], also neutralized IFN- activity, however IFN-
(95133) failed to block binding of IFN- to receptor and IFN- activity simultaneously. Antibodies to
internal peptides failed to block both antiviral activity and binding of IFN- to receptor. In studies with
recombinant murine IFN- receptor, which consisted of the entire chain except for the transmembrane
domain, the C-terminal peptide did block binding of IFN- to receptor [32]. Thus we have the interesting
paradox wherein the IFN- C-terminal peptide blocked binding of IFN- to the recombinant, soluble
receptor and yet did not block binding to the cell-surface receptor. One interpretation of these findings
has allowed us to formulate the velcro-key model of binding to receptor that involves both N- and Cterminal domains of IFN- (Figure 5). The N-terminus binds in the lock and key manner characterized
by specific ligand-receptor binding. The hydrophilic C-terminus binds to a region of the receptor distinct
from that for the N-terminus, most likely through its polycationic region, which is conserved across
species barriers. Binding of this type would exhibit high affinity and low specificity, similar to a piece of
velcro. The C-terminal peptide of IFN- would therefore act as a poor competitor for cell-surface
binding due to its low specificity alone. This interaction becomes specific in the context of the whole
IFN- molecule and may increase the affinity of receptor binding.
An alternative explanation that may also account for the inability of the C-terminal peptide to compete
for cell-surface interactions is that its binding site is located not on the extracellular domain of the
receptor, but rather on the intracellular domain. The primary differences between the cell-surface form
of the IFN- receptor and (2) the accessibility of the recombinant receptor's cytoplasmic domain. A
synthetic peptide corresponding to the membrane proximal region of the cytoplasmic domain of the
murine IFN- receptor was able to bind IFN- and specifically compete with the binding of IFN-
(95133) to fixed/permeabilized cells [33].
Studies by others have reaffirmed the importance of both the N- and C-terminal regions of IFN- in
function. Using recombinant DNA techniques, it has been shown that deletion of residues from the Nterminus of the molecule
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Figure 5
Velcro-key model of IFN- binding to its
receptor. (From Reference 25. Copyright
1992. The American Association of
Immunologists.)
results in decreased receptor binding [34]. Deletions or substitutions at the C- terminus have a direct
effect on function of the molecule [3538]. Epitope mapping of neutralizing monoclonal antibodies has
also revealed an internal region of the molecule (from residues 8494) as being functionally important
[39]. This sequence bears strong homology to the nuclear localization sequence (NLS) of the SV40 large
T antigen and has recently been demonstrated to be fully functional as an NLS for IFN- [40]. Thus,
internal regions of the IFN- molecule are also likely to play an important functional role.
B. IFN- Receptor Chain Sites of Interaction with IFN-
Both the human and the murine IFN- receptors consist of a ligand-binding subunit and a speciesspecific cofactor molecule. It is through interaction with this cell-surface receptor complex that IFN-
exerts its biological effects. The IFN- molecule and its N-terminal peptide IFN- (139) bind
specifically to the
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cell-surface receptor and to a recombinant, soluble form of the ligand-binding chain of the receptor [25].
Synthetic peptides corresponding to the sequence of the extracellular domain of the ligand-binding
subunit were used to define the region of the receptor to which the N-terminus of IFN- binds. Receptor
peptide MIR (95120) competed most strongly with IFN- binding to both cell-surface and recombinant,
soluble receptor [41]. Additionally, antisera to this peptide and the adjacent overlapping peptide, MIR
(118143), inhibited the binding of IFN- to the recombinant, soluble receptor. Therefore, the receptor
domain responsible for binding the N-terminus of IFN- is defined by the region encompassing residues
95120 of the ligand-binding subunit of the IFN- receptor and may extend further into the neighboring
sequence.
Antibodies to the C-terminal region of IFN- have been shown to be potent neutralizers of IFN- activity
[29]. However, no cell-surface binding site for the C-terminus of IFN- could be localized using either
antisera or synthetic peptides. Furthermore, as indicated above, the C-terminal IFN- peptide, IFN-
(95133), competed specifically with the intact IFN- molecule for binding to a recombinant, soluble
form of the receptor, which consists of both the extracellular and the intracellular domains [42]. It was
hypothesized that since the intracellular portion of the soluble receptor was accessible, in contrast to that
of the cell-surface receptor, the C-terminus of IFN- might indeed be binding to this region. In studies
using synthetic peptides corresponding to the cytoplasmic domain of the murine IFN- receptor, only
peptide MIR (253287) specifically bound both murine IFN- and its C-terminal peptide, MuIFN-
(95133) [33]. This peptide corresponds to the membrane proximal region of the receptor's cytoplasmic
region. Antibodies to this receptor peptide inhibited the binding of the C-terminus of murine IFN- to
the receptor in cells which had been fixed and permeabilized. Analogous binding studies with human
IFN- and its C- terminal peptide, HuIFN- (95134), yielded a similar result [43]. Surprisingly, the
binding of the IFN- C-terminal peptides to their cytoplasmic binding sites is not species restricted,
which is in contrast to the binding of the whole molecule at the cell surface. Both human and murine
IFN- and their C-terminal peptides bound equally well to receptor peptides of either human or murine
origin [43]. Thus, a receptor binding site for the C-terminus of the IFN- molecule has been localized to
the membrane proximal region of the ligand-binding subunit's cytoplasmic domain (Figure 6).
Previously, there have been several reports of human IFN- having activity on murine cells when
administered cytoplasmically [4446]. With the identification of a cytoplasmic binding site for IFN-,
which is not species restricted, the question arose as to whether this might be the basis for these earlier
observations. Thus, C-terminal IFN- peptides of both human and murine origin were used to stimulate
murine macrophage lines P388D1 WEHI-3. Macrophages were chosen particularly for their capacity to
nonspecifically endocytose material,
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Figure 6
Proposed receptor activation pathway for IFN-. (From Reference 53. Copyright 1995.
The American Association of Immunologists.)
and we took advantage of this as a means of introducing the IFN- peptides into these cells. The IFN- Cterminal peptides induced a potent antiviral state in the murine macrophages and upregulated expression
of MHC class II molecules, both in a dose-dependent fashion [43]. These effects were demonstrated to
be sequence specific, as a scrambled version of the murine C-terminal peptide lacked activity.
Furthermore, a truncated form of the murine C-terminal peptide, lacking the sequence of basic amino
acids (RKRKR), was also without activity. The absence of activity of this truncated peptide was linked
directly to a loss of its ability to bind to the receptor [43]. Therefore, interaction of IFN-, via its Cterminus, with its cytoplasmic binding site is important for function and requires the presence of a
region of basic amino acids near the C-terminus of the molecule.
C. Structural Biology of IFN- and the IFN- Receptor
Structure-function studies of IFN- carried out using the synthetic peptide approach and site-specific
antibodies indicated that both the N- and C-terminal regions of the protein were not only functionally
important, but also accessible at the surface of the molecule. The x-ray crystal structure of human IFN-
has been determined and reveals that in the IFN- homodimer both the N- and the
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Figure 7
Stereo view of IFN- [40]. Regions 139 of chain A and 95119 of chain B of the dimer
are highlighted.
C-terminus were indeed accessible [3]. Figure 7 illustrates the close proximity of the N- and C-termini
of IFN-. The subunits of the homodimer are oriented head to tail, such that the N-terminal helix-loophelix (corresponding to residues 139) of one IFN- molecule interacts with the C-terminus of the
second IFN- molecule.
As mentioned above, synthetic peptides were also instrumental in identifying the region of the receptor
to which the N-terminus of IFN- binds. Recently, the crystal structure of a complex between murine
IFN- and the murine IFN- R subunit has been determined [5]. The synthetic peptides and
corresponding antisera had predicted an interaction of murine IFN- residues (139) with receptor
region (95143). The crystal structure confirmed these observations, indicating an interaction of IFN-
residues (142) with receptor residues (108132). However, the crystal structure did not define an
extracellular binding site for the C-terminus of IFN-. There has been some speculation that the basic
amino acid residues of the C-terminus may interact with an acidic patch on the receptor's extracellular
domain, which would support the previously mentioned velcro-key model, but that the crystallization
conditions precluded this interaction [5]. It is quite possible that such an interaction may occur as a
transitional state prior to the internalization of the C-terminal portion of IFN- and interaction with the
cytoplasmic region of the receptor. An alternative explanation for the apparent lack of a binding site for
the IFN- C-terminus on the receptor's extracellular face is that its primary site of interaction is
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with the cytoplasmic portion of the receptor as described above. Thus, the orientation of the C-terminal
portion of the IFN- molecules in the receptor complex should be such that they are situated near to the
cell membrane. When one examines the structure of the receptor-ligand complex, it is easy to see that
this is indeed the case. Studies are currently under way to determine the crystal structure of the complex
between the cytoplasmic domain of the IFN- receptor and the IFN- molecule, in particular the Cterminus. With regards to the definition of sites of interaction between receptors and ligands, the
synthetic peptide approach has repeatedly proven to be an accurate indicator of structurally important
regions of the IFN-/IFN- R system.
D. Signal Transduction by IFN-
Within the past several years some of the immediate-early signal transduction events initiated in
response to IFN- stimulation have been elucidated. Treatment of cells with IFN- leads to the rapid
activation of two protein tyrosine kinases, JAK1 and JAK2 [47]. The JAK kinases are a newly emerging
family of protein kinases important in signaling via cytokines and growth factors. These proteins are
unrelated to the src family of tyrosine kinases and are characterized as being larger, having two putative
phosphotransferase domains and containing no characteristic SH2 or SH3 domains [4851]. Members of
the Janus kinase family are found associated with the cytoplasmic domains of cytokine and growth
factor receptors at or near to the membrane proximal region [51].
In the resting cell, JAK1 and JAK2 are found associated with the and /AF-1 chains of the IFN-
receptor, respectively [52]. These kinases as well as the ligand-binding chain of the IFN- receptor are
tyrosine phosphorylated in response to IFN- treatment [47,53,54]. This leads in turn to the tyrosine
phosphorylation of a latent cytoplasmic transcription factor, known variously as p91, Stat 91, or Stat 1
on tyrosine residue 701 [55]. It is interesting to note that the IFN- signal-transduction pathway partially
overlaps with that of IFN-. Stimulation of cells by IFN- leads to the activation of JAK1 and another
Janus family kinase, Tyk2 [56,57]. In turn, this cascade leads to phosphorylation of two latent
cytoplasmic transcription factors, p84 (Stat 1) and p113 (Stat 2) in addition to the p91 (Stat 1)
activated by IFN-.
The identification of tyrosine kinases that directly associate with the subunits of the IFN- receptor lead
to the question of how the binding of IFN- might affect these proteins. Recently, the synthetic peptide
method was used to identify two regions of the murine IFN- receptor's chain as being important for
interaction with the kinase JAK2 [58]. One of these regions lies in the distal portion of the cytoplasmic
tail (residues 404432), while the other (residues 283309) is nearer to the membrane proximal region to
which the C-terminal part of IFN- binds (residues 253287). The fact that there are adjacent binding
sites for JAK2 and IFN- implied a potential for interaction between the IFN-
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ligand and the machinery of signal transduction, namely JAK2. It was found that both intact murine IFN and its C-terminal peptide (95133) are capable of specifically mediating an increase in the degree of
association between the recombinant, soluble IFN- receptor and JAK2. These findings were further
supported as IFN- and IFN- (95133) caused an increase in the amount of JAK2 coprecipitating with
the receptor from intact murine macrophages [58]. This has been the first such demonstration of an
extracellular cytokine ligand participating directly in interaction with cytoplasmic signaling elements.
E. IFN- as a Candidate for Rational Drug Design
The IFN- molecule is a potentially attractive model for the rational application of drug-design
strategies. Reagents exist that are capable of either positively or negatively modulating the in vivo
effects of IFN-. An initial target is quite simply at the level of receptor-ligand interaction. Synthetic
peptide analogs of the N-terminal region have been successfully applied in vitro to inhibit interaction of
intact IFN- with cell-surface receptors [30]. Interestingly, it has been observed that the N-terminal
region of mouse IFN- has the ability to interact with the human receptor [59]. It was shown that the
mouse peptide IFN- (139) had a 10-fold greater ability to block the binding of human IFN- to cellsurface receptors. This was shown to be correlated with a more stable structure in solution for the
murine peptide and illustrates the importance of stable structure to receptor binding, which may be
exploited when designing peptide mimetics. The solution structure of this peptide has also been
determined and could provide the beginning steps for determining the structural requirements of an
antagonist [7]. Future studies could focus on cocrystallization of the peptides with receptor or NMR
studies of peptide domains of the receptor and IFN-. Recombinant, soluble forms of the extracellular
domain of the ligand-binding subunit of the receptor have also been used in analogous fashion both in
vitro to inhibit cell-surface binding and in vivo to interfere with disease progression [60]. Therefore
prevention of potentially deleterious effects of IFN- may be achieved by preventing initial interactions
with receptor molecules at the cell surface.
A second candidate region lies within amino acid residues 8494 of human IFN- and the corresponding
region of its murine homologue. This portion of the molecule functions as a nuclear localization signal
and, therefore, is also an attractive target for drug design. In cells treated with IFN-, the IFN- molecule
traffics rapidly to the nucleus of the cell, usually within one to two minutes. When the IFN- molecule is
crosslinked to its receptor, the resultant receptor-ligand complex migrates to the nucleus [40]. The
implication is that this sequence may therefore be of use in artificially targeting proteins from the
cytoplasm directly to the nucleus. This is potentially a very attractive method
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for directed subcellular targeting of synthetic transcriptional activators or repressors that might not
otherwise be directed to the nucleus.
Finally, the C-terminal region of IFN- offers another possibility. With respect to the induction of an
antiviral state, or the upregulation of MHC class II molecule expression, synthetic peptides
corresponding to the C-terminal 39 amino acids of either human or murine IFN- function as potent
agonists. This activity is due to the interaction of these peptides with the cytoplasmic domain of the IFN receptor and the associated protein tyrosine kinases. Furthermore, in contrast to the stringent species
specificity of intact IFN-, the action of the C-terminal peptides agonists is not limited by species
constraints. This property therefore renders the C-terminal portion of the IFN- molecule an attractive
model for the development of IFN- agonists and antagonists. The identification of the C-terminus of
IFN- as that part of the molecule that contacts the cytoplasmic portion of the receptor implies that in
developing IFN- agonists, the primary focus should be on this region of the protein. Corresponding
antagonists may be developed based upon the portion of the receptor to which the IFN- C-terminus
binds.
IV. Conclusion
The interest in IFNs as therapeutics has existed from their initial discovery in 1957. Since then scientists
have been trying to understand the mechanism of their action and apply that knowledge to the treatment
of many different diseases, meeting with some success. The effort now is to understand how IFNs work
at the molecular level, with the goal being to design better, more specific therapeutics. Through
structure/function studies, we now know where the functional sites lie on many of the IFNs. We also
know the sites of interaction with their receptors and second messenger systems. From these studies,
initial candidates for structure-based drug design have been identified. Although more work is needed to
further characterize the IFNs and their receptor systems, the challenge now is to apply our existing
knowledge and create second generation molecules that can modulate the many activities of these
fascinating proteins.
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18
The Design of Anti-Influenza Virus Drugs from the X-ray Molecular
Structure of Influenza Virus Neuraminidase
Joseph N. Varghese
Biomolecular Research Institute, Melbourne, Victoria, Australia
I. Introduction
Influenza has plagued humankind since the dawn of history and continues to affect a significant
proportion of the population irrespective of age or previous infection history. These periodic epidemics
that reinfect otherwise healthy people have devastated communities world wide. Some pandemics like
the 19171919 Spanish flu were responsible for the death of tens of millions of people throughout the
world. The origins, spread, and severity of influenza epidemics have been a puzzle that has only in the
last two decades been adequately addressed. In early times it was thought that the disease was the evil
influence (sic) of the stars, and other extraterrestial objects. At present it is generally accepted that the
disease is of viral origin, spread by aerosols produced by infected animals, and the continual production
of new strains of the virus results in reinfection of the disease (reviewed in Reference 1).
There are three types of influenza virus classified on their serological cross-reactivity with viral matrix
proteins and soluble nucleoprotein (A, B, and C). Only type A and B are known to cause severe human
disease. Type B is only found in humans, while type A has a natural reservoir in birds and some
mammals like pigs and horses [2]. Influenza, an orthomyxovirus, is a 100 nm lipid-enveloped virus
(Figure 1) containing an eight-segment negative single-stranded genome [3]. Two of the segments code
for the surfaces glycoproteins, hemagglutinin (which binds to terminal sialic acid), and neuraminidase
(which
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Figure 1
A schematic diagram of an influenza virus particle that illustrates
its constituent components and morphology. The surface antigens hemagglutinin
and neuraminidase are attached to the lipid and matrix protein shell that
encapsulates the eight negative-stranded RNA genes of the virus and
associated nucleoprotein and polymerase.
cleaves terminal sialic acid) and which appear as spikes protruding out of the viral envelope. The viral
target in humans is the upper respiratory tract epithelial cells. Replication (see Figure 2) begins with
penetration of the virion through the mucin layer covering the epithelial surface, followed by attachment
to the viral receptor by the hemagglutinin. Penetration of the cell is achieved by endocytosis and the
virion core is released after the fusion of the virion and vesicle membrane mediated by the
hemagglutinin. Fusion is enabled by a conformational change in the hemagglutinin made possible by
lowering the pH of the endosome by the M2 ion channel protein. Following replication, the progeny
virions are released by budding off the cell membrane [4,5].
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Figure 2
A simplified schematic of the replication cycle of an influenza virion in the host
respiratory epithelia. Details of viral transport through mucin and pathways of
viral spread on budding from the epithelial cell membrane during replication is
not understood. Neuraminidase activity is important for release of budding
progeny virions, desialyation of viral glycoproteins, and probably facilitates
transport through sialic-acidrich mucin.
Release of virions occur 8 hours post infection and the onset of infection is sudden, resulting in pyroxia,
muscular and joint pain, and a dry cough [6]. Virus shedding continues for up to a week, when a rise in
virus-specific antibody clears the virus from the host. The vulnerability of the host succumbing to
viremea during this week of rising viral titer is mediated by interferon induction [7] 48 hours post
infection, which attenuates viral replication until the cell-mediated immune response begins to clear the
virus. The severity of the illness is thought to depend on the level of cross protection arising from
antibodies raised from previous influenza infections [19]. The course of the illness can be debilitating,
and no effective treatment is available at present to halt the progression of the disease. Death can result
for susceptible populations (neonate and elderly) primarily as a result of secondary infections [8]. This
chapter shall examine a structural basis for the continual emergence of new influenza strains, and the
reasons current vaccines against influenza fail to protect against all
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strains of influenza. The discovery of the active site of influenza neuraminidase and the exploitation of
its structural conservation shall be discussed in terms of the design of potent neuraminidase inhibitors.
The potential therapeutic use of these inhibitors as antiviral drugs against influenza virus infections shall
be examined.
A. Antigenic Variation
The plethora of different strains of virus that are responsible for the continued reinfection of virus in
humans is primarily related to mutations in the viral genes of two surface glycoproteins, hemagglutinin
and neuraminidase [9]. The current paradigm for this genetic variation [10,11] is that these mutations
arise primarily from incremental changes in the amino acid sequences of these glycoproteins by
selection pressure of the immune system of the infected host. This mechanism termed Antigenic Drift
accounts for most of the strain variation within a particular subtype of influenza.
However, infrequently a mutation arises by genetic reassortment of viruses from different animal hosts
(Antigenic Shift) whereby an entirely new gene for one of the surface glycoproteins is generated that
is significantly different (~50%) in amino acid sequence from the parent virus. This is the mechanism by
which new subtypes of influenza arise and are primarily responsible for the major pandemics that occur.
Strains of influenza virus are classified by type (A, B, or C), geographic location, date of original
isolation, and the subtype of the hemagglutinin and neuraminidase antigens. There exist 9 known
subtypes (N1 to N9) of neuraminidase and 13 known subtypes (H1 to H13) of hemagglutinin for
influenza A in all animal populations. Two neuraminidase (N1 and N2) and three hemagglutinin (H1,
H2, and H3) subtypes of influenza A have occurred in strains that have infected humans since 1933
when isolates were first characterized [12]. Prior to 1933 there is indirect evidence of antigenic shift
occurring in human populations [13]. The N1 subtype was associated with virus isolated between 1933
and 1957, after which time the N2 subtype appeared in the Asian influenza. No major change in the
structure of neuraminidase has occurred since, although the hemagglutinin subtype has changed from H2
to H3 in 1968 in the Hong Kong pandemic, and H1N1 reappeared in 1978 as the Russian pandemic.
Influenza B, which infects only human hosts, has only one subtype, but like type A undergoes continual
antigenic drift.
B. Current Therapeutics
Amantidine and Rimantidine are the only class of drugs that have been approved for therapy. At high
concentration (>50 mg/mL) Amantidine is thought to buffer the pH of the endosome and prevent the
conformational
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change of the hemagglutinin necessary for fusion. Drug-resistant mutants arise where the hemagglutinin
trimers are thought to be less stable than the wild type [14]. At low concentrations (<1 mg/mL)
Amantadine blocks the activity of the viral M2 ion channel protein, which plays a role in virus uncoating
and glycoprotein maturation [15]. Amatadine prevents fusion by altering the ability of the virus to
change the ion balance [16] within the endosome and the trans-Golgi network. However Amatadine
rapidly gives rise to drug-resistant strains by mutations in the hemagglutinin and the M2 protein that
circumvent or block the activity of the drug. Rimantadine has been shown to lead to drug resistance in
humans 2 days post treatment [17].
C. Influenza Vaccines
Influenza virus vaccines [18,6] prepared from killed (formalin inactivated) virus are used currently
worldwide as the only prophylactic treatment available against the disease. Killed vaccines contain
whole virus or fractionated subunits. These vaccines attempt to incorporate antigens from influenza
strains that will circulate in the community during an expected outbreak, conferring immunity to viral
strains that are closely related to the strains the vaccine is made from. However antigenic drift reduces
the susceptibility of the virus to neutralization by antibodies raised by immunization. For example,
commercial vaccines containing inactivated A/Beijing/353/89 H3N2 strain circulating among humans
from 1990 to 1993 provided significantly less protection [20] against the antigenic drift [21] variant
A/Georgia/03/93.
A more radical vaccination treatment, involving the direct injection into muscle of plasmid, DNAencoding hemagglutinin and M1 influenza viral proteins has been attempted successfully on animal
models [22]. This is thought to involve incorporating the plasmid into muscle cells and eliciting a cellmediated immune (CMI) response that offers cross protection overcoming antigenic drift in the virus.
The success of the method arises from a CMI response to segments of conserved amino acids in these
proteins. Problems arising from possible autoimmune response to DNA and problems in ensuring the
exogenous DNA is not integrated into the cell genome or sensitive cell lines elsewhere in the host have
precluded testing in humans at present. Furthermore, mutations in the nuclear proteins of influenza
(which have been relatively stable to date) arising from CMI selection pressure could undermine the
efficacy of this technique.
II. Viral Neuraminidase Activity
Enzyme activity on the surface of influenza virus was first detected by Hirst [23], who observed that red
blood cells once agglutinated by influenza virus
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could not again be agglutinated by either the eluted virus or fresh virus preparations. This activity is now
attributed to neuraminidase, which is one of the two integral membrane glycoproteins of influenza virus
(for reviews see References 24 and 25).
A. Function
Neuraminidase is an exoglycosidase that destroys the hemagglutinin receptor by cleaving the ketosidic linkage of terminal sialic acid [(N-actylneuraminic acid (Neu5Ac))] to an adjacent sugar
[26,27]. Viral hemagglutinin binds specifically to Neu5Ac-containing receptors on the surface of
susceptible cells [28]. Neuraminidase, which also removes terminal sialic acid from a range of
glycoconjugates, plays an important, but not completely understood, role in the viral replication cycle.
Without neuraminidase activity viruses [29] were thought to be immobilized by mucosal secretions in
the upper respiratory tract. By removing terminal sialic acid from the sialic-acid-rich mucous layer
[27,30] protecting target cells, neuraminidase could facilitate penetration of the virus to the cell surface.
It has been shown that neuraminidase-deficient virus [31] can still replicate in vivo, albiet at a much
reduced rate [32]. This shows that neuraminidase does not play an essential role in viral entry,
replication, assembly or budding in mice, but has an important role in the spread of the infection by
preventing aggregation at the cell surface and possible immobilization in the mucin by hemagglutinin.
Once replication is initiated in the infected cell, the freshly synthesised viral glycoproteins have to be
desialylated to prevent self-aggregation at the infected host cell surface by hemagglutinin binding to
terminal sialic acid on these glycoproteins. Finally on elution of progeny virions from infected cells,
neuraminidase activity is required to facilitate viral escape from the cell surface.
Inactivation or inhibition of neuraminidase during budding has been observed to result in aggregation of
virions on the cell surface [3335]. Inhibition of this glycohydrolase could provide a means of
controlling this disease by slowing the rate of viral attachment and subsequent release of progeny virions
allowing the host immune system to eliminate the virus while the number of infected cells is low.
B. Morphology
There are between 50 to 100 neuraminidase spikes per virion [36] which is approximately 10% of the
visible spikes projecting out of the surface of the virion [37]. These spikes can be removed from the
virus by treatment with detergent [38]. Electron microscopic images of the neuraminidase spikes [39]
reveal a mushroom-shaped molecule made up of a boxlike head of about 80
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80 40 with a narrow centrally attached stalk (15 wide and 100 long), which terminates into a
hydrophobic knob anchored in the viral envelope. The detergent-released spikes can be digested by
pronase to release the neuraminidase heads, which retain full antigenic and enzyme activity [40,41].
The molecule was found to be a tetramer of molecular weight 240,000, reducing to 200,000 when
treated with pronase [42]. A low resolution x-ray image of crystallized neuraminidase heads [43]
established that the enzyme had circular 4-fold symmetry.
III. Molecular Structure of Neuraminidase
Crystals of pronase-released heads of the N2 human strains of A/Tokyo/3/67 [44] and A/RI/5+/57 were
used for an x-ray structure determination. The x-ray 3-dimensional molecular structure of neuraminidase
heads was determined [45] for these two N2 subtypes by a novel technique of molecular electron density
averaging from two different crystal systems, using a combination of multiple isomorphous replacement
and noncrystallographic symmetry averaging. The structure of A/Tokyo/3/67 N2 has been refined [46]
to 2.2 as has the structures of two avian N9 subtypes [4749]. Three influenza type B structures [50]
have also been determined and found to have an identical fold with 60 residues (including 16 conserved
cysteine residues) being invariant. Bacterial sialidases from salmonella [51] and cholera [52] have
homologous structures to influenza neuraminidase, but few of the residues are structurally invariant.
A. Structural Topology
The protein fold consists of a symmetric arrangement of six four-stranded antiparallel sheets arrange
as blades of a propeller (Figure 3), the propeller axis being approximately parallel to but titled away
from the circular 4-fold axis of the tetramer. This tilt angle varies between the known subtypes. This
topology has now also been found in the seven sheet propeller structure of bacterial methylamine
dehydrogenase [53] and galactose oxidase [54], and the eight sheet propeller structure of methanol
dehydrogenase [55].
Each sheet of neuraminidase has a W topology (+1,+1,+1) [56] with four strands connected by reverse
turns (Figure 3). The first strand of each sheet enters from the top, approximately parallel to and near the
propeller axis; the fourth strand exits from the bottom, approximately perpendicular to the propeller axis.
Top and bottom surfaces of the head refer to the faces of the tetramer away and towards the viral
membrane respectively. Each sheet thus has a characteristic 90 right-hand twist between the inner- and
outermost strand. The six sheets and their connections to each other are topologically identical.
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Figure 3
(a) A MOLSCRIPT [107] stereo diagram of the neuraminidase tetramer viewed
from above down the symmetry axis. The different shaded arrows represent
strands comprising the six sheets that form the propeller framework of
each subunit. (b) A stereo diagram of a neuraminidase monomer viewed down
the 4-fold axis, and -sialic acid is shown bound in the active site
of the enzyme, which lies in a pocket formed by the six
sheets near the pseudo six-fold axis of each subunit.
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The first strand of each sheet is connected across the top of the submit to the fourth strand of the
preceding sheet. The N-terminus lies across the bottom of the subunit, and builds the fourth strand of the
sixth sheet before entering the first sheet. The C-terminus strand builds the third strand of the sixth
sheet, enters the subunit interface from the bottom, and runs parallel with the outermost (fourth) strand
of the sixth sheet.
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B. Protein Structure
The 6 sheets of the subunit, although topologically identical, vary in size and detailed structure.
However, the six sheets can be considered as maintaining an approximate 6-fold symmetry relationship
to each other about an axis parallel to the mean direction of the first strand of each sheet and through the
centroid of these directions. Four disulfide bridges are formed between adjacent sheets as well as two
between the sheets, which distort the sheet structure. Similar pairing has now been found in one of the
domains of CD4 [57]. Also short bulges occur on the inner and outer strands of most of the sheets. The
packing of the sheets is stabilized by hydrophobic interactions at the sheet interfaces and hydrogen
bonding at the periphery of the sheets. In addition the intersheet disulfide bonds and ion pairs stabilize
the structure.
In general the loops connectioning the sheets are shorter and tighter on the bottom of the subunit
compared to the outer loops, which tends to be more elaborate. The intersheet loop connecting the fourth
and fifth loop is the most extensive in the structure and is stabilized primarily by a disulfide bridge
between Cys318 (sequence numbering of A/Tokyo/3/67 will be used throughout) and Cys337, a
conserved ion pair Asp330Arg364, and a putative Ca2+ ion binding site. This calcium binding site has
approximate octahedral coordination with the main-chain oxygens of residues 293, 297, 345, and 348, a
carboxylate oxygen of Asp324, and a water molecule in N2 and type B neuraminidases. In N9, the mainchain oxygen at 343 is replaced by a water molecule. Calcium has been shown to be necessary for
neuraminidase activity [58] and this site is connected to the active site via conserved residues; however,
the functional role of calcium in the structure is unknown, although it has been shown that calcium is
essential for the thermostability of the molecule [59].
C. Carbohydrate Structure
Carbohydrate at four N-linked glycosylation sites were observed in N2 neuraminidase at residues 86,
146, 200, and 234 in the x-ray structure. Two N-acetylglucosamines were resolved at Asp86 and
Asp234, both at the bottom surface of the monomer. The carbohydrate at Asp200 consists of eight sugar
residues with linkages consistent with known mannose-rich simple N-linked carbohydrates [60]. This
oligosaccharide emerges from the side of the monomer and covers a neighboring subunit (see Figure 4).
The oligosaccharide site at Asn146 is the most conserved of all neuraminidase glucosylation sites,
except that of the neurovirulent virus A/WSN/33 [61]. The absence of this glycosylation site in
A/WSN/33 has been shown to confer neurovirulence in mice [62]. It is a complex sugar containing Nacetylgalactosamine [63] that is not found in any other of the known oligosaccharides of influenza virus
glycoproteins and is the only glycopeptide antigenically related to chick embryo host antigen
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Figure 4
A CPK model of two tetramers of A/Tokyo/3/67 neuraminidase heads as seen
in the crystal structure. The four carbohydrate spikes emanating from the top
of the molecule (at Asn146) interdigitate and form an open barrel structure.
They form an unusual crystal contact. The dark spheres represent carbohydrate
and the light spheres represent protein. The carbohydrate at Asn200 starts
from one subunit and covers a neighboring subunit on the same tetramer. The
other carbohydrates lie on the
underside of the tetramer.
[63,64]. The oligosaccharide appears as a spike emanating from the top of the monomer, forming a
crystal contact with a neighboring tetramer in crystals of A/Tokyo/3/67 neuraminidase. The four
carbohydrate spikes of a tetramer form an open barrel structure of eight carbohydrate chains with the
neighboring tetramer, with no apparent intercarbohydrate contacts. This oligosaccharide may play an
important but as yet unidentified role in neuraminidase structure or activity.
D. Antigenic Variation in Neuraminidase Structures
Comparison of all known sequences of approximately 390 residues of the neuraminidase globular head
[24], indicates that only 54 (excluding 16 conserved cysteine residues) are invariant (Figure 5a). Apart
from 21 residues involved
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Figure 5
(a) Stereo image of a CPK atomic model of an influenza virus neuraminidase
tetrameric head viewed distal to the viral membrane. The darker-shaded atoms
represent totally conserved residues that for the most part form the enzyme
active-site pocket. The lighter shaded atoms represent strain-variable
residues and carbohydrate. (b) A stereo image of the enzyme active-site
pocket of a subunit of neuraminidase with the same shading scheme.
in preserving the structural integrity of the molecule [46], the main clustering of these invariant residues
is within the enzyme active site (Figure 5b), where 17 are in the active site and 16 are neighboring the
active site. This is a cavity on the upper surface of the molecule into which sialic acid has been observed
to bind [65,66,50]. Excluding the active-site pocket, strain variation occurs over the entire surface of the
neuraminidase heads. The active site was found to be in
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Figure 6
Stereo image of a ball-and-stick model of sialic acid bound to the active site of
Tern/N9 neuraminidase. The hydrogen-bond interactions with conserved residues
are shown as dotted lines. Nitrogen atoms are shaded black, oxygen atoms
are shaded dark gray, and carbon atom are shaded light gray.
a pocket of totally conserved (over all animal subtypes) residues [65]. In this way the enzyme active-site
pocket is surrounded by highly variable surface residues that prevent immune recognition of the active
site by antibody molecules [25]. The x-ray diffraction studies of neuraminidaseantibody complexes
have shown that the footprint of an antibody in the complex is larger than the exposed surface of the
conserved region of the active site [6769]. These structural results indicate that antibodies are unable to
exert mutational pressure on the conserved active site because they cannot bind there without engaging
strain-variable residues as part of the binding surface. However, as antibodies bind to strain-variable
elements of the structure, the virus can overcome host immune pressure by point mutations of the
residues that do not have a catalytic or structural role [70,48] but are able to disrupt the
antigenantibody binding interface. The rapid emergence of these escape mutants explains the failure
in producing a universal vaccine for influenza.
E. The Enzyme Active Site
The structures of N-acetyl neuraminic acid (sialic acid Neu5Ac) and the 2-deoxy-2,3-dehydro-N-acetyl
neuraminic acid (Neu5Ac2en) inhibitor complexed with N2 neuraminidase [66] revealed the nature of
the interactions of the
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molecules in the active-site pocket (Figure 6). Sialic acid binds in the active site in the -anomer and, in
a distorted half-chair conformation, through the same face as used in its interaction with hemagglutinin
[71]. The carboxylate group of the sugar interacts with three guanidinium groups of argine residues 118,
292, and 371 and has an equatorial conformation with respect to the sugar ring (this group, axial toward
the floor in the undistorted structure, is probably held equatorial by interactions with these arginine
residues). The NH group of the 5-N-acetyl side chain interacts with the floor of the active-site cavity via
a bound water molecule. The oxygen of the 5-N-acetyl side chain is hydrogen bonded to the N of Arg
152, while the methyl group lies in a hydrophobic pocket near Ile222 and Trp178. The last two hydroxyl
groups of the 6-glycerol side chain are hydrogen bonded to carboxylate oxygens of Glu276 and the 4hydroxyl is directed to a carboxylate oxygen of Glu119. The glycosidic oxygen O2 interacts with a
carboxylate oxygen of Asp151. Similar binding of sialic acid in the active site was observed in type B
virus [50].
Comparison of active sites of N2, N9, and type B neuraminidase [72] show there are no significant
differences between active-site orientations, except for some minor displacements of Arg224 and
Glu276, where the major interactions with the 6-glycerol group of sialic acid occur. However there are
differences in the water structure in the active sites of the different subtypes. The Gly405 residue (in N2
and N9) is replaced by a tryptophan in type B, which displaces four water molecules that lie in a solvent
pocket bounded by arginines at residues 371 and 118. The Val240 residue (in N2 and N9) is replaced by
a methionine in type B, which displaces two water molecules that form a channel under Arg 224,
decreasing the flexibility of the active site of type B in this region. These waters are not displaced in the
sialic acid/neuraminidase complex in N9 and N2 and would alter the hydrogen-bonding pattern of the
complexes when compared to type B.
Comparison of the active sites of influenza neuraminidases and bacterial sialidases [51,52] indicates that
there is considerable conservation of the catalytic site at the carboxylate-binding end. The residues
Asp151, Arg118, Glu277, Arg292, Val or Ile349, Arg371, Tyr406, and Glu425 are conserved over all
known viral and bacterial strains. The arginyl residues 118, 292, and 371 position the 2-carboxylate
group and the Val(or Ile)349, Glu425, and Glu277 are important in positioning the triarginyl cluster. The
residues Asp151 and Tyr406 are presumably important in bond cleavage, but the precise mechanism is
still unclear. These eight residues (Figure 7) are thus most likely to be conserved in all neuraminidases.
Differences between viral, bacterial, and mammalian neuraminidase structures may correspond with the
different role these enzymes have in vivo. These differences are likely to be in the interactions of the 6glycerol, 5-N-acetyl, and 4-OH groups of silaic acid. In the influenza virus, the turnover rate must be
balanced against the requirement to maintain
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Figure 7
Stereo image of the active site of Tern/N9 showing the active-site residues
surrounding 4-guanidino-Neu5Ac2en (black). Those residues that are conserved
in both influenza and bacterial neuraminidase are shaded gray, and those that are
conserved only in influenza virus neuraminidase are not shaded.
sufficient sialic acid at the cell surface to enable attachment via the hemagglutinin. This balance may
require some configuration of residues in the active site not directly responsible for catalysis but only
involved in the binding and release of sialic acid.
IV. Neuraminidase Inhibitor Design
Earlier screening programs [73] failed to identify potent inhibitors of viral neuraminidase. The first
inhibitor synthesized [74] with a Ki value in the micromolar range was Neu5Ac2en. This was based on
the proposed transition state of the reaction, where the anomeric carbon (C2) bound to the ketosidic
oxygen has a trigonal state. Several analogues of Neu5Ac2en were synthesized soon after, and the most
potent of these, a trifluoracetyl derivative, had a Ki of only 0.8 M [75]. While this compound showed
that in cell culture it retarded virus shedding [34,76], it failed as an effective antiviral agent in animals
[77].
The x-ray structure of Neu5Ac2en/neuraminidase complexes have been determined for N2 [66], type B
[78], and N9 [79]. The Neu5Ac2en molecule binds in the active site of neuraminidase with the
carboxylate oxygen atoms placed in the same location as the carboxylate of sialic acid. The 5-N-acetyl, 4-
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hydroxy, and 6 Neu5Ac2en -glycerol are positioned isosterically in the two molecules.
An alternative approach to develop sialidase inhibitors has been made using synthetic thioglycoside
analogs of gangliosides such as Neu5Aca(2-S-6)Glcb(1-1)Ceramide [80] were shown to inhibit different
subtypes of human and animal influenza virus with Ki values of up to 2.8 M. These metabolically
stable ganglioside analogs contain a thioglycosidic linkage to the terminal neuraminic acid that resists
cleavage by the enzyme. Several flavonoid neuraminidase inhibitors have been isolated from plant
extracts [81], one of which 5,7,4'-trihydroxy-8-methoxyflavone, was a more potent inhibitor than
Neu5Ac2en. Recently, in vivo anti-influenza virus activity of a Kampo (Japanese herbal medicine)
preparation has shown promising results in inhibiting influenza virus replication in mice [82], but the
mode of action of these compounds is unclear.
A. Enzyme Mechanism
The similar positioning of the carboxylate oxygens and ring of Neu5Ac2en and sialic acid suggests that
Neu5Ac2en is probably a transition state analog. As Neu5Ac2en has a higher affinity for neuraminidase
than sialic acid, a mechanism of catalysis was proposed [83] that involves the distortion of the substrate
by the formation of a oxycarbonium ion intermediate, which has a similar structure to Neu5Ac2en.
However the structural results from sialic acid/neuraminidase complexes suggest that the tighter binding
of Neu5Ac2en more likely comes from the relaxation of the conformational strain arising from the
transition from chair to boat of the pyranose ring of sialic acid in the active site [66].
Evidence for a sialyl cation transition state by isotopic effects [84] support the existence of a
oxycarbonium ion intermediate. However the structural basis for neuraminidase activity is still unclear.
It has been suggested [66] that the tyrosyl oxygen of Tyr406, assisted by the sialic acid carboxylate
itself, could stabilize the developing charge on the oxycarbonium ion intermediate. The reaction would
be completed by the activation of a water molecule by a deprotonated Asp151 and its attack on the
carbonium, resulting in the formation of the -anomer of sialic acid [85]. However the pH-activity
profile of neuraminidase [86,87,78] suggests a bell-shaped profile, which indicates normal activity from
a pH range of about 4.5 to 9. This would indicate that the role of Asp151 as the acid group in the
catalysis is unclear. A nonspecific proton donor has been proposed [78], probably a water molecule as
the acid group, with a deprotonated Tyr406 stabilizing the oxycarbonium ion, and a proton transferred
from water to the departing aglycon group. It has also been postulated that a proton is eliminated at C3
leading to the transformation
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of the oxycarbonium ion into Neu5Ac2en, which is produced irreversibly at low levels from sialic acid
by the enzyme [78]. An SN1-type mechanism has been suggested [88] that is facilitated by an activated
water molecule, which can be expelled upon inhibitor binding. The catalytic mechanism could possibly
proceed without an acid group: the electrostatic potential of the enzyme could lower the barrier
preventing the breaking of the ketosidic bond and the solvent could protonate the aglycon after release
[89]. Clearly details of the enzyme mechanism have yet to be elucidated definitively. Structural
considerations indicate that only Tyr406 (and possibly Glu277) and the triarginyl cluster are essential in
the enzyme mechanism and that Asp151 is implicated.
B. Inhibitor Design Principles
All the nearest-neighbor interactions between sialic acid or Neu5Ac2en and the protein are with totally
conserved amino acids. Thus an inhibitor designed to bind only to the conserved active-site residues of
neuraminidase would inhibit neuraminidase activity across all strains of influenza. This would enable
the development of an antiviral drug that would affect the spread of viral replication potentially in three
ways, i.e., transport through the protective mucosal layer, desialyation of freshly synthesized viral
glycoproteins, and elution of progeny virions from infected cells.
The development of potent inhibitors was based on the structural information of the N2 neuraminidase
conserved active site and its complex with sialic acid and Neu5Ac2en [66]. There are no reports of de
novo molecules designed to fit into the cavity, and the most useful approach was to consider molecules
that were structurally related to Neu5Ac2en. This involved the design of molecules that would bind
isosterically to Neu5Ac2en but which were modified to increase the number of favorable interaction
with the protein. The method of Goodford [90] enabled the calculation of favorable binding sites for a
variety of chemical probes. The validity of this method was indicated by its ability to identify the
positions of the carboxylate binding site of sialic acid as an energy minima for a carboxylate probe
(Figure 8a), and the successful prediction of known bound-water sites in the active sites by a water
probe. Utilizing this methodology, predictions of energetically favorable substitutions to Neu5Ac2en
were examined [91]. A replacement of the hydroxyl at the 4-position of the pyranose ring of Neu5Ac2en
by an amino group was identified by this procedure as an energetically favorable substitution. A
protonated primary amine probe identified a favorable binding site of -16 kcal mol-1 at this location and
in a pocket in the active site near two conserved glutamate residues Glu119 and Glu227 (Figure 8b).
This suggested that the substitution of the 4-hydroxyl group by an amino group would increase the
overall binding interactions by forming a salt link with Glu119. Furthermore the substitution of the 4hydroxyl group with a much bulkier guanidinyl group would lead to even tighter binding as a result
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Figure 8
Stereo image of the residues in the active site of Tern/N9 complexed with
4-guanidinoNeu5Ac2en overlayed with GRID maps [90] (caged mesh contours)
for (a) a carboxylate oxygen probe, contoured at -12 kcal/mol; (b) an amino
nitrogen probe, contoured at -12 kcal/mol.
of lateral interactions of the terminal nitrogens of the guanidinyl group and the carboxylate groups of
Glu119 and Glu227.
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This led to the design and syntheses of 4-amino-Neu5Ac2en and 4-guanidino-Neu4Ac2en [91] which
bound to A/Tokyo/3/67 with a Ki of 50 nM and 0.2
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nM, respectively. These compounds were later shown by x-ray studies of 4-guanidino and 4-aminoNeu4Ac2en complexed with A/Tokyo/3/67 neuraminidase to bind close to that predicted by the design
studies. However details of the interactions of the guanidinyl group of the 4-guanidino-Neu4Ac2en with
the glutamic acid groups (Glu119 and Glu277) in the floor of the active site were slightly different. This
was confirmed on a higher resolution x-ray study (Figure 9) of a 4-guanidino-Neu4Ac2en complexed
with Tern N9 neuraminidase [72]. One of the primary guanidinyl nitrogens of 4-guanidino-Neu4Ac2en
is hydrogen bonded to the main-chain oxygen at residue 178, a carboxylate oxygen of Glu227, and a
water molecule. The other primary guanidinyl nitrogen interacts with the main-chain oxygen of residues
178 and 151. The secondary guanidinyl nitrogen interacts with the carboxylate of Glu119 and Asp151.
The interactions with Glu119 are electrostatic and van der Waals in character and lack hydrogenbonding geometry (postulated in the design study) as the carboxylate group of Glu119 stacks parallel to
the guanidinyl group. Furthermore, theoretical energy-minimized structures of the complex using
AMBER [92] converged to the x-ray structure only if the protein nonhydrogen atoms were kept rigid in
the x-ray structure [72]. Otherwise this resulted in active site residues showing large distortions in their
conformation. This is an example of the difficulty in correctly modeling even modest changes in the
interactions of an inhibitor/active site complex.
The 4-guanidino analog shows potent inhibition of neuraminidase activity in all known wild strains of
influenza. Furthermore it is very specific to in-
Figure 9
Stereo image of the Tern/N9 4-guanidino-Neu5Ac2en complex showing the
hydrogen-bond interactions (dotted lines) of the inhibitor with conserved residues
in the active site of the enzyme. Nitrogen, oxygen, and carbon atoms are
shaded black, dark gray, and light gray, respectively.
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fluenza, as it shows weak inhibition to bacterial, para influenza, and mammalian neuraminidases
[91,93]. This is possibly due to the specific interactions of the 4-guanidino group within the subpocket
of the active site of influenza neuraminidase that is not conserved in other neuraminidases. In bacterial
neuraminidases [51] this pocket is much smaller, and would prevent the binding of the 4-guanidino
group in this region of the active site. This is consistent with the proposition that the interactions of
sialic acid with the active site of neuraminidases are function specific at the C4, C5, and C6 position of
sialic acid and that modification at these positions confer specificity to the target enzyme [72]. Other
approaches [94] to structure-based design of inhibitors have to date produced only millimolar inhibition
of neuraminidase activity.
V. Antiviral Activity
In vitro inhibition of viral replication in tissue culture was demonstrated earlier [34] for the trifluro
derivative of Neu5Ac2en, but its antiviral activity in vivo was not demonstrated [77]. As a consequence
of this, efforts were directed towards hemagglutinin, which was then considered a better target for
antiinfluenza drugs. The interest in neuraminidase inhibitors as anti-influenza drugs has only been
revived with the success of 4-guanidino-Neu5Ac2en and its analogs in attenuating viral titer in mice
when administered directly into the lungs [91,95].
A. Inhibition In Vitro
Von Itzstein and co-workers [91] have shown that the 4-amino-and 4-guanidino-Neu5Ac2en inhibit
influenza strains A/Singapore/1/57 and B/Victoria/102/95 in MDCK cells with IC50 values (the
concentration required to inhibit plaque formation in MDCK cells by 50%) of 1.5 mM and 0.065 mM (4amino) and 0.014 mM and 0.005 mM (4-guanidino) respectively. These IC50 values, in particular for the
4-guanidino compound, are well below those found for amantadine, ribovarin, and Neu5Ac2en.
Furthermore in comparison to Neu5Ac2en, the 4-guanidino-Neu5Ac2en inhibitor was 100-fold less
active against human lysosomal sialidase and over 1000-fold more active against a wide range of
clinical isolates of influenza A and B, including amantidine and rimantadine resistant variants [93].
The inhibition of virus replication in MDCK cells has been confirmed [96] and this knowledge
prompted the extension of the inhibition studies to human respiratory epithelium cells in vitro [97],
which indicated high antiviral activity for strains of A(HINI) and A(N3N2) isolates. They also found
that delayed administration of the drugafter viral replication was well estab-
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lishedwas associated with inhibition of virus replication. However viral titer was higher (1.3 log10
compared to 4.0 log10 at 10 mg/mL concentration of the drug) for the delayed administration compared
with viral titer when the drug was present throughout the period of viral exposure. The clinical
significance of this in the treatment of established infections has yet to be explored in detail, although
preliminary clinical trails [98] have indicated some positive results.
B. Administration and Inhibition In Vivo
The earlier work of Palase and Schulman [77] indicated that the failure in inhibiting viral replication in
mice after intranasal and subcutaneous treatment with Neu5aC2en would also occur for Neu5Ac2en
analogs. This failure of Neu5Ac2en as an antiviral treatment in animal models can now be ascribed to
the rapid excretion of the compound [99] thereby not delivering sufficient concentration of the inhibitor
to the infected tissue. It had been shown [91] that the antiviral activity in mice is considerably more,
when the 4-guanidino compound is administered intranasally than when the drug is injected
intraperitoneally. This can be attributed to the localization of sufficiently high concentration of inhibitor
in the lining of the nasal and respiratory epithelia where influenza virus replication is believed to occur.
Animal trials with ferrets challenged with influenza virus have shown the 4-guanidino Neu5Ac2en
compound is effective in studies [91] involving prophylactic administration of the drug. When the drug
is administrated intranasally, 50 g/kg, twice daily, one day before infection with the virus and the
succeeding six days, it substantially reduces virus titer in nasal washing and abolishes fever that usually
appears 3 days after infection.
The drug is currently undergoing clinical trials, and the initial results from a double blind, randomized,
placebo-controlled trial using this compound have been positive both for early treatment and
prophylaxis of experimental inoculation of human volunteers [98] with influenza A/Texas/91.
C. Drug Resistance
Although the active site of influenza virus has been conserved in all known field strains of the virus, the
possibility of drug resistance needs to be addressed. Experience with influenza and other viruses, in
particular HIV, have shown [100] that drug-resistant mutants arise very rapidly, resulting in the
effectiveness of antiviral drugs being short lived. One attempted solution to the problem is the use of
several drugs during therapy [101], making it more difficult for the virus the develop resistance.
In the case of influenza virus, there have to date been no reports of drug resistance from field strains.
However it has recently been reported [102,103]
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that 4-guanidino-Neu5Ac2en-resistant mutants can arise from multiple serial passages of virus in
MDCK cells in the presence of the inhibitor. It has been demonstrated [104] that almost all of the
mutations arise in the hemagglutinin receptor binding site and not on the neuraminidase. These altered
HA variants, which have weaker binding to HA receptors, appear to arise as a result of increased
inhibition of the neuraminidase by the drug. This is consistent with the earlier proposition that the rate of
desialylation of receptor is critically related to the rate of attachment to receptor, for the virus infection
and elution. The decreased activity of the neuraminidase by the drug selects HA mutants with decreased
binding to receptors.
However a drug-resistant neuraminidase mutation has been isolated [103,105] that results in a single
active-site residue mutationwith apparently unaltered activityof glutamic acid 119 to glycine. The
crystal structure of this mutant and its complex (Figure 10) with 4-guanidino-Neu5Ac2en has been
determined [105] and the structure suggests that the decrease in inhibitor binding arises from the loss of
stabilizing interaction with the 4-guanidino group of the drug [72] and alterations in the solvent structure
of the active site. This alteration arises from a water molecule that binds near the location of one of the
carboxylate oxygens of the glutamic acid in the wild type molecule. The location of the 4-guanidinoNeu5Ac2en drug in the complex with the mutant enzyme is isosteric compared to the drug/wildtype
complex [72]. The only differences are the interactions with residue 119.
Figure 10
Stereo image of the Glu119Gly Tern/N9 mutant complexed with
4-guanidino-Neu5Ac2en. The inhibitor binds in a similar conformation as with
the wild type enzyme. The atom labeled Wat represents the water
molecule found in the mutant enzyme that assumes the role of the glutamic acid
in the wild type enzyme. Nitrogen, oxygen, and carbon atoms are shaded
black, dark gray and light gray, respectively.
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VI. Conclusion
It has now been over a decade since the structure of influenza virus neuraminidase was determined
[45,65]. The development of the 4-substituted Neu5ac2en analog inhibitors dates from 1987 when the
structure of sialic acid and Neu5Ac2en complexed with neuraminidase were determined to sufficient
accuracy to permit modeling of potential inhibitors [66]. The development was a multidisciplinary
collaboration of biochemists, crystallographers, molecular modelers, and synthetic chemists and
culminated in the synthesis [106] and biological testing of the compounds [91]. Preliminary data on the
efficacy of these drugs on humans indicate its effectiveness in both prophylaxis and treatment of the
disease [98]. The 4-guanidino compound is in Phase 2 trials (October, 1995). While drug resistance of
the virus has been observed in vitro, it will be interesting to see if variants arise in animal studies as they
do for Amantidine. This is one of the first rationally designed antiviral drugs to be synthesized and
portends well for this methodology to be used as an additional weapon in controling the many pathogens
that have plagued humanity for so long.
Acknowledgments
The author would like to acknowledge Peter Colman, my collaborator in neuraminidase crystallography,
Mike Lawrence for the GRID maps shown here and reading this manuscript, Brian Smith for discussions
on enzyme mechanisms, Jenny McKimm-Breschkin for discussions on drug resistance, Bert van
Donkelaar for technical support, and Paul Davis for computing support.
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19
Rhinoviral Capsid-Binding Inhibitors: Structural Basis for Understanding
Rhinoviral Biology and for Drug Design
Vincent L. Giranda
Abbott Laboratories, Abbott Park, Illinois
Guy D. Diana
ViroPharma, Inc., Malvern, Pennsylvania
I. Introduction
A cure for the common cold has been sought after so long that it has become a cliche: We can
_____(do something difficult) but we can't cure the common cold. There are, unfortunately, a number
of factors that conspire to make the cure for the common cold ephemeral. In spite of these difficulties,
dramatic progress has been made in producing chemotherapies for human rhinoviruses (HRVs), which
are the major cause of the common cold in humans [1]. Although most colds are generally both mild and
self-limiting, they are responsible for both a large proportion of visits to physicians and lost work time
[2]. Billions of dollars are spent in the United States alone on symptomatic relief from this disease. The
ubiquitousness of this disease has led many people to seek a cure for many years (the discovery of the
class of HRV inhibitors described here is over 20 years old). This chapter will describe the influence of
structure-based approaches in the design of a class of antipicornaviral agents called capsid-binding
inhibitors.
Any effort to inhibit HRV replication, and thus cure many common colds, is made more difficult by
three factors. First, there are at least 102 described serotypes of HRVs, and it seems likely that there are
many more serotypes not yet described [3]. Rhinoviruses are responsible for about 4060% of the colds
in humans [1,4]. Therefore, a chemotherapeutic agent would need to be effective
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against most of the HRV serotypes to be useful in approximately 50% of common colds. This
percentage may be somewhat higher because some of the other causes of cold-like illnesses, particularly
the enteroviruses (e.g., Coxsackie and echoviruses), are also inhibited by capsid-binding compounds
[57]. In order to be efficacious, drugs must necessarily have a broad spectrum of activity.
The second difficulty in producing HRV chemotherapy stems from the relatively innocuous nature of
HRV infection. Compounds must be able to be very safely administered, with a minimum of drug-drug
interactions, if therapy is to be acceptable. An analogy may be drawn between common headache
remedies and common cold chemotherapy. Common headache cures such as nonsteroidal
antiinflammatory agents and acetaminophen clearly can cause serious side effects (gastrointestinal
bleeding or catastrophic liver failure) particularly if misused [8]. In spite of this possibility, serious
complications from these agents are quite rare. One would suspect that an antirhinoviral agent would
need to be at least as safe as headache remedies.
The third major difficulty in developing cold cures arises from the fact that the HRVs are RNA viruses.
When presented with any selective pressure, including chemotherapeutic or antibody challenge, RNA
viruses mutate rapidly [9]. This ability to mutate is most clearly illustrated in influenza viruses (RNA
viruses), where new strains continuously arise to circumvent immunity in a population. Influenza A
viruses have been shown to mutate around the anti-influenza drug Amantadine, after a single passage
through a susceptible human host. The mutated viruses shed from a host treated with Amantadine are
now resistant to Amantadine. These mutated viruses appear to be as virulent as the parent strain of virus
[10].
Any effort at antirhinoviral therapy must attend to these three issues: (1) serotypic diversity; (2)
exceptional safety; and (3) viral resistance. Therefore, any structure-based approach cannot concentrate
on potency alone, but must also attend to these three issues as well. The requirement for inhibiting
multiple targets has been addressed in tangible ways using structure-based design and will be discussed
here. Safety issues have been addressed in limited published data from clinical and preclinical studies,
but structure-based design has not played any significant role in addressing these problems. One might
argue that structure-based approaches have aided in the design of clinical backups. These backups are
then brought forward after an initial drug fails for safety reasons. Drug-resistant mutations created in the
laboratory have also been examined structurally and will be discussed. The importance of resistance
developing in the clinical setting has not yet been answered.
This chapter will first introduce the target, the HRVs, and describe their anatomy and life cycle.
Emphasis will be placed on the viral capsid and its disassembly or uncoating. How structural
information has helped in our under-
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standing viral physiology will be highlighted. This will be followed by a description of capsid-binding
antirhinoviral compounds. We will leave discussions of other HRV targets, e.g., proteases, to other
authors more knowledgeable in these subjects. Drug structureactivity relationships will be discussed,
followed by a discussion of drug resistance. Finally clinical trials and future prospects for capsidbinding inhibitors in any antirhinoviral armamentarium will be discussed.
II. The Human Rhinovirus Anatomy
The human rhinoviruses are picornaviruses (pico = small; rna = RNA), a family of small (300 in
diameter), positive sense, single-stranded RNA viruses. Other generas in this family include the
enteroviruses (e.g., polioviruses); the aphthoviruses (e.g., foot-and-mouth disease virus); the
cardioviruses (e.g., mengovirus, EMC virus); and the heparnaviruses (e.g., hepatitis A virus). With the
exception of the heparnaviruses, a crystallographic structure is known for at least one of the viruses in
each genera [5,1117]. These structures show remarkable similarities, which will be described. In spite
of these similarities, caution should be exercised when trying to generalize data gathered in one genus to
other members of the picornavirus family.
The picornaviruses share an icosahedral structure (Figure 1). The icosahedral protein coat that
encapsidates the viral RNA is made up of 60 symmetrically arranged protomers. Each of these
protomers is comprised of four viral polypeptides, termed VP1 through VP4. This was the extent of our
structural knowledge of the picornaviruses until the 1985 structure of HRV14 was published by Michael
Rossmann and coworkers [16]. This was followed rapidly by structural determination of a variety of
other picornaviruses. These structures provided the framework on which to base current paradigms for
the picornaviral life cycles, particularly with regard to their assembly, attachment, and uncoating.
The VP1, VP2, and VP3 all contain a core eight-stranded antiparallel barrel (Figure 2). These
polypeptides have surfaces on both the exterior and interior of the virion particle, facing both solvent
and viral RNA. The fourth polypeptide, VP4, is considerably smaller than the others and does not
contain the eight-stranded barrel motif. The VP4 resides entirely on the interior surface of the virion, in
close association with the viral RNA. The N-terminus of VP4 is known to be myristoylated in both rhinoand polioviruses [18,19].
Three regions of the picornavirus structure deserve special attention because they appear to play crucial
roles in the viral life cycle as well as bear on the function of the capsid-binding compounds. These
regions are the canyon, the VP1 hydrophobic pocket, and the cylinder.
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Figure 1
Schematic illustration of the icosahedral rhinovirus 14.
(a) Shown is the icosahedron comprised of 60 copies
each of VP1 (light gray), VP2 (black), and VP3 (gray).
The shaded circles around each five-fold axis indicate
the canyon positions. Also indicated is the approximate
position of the VP1 hydrophobic pocket that lies
underneath the surface of the virion. (b) An
icosahedral pentamer is expanded with one viral protomer
shown as a protein ribbon diagram. (c) This pentamer is
seen in a cutaway view. Here VP1 is white, VP2 and VP4
black, and VP3 gray. A capsid-binding compound is
depicted as black spheres inside the VP1 ribbon diagram.
The cross hatched regions on the (c) schematic (right)
indicate areas that disorder when HRV14 crystals are
exposed to acid.
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A. The Canyon
The HRVs have been divided into the major and minor receptor groups based on two identified cellular
receptors [3]. The major group, which is comprised of approximately 90 serotypes, binds to the
intercellular adhesion molecule 1 (ICAM-1) [20]. The minor group, about 10 serotypes, binds to the low
density lipoprotein receptor family [21].
The canyons are depressions approximately 15 to 20 deep that encircle each icosahedral five-fold axis
(Figure 1). When first seen in HRV 14, these canyons were postulated to be the site at which a cellular
receptor would bind. Subsequent electron-microscopic data revealed that ICAM-1 does indeed bind in
the canyon as predicted, although in a somewhat different orientation than early models [22,23]. These
canyons allow the receptor binding sites to escape immunological surveillance because the canyons are
too narrow to allow an immunoglobulin to contact the canyon floor. Directly underneath the floor of the
canyon lies a second important structure, the VP1 hydrophobic pocket.
B. The VP1 Hydrophobic Pocket
The VP1 hydrophobic pocket is the site where the capsid-binding compounds reside. This hydrophobic
pocket in VP1 was not initially apparent in the HRV14 structure because it exists in a closed
conformation in the native HRV14. The addition of a capsid-binding antiviral agent induces the pocket
to open. This was first seen by Smith and coworkers when the first crystal structure of a capsid-binding
drug, WIN 51711, was solved bound in HRV14 [24]. (WIN is the designation for a Sterling Winthrop
compound.) This was a seminal event in the structure-based design of these capsid-binding compounds;
before this structure was solved the exact site at which the compounds bound was unknown.
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Figure 2
Panels (a), (b), and (c) depict VP1, VP2, and VP3 barrels, respectively. In all
cases the view is such that the virion exterior would be on the right side of
the page. A capsid-binding compound is depicted as gray spheres as it is
found inside VP1.
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Subsequent studies have shown this pocket is present in every known HRV and enterovirus structure.
This pocket is inside the barrel of VP1, directly underneath the canyon floor where ICAM-1 binds
(Figure 1c). The proximity of the hydrophobic drug-binding site to the receptor-binding site explains the
effects these compounds have on viral attachment in specific HRV serotypes (see below).
C. The Cylinder
The cylinder is a complicated structure that lies on the inside of the viral coat (Figure 1c). There is one
cylinder at each icosahedral five-fold axis. The cylinder is formed by winding the five-fold related
VP3 N-termini around the symmetry axis. In close association with this cylinder is the N-terminal
regions of VP1 and VP4.
The myristoyl moiety attached to VP4 is observed in the poliovirus structures. This moiety is in close
association with the cylinder formed by VP3 [19]. In HRVs, density consistent with the myristoyl
moiety is also seen near the cylinder [25]. In the HRVs, structural disorder of the first 2528 Nterminal residues of VP4 obscures the connection between VP4 and the myristoyl group. The addition of
myristic acid to protein is seen in many viral and cellular
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proteins. It is typically a signal moiety that directs the protein to which it is attached towards cellular
membranes [26].
Above the cylinder is an ion, thought to be Ca2+, which lies right on the five-fold axis and coordinates
to the five adjacent VP1 polypeptides [25,27]. The proximity of the cylinder to structures shown later
to become external during uncoating (portions of VP1, VP4) suggests that it may be important in
uncoating.
III. The Human Rhinovirus Life Cycle
The HRVs must undergo a number of transitions to replicate (Figure 3). First, they must attach to the
cell surface at a cellular receptor. They are then internalized into the cell via the endosomal
compartment. Following internalization, they must eject their RNA out of the viral capsid, through a
lipid bilayer, and into the cytosol in a manner that preserves the integrity of the RNA. Replication
Figure 3
A schematic diagram depicting some of the
required steps in viral replication.
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of the RNA and production of a polyprotein ensues. The polyprotein is then processed by viral proteases
to form the viral polypeptides. The coat must then assemble, package RNA, and leave the cell. The
capsid-binding compounds' effect on propagation have been shown to occur at the attachment,
uncoating, and assembly steps (25,2730). The relative effect on each of these steps appears to be
variable and has not yet been extensively characterized. The most universal effect appears to be on the
uncoating step.
During uncoating several different rhinovirus subparticles are observed. These are thought to be
intermediates in the uncoating process [3233]. The fully infectious 149S particle becomes an 125S Aparticle, which has lost VP4 but still maintains viral RNA. This A-particle is no longer infectious. The Aparticle then releases RNA to become an 80S empty shell.
The formation of these types of particles can be induced by association of the virion with the cell
receptor or acidification [32,34]. In poliovirus, attachment to cells has been shown to lead to a particle
that has lost VP4 and has externalized the N-terminal region of VP1, which normally resides on the
virion interior.
There has been considerable debate about how the HRVs accomplish the transfer of RNA from inside
the virion into the cell cytosol. This step is crucial for productive uncoating. An important question
concerns the requirement for acidification of the endosome for HRVs to release their RNA. Evidence
that appeared to conflict was found in a number of studies using either entero- or rhinoviruses (3539).
This question was later addressed by experiments that specifically separated entero- and rhinovirus
behavior [40]. These experiments showed that HRVs, unlike poliovirus, require a pH-lowering step for
productive infection. This pH lowering is likely to occur in the endosomal compartment. It should be
noted that HRV and enteroviruses have been classified historically based on their resistance to acid:
HRVs are acid-labile, while enteroviruses are stable in acid. Consequently, differences in behavior
between the rhino- and enteroviruses in an acidic environment within the cell are not surprising.
To replicate the changes in HRV that might be induced by acidification in the endosome, HRV14 was
acidified in the crystalline state and examined via x-ray diffraction. When compared to the native
HRV14, the acidified HRV14 capsid becomes disordered in three regions: the Ca2+ ion on the five-fold
axis; a region of the cylinder and the adjacent portion of VP4; and the GH loop [41]. The GH loop is
the region of structure that connects -strands G and H and lies directly between the hydrophobic pocket
and the receptor binding site (Figure 4). It forms the roof of the VP1 hydrophobic pocket and the floor of
the canyon at the receptor binding site.
Mutants that are resistant to acid in vitro were isolated [41,42]. These mutants cluster about the GH loop
(Table 1, Figure 4), and typically would be thought of as mutants that stabilize protein structure (e.g.,
larger or more
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Table 1 VP1 Acid- and Drug-Resistant Mutations
Phenotype
Mutations in VP1
Acid resistant
H1078L, H1078Y, N1100K, N1100T, D1101E, N1145S,

W1163R, V1188A, V1191A, T1216I, M1221L, M1224L,
A1225V
Drug resistant (compensation)
N1100S, N1105S, V11531, N1219S, S1223G
Drug Resistant (exclusion)
V1188L, V1188M, C1199F, C1199Y, C1199W, C1199R
branched amino acids) [4346]. The proximity of these mutations to the GH loop coupled with the
loop's movement on acidification suggest that the GH-loop movement plays an important role in
uncoating.
It has also been suggested that binding of ICAM-1 to the virions may also play a role in uncoating. In
support of this hypothesis, it has also been observed
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Figure 4
A ribbon diagram of HRV14 VP1 bound to WIN 61605 (small gray spheres
with black bonds). The VP1 region that disorders under acid conditions is
depicted by black color on the ribbon diagram. Residues that can be
mutated to be acid stable, drug resistant (compensation type) or to either
phenotype are shown as black, white, and gray ball-and-stick models,
respectively. The majority of mutations are near the site of drug binding
as well as the site of acid-induced disorder in VP1.
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that in some HRVs (particularly HRV14 and to a lesser extent in HRV3, but not HRV16) the addition of
ICAM-1 itself, in the absence of acid, can induce structural changes that mimic uncoating in the capsid
[34]. However, these changes are unlikely to lead to productive uncoating because they occur in the
extracellular space. Therefore, the binding of ICAM alone, at the cell surface, is unlikely to be sufficient
to cause productive uncoating of HRVs.
It seems that the hydrophobic pocket in HRVs is maintained to allow an induced transition around the
GH loop that is required for productive uncoating. This transition could be induced by acidification in
the endosome, receptor binding, or a combination of the two. Filling this pocket in VP1 with a drug or
naturally occurring factor would inhibit this transition, thus inhibit uncoating. As expected, binding of
compounds in the VP1 pocket has also been shown to inhibit intracellular uncoating as well as either
acid-or heat-induced uncoating of the virus [28,29,47].
An attractive hypothesis suggests that the GH loop transition precedes or allows the externalization of
VP4, which would be required for the formation of the uncoating intermediate particles. Remember,
VP4 contains the myristoyl moiety, which can signal VP4 to associate with a membrane. The VP4
would drag the N-terminal region of VP1 (to which it is closely associated) to the exterior of the virion.
This would result in a particle with the N-terminus of VP1 exposed, as has been observed in poliovirus
[4850]. The sequence of this exposed region of VP1 suggests that it can form an amphipathic helix.
The VP1 helices could then insert into the membrane and form a pore, which could allow the passage of
RNA through the lipid bilayer into the cytosol. This is reminiscent of the pore formation by colicin [51].
The observation that both the Ca2+ ion plus the cylinder and VP4 become disordered under acidic
conditions are consistent with this hypothesis. These are regions that would need to disorder to allow the
externalization of VP4 and the N-terminus of VP1.
IV. Capsid-Binding Compounds
Capsid-binding compounds were discovered long before the emergence of the HRV crystal structure
[52]. They were initially discovered on screening of compounds that had been produced by an insect
pheromone project at Sterling Winthrop. Examples of compounds known or presumed to bind in this
pocket are shown in Figure 5 [5269]. The prototypical WIN drug contains an oxazoline ring attached to
a phenoxy group, which is in turn linked by an aliphatic chain to an isoxazole ring. The three rings will
be termed A, B, and C here (Figure 6). Compounds of this type were the first to be shown to inhibit the
viral uncoating and also to stabilize the virion to heat-induced denaturation [29].
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Figure 5
Some compounds which have been shown to inhibit picornavirus
replication. These are thought to bind in the VP1 hydrophobic
pocket. References are indicated on the figure.
The structure of the first compound to be solved in complex with HRV 14 was found to be bound in an
extended conformation within the VP1 hydrophobic pocket [24]. The compound is almost entirely
buried within the capsid of the virion. Since in the native HRV14 the pocket exists in a closed
configuration, binding required large motions in VP1 to accommodate the drug. These
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Figure 6
Some WIN compounds depicting different rings
and linkers. Note WIN 52452 has no C ring.
motions, of up to approximately 4.5 , are most pronounced in the region of the GH loop.
In contrast, HRV1A and HRV16, a minor and major receptor group virus respectively, have their
pockets in open conformations even in the absence of drug [13,15]. For these serotypes, drug binding
typically shifts positions of capsid atoms a maximum of 1 to 2 , smaller than those shifts seen in
HRV14. Drugs bind in these pockets in a fashion similar to that seen in HRV14, extended and almost
entirely buried by VP1.
The structures of five HRVs have been solved to date: HRVs 1A, 3, 14, 16, and 50. In all of these
HRVs, as well as the polio- and coxsackie viruses, VP1 hydrophobic pockets have been observed
[5,12,13,15,17,24,70,71] (HRV3, Zhao, R. et al., personal correspondence; HRV50, Giranda V. L. et al.,
unpubhttp://legacy.netlibrary.com/nlreader/nlReader.dll?bookid=12640&filename=Page_499.html (1 of 2) [4/9/2004 12:36:54 AM]
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Figure 7
HRV14 VP1 hydrophobic pocket schematic with WIN 61605
illustrating some of the terminology commonly used to describe
this pocket. Notice the GH loop separates the pocket from the
canyon floor.
lished data). These pockets all share similar features and have been described as foot shaped, with a
hydrophobic toe region, a heel region capable of hydrogen bonding, and a pore region near the ankle of
the foot (Figure 7).
Many of the picornavirus structures have been shown to have electron density in their VP1 pockets even
in the absence of any added drug. These densities have been modeled as fatty acids or similar
compounds [12,15,56,7072]. The occurrence of these pocket factors have led some to hypothesize that
these factors perform a similar function as do capsid-binding inhibitors, that is, to stabilize the virions
[15,24,41,73,74].
Teleologically one could argue that the virion would pick up a fatty acid in its VP1 pocket before its
egress from the cell, which would then stabilize the virion in transit to new hosts. The HRVs are known
to be stable for long periods of time on surfaces and the dominant mode of transmission is thought to be
hand-to-hand contact [75,76]. When a new host cell is reached, the stabilization factor might exit the
pocket. This would allow the necessary conformational transition (probably at the GH loop) to occur and
allow productive uncoating.
A. Multiple Targets
The use of these structures in a traditional structure-based drug design approach has been limited by the
large number of unknown target serotype structures. There have been useful studies that have grouped
picornaviruses based on their susceptibility to various antiviral agents (see below) [7,77]. However, in
order to design a truly broad-spectrum single drug, the structural elements common among many
serotypes must be considered.
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Because of the large number of serotypes, a successful inhibition strategy needs to consider whether it is
better to have a drug that is exceedingly potent against a small subset of HRV serotypes (e.g. 30%) or a
drug that is somewhat less potent but effective against most serotypes (e.g. 90%). Because of these
considerations, different parameters are required to describe viral inhibition.
Two important values that have been used extensively to describe potency for antipicornaviral
compounds include the mean inhibitory concentration (MIC) and the MIC80. The MIC is the
concentration that inhibits the viral progeny production by 50% in a cell-based plaque assay. The mean
MIC is the average MIC over the number of serotypes against which the compounds have been tested.
The MIC80 is the MIC at which at least 80% of the serotypes tested will be inhibited by at least 50%.
Another way to think about the MIC80 is that it is the MIC value for the serotype that is at the 80th
percentile rank for viruses inhibited. For example, if ten viruses were tested, the MIC80 would be the
MIC concentration of the drug that inhibits the 8th most sensitive virus [15].
B. Potency and Binding Energetics
It would be reasonable to assume that the activity of a drug (MIC) against a specific serotype would be
related to its binding energy. This is an important consideration because algorithms that are used to
predict potency rely on estimations of binding energy. The only experiment completed to directly study
this correlation suggests a rough correlation in a small number of samples. This study however was
limited to a small number of compounds in a single chemically similar series [78]. It is unclear whether
this relationship will hold over diverse chemical entities.
One could imagine a series of compounds that binds, but is ineffective at stabilizing the virion to any
extent, thus ineffective in inhibiting viral replication. This appears to be what was observed when
fragments of WIN compounds that only contained the A and B rings were examined. These fragments
were less able to stabilize the virus to heat-induced denaturation than intact compounds. Although the
binding constant for these compounds has not been determined, the diminished thermostabilization
occurred at concentrations of compound sufficient to allow the compounds to be seen bound in the VP1
pocket via x-ray crystallography [79]. This observation suggests that the drug binding and inhibition
have been decoupled to some degree.
Another question concerning correlating binding affinity to viral inhibition is the number of sites per
virion required to be occupied before the virion can no longer uncoat: all 60, or a few? Further, is there
any positive or negative cooperativity in the interaction? These questions have not yet been answered.
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C. Structure-Activity Relationships
Structural features, as stated above, have been found that are common to the known HRV structures.
These features place constraints on compounds that have been demonstrated in a large number of
structureactivity relationships. Shape considerations, hydrophobicity requirements, hydrogen-bonding
requirements and compound flexibility will be discussed.
Pocket-Shape Considerations
Compound Length. The results of x-ray studies on several HRV serotypes have shown that the
hydrophobic pockets vary in size, but all pockets impose structural constraints on compounds. The
pockets, which almost completely enclose the compounds, require that both drug length and width must
be limited. The results of structureactivity relationships for one series of WIN compounds have shown
that the optimum length of the aliphatic linking region between the phenoxy (B ring) and isoxazole (C
ring) ring is between 3 and 6 carbons (Table 2). This optimum length will vary in other series based on
the extent of substitution on the A and C rings (see below) [54,8082]. This effect on linker length is
obvious both for individual serotypes like HRV14 and for the MIC80, which measures many serotypes.
This effect of pocket length can also be seen when substituents are added to either the A or C ring which
would lengthen the compounds. Additions of alkyl chains of four or more carbons tend to decrease
potency, whereas shorter chains may increase potency (Table 3) [54,83].
In studies on compound length, HRV14 appears to be more sensitive to longer compounds than are
many other rhinoviruses. This is consistent with the observation that the hydrophobic pocket of HRV14
is longer than that of HRVs
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1A, 16, and 50. Cluster analysis examining the sensitivity of 100 different picornaviruses against 15
different inhibitors showed that the sensitivities of different viruses depends on the shape of the pocket,
with shorter pockets preferring shorter compounds [7]. This analysis is consistent with the structural
observations, i.e., the viruses with long pockets (e.g., HRV14 and polioviruses) cluster together. A
cluster distinct from that which contains HRV14, and prefers shorter compounds, is the group that
contains the shorter pocket viruses: HRVs 1A, 16, and 50 (Figure 8).
It is interesting that these drug sensitivity groups do not correspond to the receptor binding groups of the
viruses. For example, both HRV14 and 50 are major receptor group viruses, but the pocket of HRV50 is
clearly shaped more like the pocket of the minor receptor group virus 1A.
The argument that the two different drug sensitivity groups may constitute distinct classes of HRVs has
been bolstered by genetic examination, which has shown that clusters with similar amino acid sequences
correlate well with the drug sensitivity [84]. As with the pocket shapes, these genetic clusters do not
correlate with the two receptor groups into which the HRVs are placed.
Attempts have been made to break through the length barrier imposed by the pocket structure. One
could conceive of a drug that could pass through the pore at the heel of the pocket and connect the VP1
hydrophobic pocket to the canyon floor. Such a drug may have activity that could inhibit uncoating via
stabilization of VP1 as well as inhibit attachment by directly blocking the receptor site. Attempts have
been made to synthesize just such compounds, with extended tails attached to the C-ring. While these
compounds bind, their tail does not extend through the pocket pore, but rather coils up within the pocket
[85].
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Figure 8
Solvent accessible surfaces (dot surfaces) of (a) HRV1A, (b) 14,
(c) 16, (d) 50 filled with WINs 56291, 61605, 56291, 61209
respectively (ball and stick). In all cases the cutaway view of the
pocket has the viewer looking from inside the virion. The pocket
of HRV14 is narrower and longer than the others and this
is reflected in the drug structureactivity relationships (Tables 13).
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Compound Width. Bulk considerations have also been examined for the phenoxy or B ring [80]. In
HRV14, which has a long narrow pocket, compounds with no substituents on the phenoxy ring were the
most potent. However most other serotypes were more sensitive to compounds with substituents on the
phenoxy ring, either a dimethyl or a dichloro. The serotypes that preferred disubstituted compounds
included HRV1A and 50, both known to have wider, shorter pockets (Table 4). This is also in agreement
with the drug-clustering analysis discussed above [7].
Induced VP1 Pocket Changes. The shape constrains discussed above are by nature somewhat
qualitative. The precision with which one could predict the binding energy of a compound based on its
shape is limited by the ability of the pocket to conform to its occupant. Conformational changes within
the pocket are most pronounced between native and drug-bound HRV14, but present to a lesser extent in
both HRV1A and 16. The extent of conformational changes produced by different drugs on the same
virus may differ. This observation is illustrated in a study of a compound SCH 38057 in HRV14. This
compound has a substantially different structure than the WIN compounds, and when bound to HRV14,
induces changes in HRV14 that are also quite different from changes resulting from the binding of a
WIN compound [62].
The ability of compounds to effect conformational changes in the capsid when bound and the variability
of these changes both between drug classes and viral serotypes have made a priori predictions of
potency based on shape considerations very difficult. Comparative Molecular Field Analysis (CoMFA)
studies has been useful, when examining similar compounds in a single serotype (HRV14), in
demonstrating spatial constraints [85], but generalizing these constraints over many serotypes and drug
classes is much more difficult.
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Hydrophobicity Requirements
Drug binding is enhanced by hydrophobicity in that portion of the drug that binds to the pocket toe.
Quantitative structure-activity relationship (QSAR) analysis of these compounds have consistently
shown that the most predictive parameter of antiviral activity is a measure of hydrophobicity, the
octanol:water partition coefficient (logP) [80,82,85]. These studies have also consistently shown that
there is no apparent correlation between electrostatic potential or dipole moment and potency.
This evidence suggests that the drugpocket interactions in the toe of the pocket are low-intensity
hydrophobic interactions. Supporting this hypothesis is the finding that many structurally diverse
molecules can be accommodated in this region, and even structurally similar molecules can bind in
distinctly different conformations. Closely related structures have been shown to shift along their long
axis by as much as 1.6 with respect to their phenoxy substituents [55]. There is even more variability
with respect to the isoxazole placement (Table 5, Figure 9). No single hydrogen-bonding or electrostatic
interaction appears to predominate within the pocket toe.
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Figure 9
A solvent-accessible surface of HRV14 (from the 61605 bound
structure, dot surface) overlaid with WIN 61605 (ball and stick),
51711, 56826, and 56291 (a). The atoms closest to the toe pocket
wall (top) for each compound are in approximately the same
position, while the isoxazole ring (C ring) positions vary significantly.
The atom closest to the toe pocket wall is quite different in (b),
which compares WIN 61605 (ball and stick) to R61837 (tubes) and
SCH 38057 (lines).
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The correlation of potency with logP might suggest that A rings with the lowest hydrogen-bonding
potential would be the most potent. This has not been shown to be true. In fact, rings with heterocyclic
nitrogen atoms are preferred to furan and thiophene rings, which would be expected to have less
hydrogen-bonding potential [86,87]. This observation even extends to tetrazole rings, which have often
been used in pharmaceutical design to replace hydrophilic groups such as carboxylates or esters.
Explanations for the variability in potency due to A-ring changes have not been satisfactory. These
heterocycles lack any consistent pattern of hydrogen bonds with the pocket residues (Table 6) [88]. Like
QSAR analysis, structural analysis has not been able to show any relationship between hydrogenbonding groups in the A ring, or dipole moment, and potency [80,85].
Extensive structural characterization of many different A-ring heterocycles has not yet been done.
Difficulties predicting relative potency of these compounds a priori stem from the lack of understanding
of solvation/desolvation effects as well as difficulties in characterizing the low-intensity hydrophobic
interactions. Consequently, it seems likely that new structureactivity relationships about the A-ring
heterocycle will continue to be determined based on empirical findings.
Hydrogen-Bonding Requirements
Capsid-binding compounds with a hydrogen-bond accepting atom in the region of the drug that binds to
the VP1 pocket heel appear to demonstrate greater potency than do their non-hydrogen-bonding
counterparts (Table 7) [55,89]. Studies using HRV14 where Asn1219, a hydrogen-bond donor in the
pore region, has been mutated to Ala have shown that the compounds bind as well in the mutated virus
as in the native virus [27]. This might suggest that the hydrogen bonding to Asn1219 at the pocket pore
is unimportant. Recent structures of compounds in HRV14 have shown that other hydrogen-bond donors
can function in place of Asn1219, even if Asn1219 is still present. These groups in HRV14 are the
hydroxyl of Ser1107 and the backbone nitrogen of Leu1106. Other hydrogen-bonding groups are present
in other rhinoviruses that coordinate to tightly bound waters, which can also act as hydrogen-bond
donors [55,56]. This provides a flexible hydrogen-bonding network that can then
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accommodate a variety of different hydrogen-bonding groups in the C ring of the drug (Figure 10).
This hydrogen-bonding network has also been observed in all classes of capsid-binding compounds that
have had their structures determined in HRVs [58,62]. Reports of compounds that bind to the capsid and
inhibit picornavirus uncoating, but would seem to have little hydrogen-bonding potential (e.g.,
dichloroflavan) have not been structurally examined [60]. The effect of adding a hydrogen-bonding
group to these compounds near the pocket pore is unknown, but one might expect that the potency of the
compound would improve. Remember that WIN compounds without a hydrogen-bonding group at the
pore region still have antiviral activity, albeit this activity is much weaker than that of an analogous drug
with the hydrogen-bonding group present.
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Figure 10
Possible hydrogen bonds in the pore region of HRV14 bound to WIN
61605 (ball and stick). The waters (spheres W1 and W2) could
potentially form hydrogen bonds to WIN 61605 as well as the side
chains of Asn1219 and Ser1107 as well as the backbone of Leu1106
(residues highlighted as tubes). The viewer is looking
from the virion exterior.
The fluidity of the hydrogen-bond donation network at the pore could allow a large number of
structurally distinct molecules to bind in this location. If the function of the pocket is to bind fatty acids
or other structurally diverse pocket factors and thus stabilize the virion. The flexibility of the hydrogenbonding network at the pore seems ideally suited for such a purpose.
Drug Flexibility
The WIN compounds all contain a flexible linking region that allows them to conform to differently
shaped interiors of VP1 hydrophobic pockets. This flexibility is seen in many compounds that share the
WIN-drug mechanism of action. However, some compounds do not contain a region as obviously
flexible as an aliphatic linker (e.g., those with unsaturated rings as linkers, Figure 5).
Flexibility in the aliphatic linking region of the WIN compounds has been explicitly studied and the
results suggest that such a property is important for
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broad spectrum activity (Table 8) [56,90]. While certain less-flexible compounds may have increased
potency for a particular serotype of HRV, these compounds have lower potency versus other serotypes.
It is not known if the effect of flexibility is an equilibrium or kinetic effect. The flexibility might allow
the compounds to expand or contract to fill available space in the VP1 hydrophobic pocket.
Alternatively, the flexibility may allow the compounds to achieve a conformation required to enter or
leave the pocket, but this conformation would not be seen in the crystallographic experiment. If this is
true, modeling of the equilibrium structure of compounds in the pocket will not be accurate predictors of
compound potency.
StructureActivity Relationship Summary
The combination of classical structureactivity relationships combine with the structures of several
compounds in a number of HRV serotypes have led to a paradigm for designing potent compounds. The
effects of pocket shape, requirements for hydrophobicity in the toe of the pocket, as well as the potential
for hydrogen bonding in the heel region appear to be strong determinants of antiviral potency. The
requirement for a broad spectrum has so far limited a more detailed paradigm, such as one that might
exist for designing inhibitors to a specific serine protease. In spite of this vagueness of antirhinoviral
structureactivity relationships, they have been useful in guiding new compound synthe-
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sis. Large numbers of compounds, which might have been synthesized in the absence of any structural
knowledge, have been eliminated from the list of inhibitors to be created, because these compounds
would not have fit well into the VP1 pocket due to its finite size and hydrophobic nature.
The goal of being able to predict with greater accuracy the potency and spectrum of compounds before
they are synthesized awaits three developments: the structures of more HRVs and compounds, the
ability to more accurately model hydrophobic interactions, and probably the most difficult, the ability to
predict changes in the HRVs that occur due to drug binding.
V. Viral Resistance
As would be expected for any virus, particularly an RNA virus, resistance to capsid-binding compounds
has been observed in the laboratory. The effect such mutations have on the life cycle of the virus is
important. Mutations that occur in regions where changes are poorly tolerated, because that region
serves a vital function in the viral life cycle, are likely to lead to less virulent viruses. The almost
universal inhibition of rhino- and enteroviruses by capsid-binding compounds, coupled with the
observation that all of the viruses with determined structures have a VP1 hydrophobic pocket, suggests
that these pockets serve an important and similar function. It seems unlikely that so many viruses would
maintain such a pocket if it were not a selective advantage. Drug-resistance mutants have been classified
into two groups, exclusion mutants and compensation mutants.
A. Exclusion Mutants
The exclusion mutants' behavior has been readily and adequately determined by biochemical and
crystallographic means [91,92]. The mutations occur within the hydrophobic pocket of VP1 and
thermostabilization studies have shown that these mutations preclude the binding of drug in the VP1
pocket. One mutation site in HRV14 has been located at position 1188, which is on the side of the
pocket closest to the viral interior, away from the canyon. Mutations at this site that convey resistance
are Val rarrow.gif Leu or Val rarrow.gif Met. In both cases the mutation is to a larger side chain,
which would be expected to fill the pocket. The crystal structure of the Val rarrow.gif Leu mutation
confirms this hypothesis and demonstrates that the Leu side chain occupies space that would normally
be occupied by an antiviral drug.
The second site found in HRV14 is at Cys1199. Mutations to Phe, Tyr, Trp, and Arg all confer
resistance at this site. Again, all of these mutations are to larger side chains. The hypothesis that these
mutations function by excluding
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drug from the pocket is confirmed by crystallographic analysis showing that the Phe side chain in the
Cys rarrow.gif Phe mutation occupies the site which would be occupied by a drug if it were bound.
B. Compensation Mutants
More intriguing and more difficult to understand are the compensation mutants. These viruses bind
compounds within their hydrophobic pockets, but are still able to replicate and are therefore able to
compensate for the bound drug (Table 1, Figure 4). These mutations, like the mutations that convey acidresistance phenotypes, cluster about the hydrophobic pocket of VP1 [27,28,92].
Two hypothesis have been presented in order to explain the behavior of the compensation mutants. The
first suggests that there is a link between the binding of receptor by virion and its ability to bind
compounds in the VP1 pocket. The second hypothesis suggests that the effect of a mutation on protein
stability leads to the drug-resistant phenotype. These two hypotheses are not mutually exclusive.
Linked-Binding Hypothesis
The compensation mutants can be subdivided into those that occur on the canyon floor and those that are
inside the VP1 hydrophobic pocket. The linked-binding hypothesis suggests that the subset of
compensation mutations that occur on the canyon floor increase the binding affinity of receptor. An
increase in binding for one of these mutants, Val1153 rarrow.gif Ile, has been observed [28]. This
increased binding would then in turn cause a decrease in the affinity of the capsid for the drug. This
would result in the drug being less effective in inhibiting uncoating.
The second subset of compensation mutants are those that occur inside the hydrophobic pocket but do
not interact with the receptor binding site. These mutations might directly decrease drug-binding affinity
in the VP1 pocket due to decreased hydrophobic interactions caused by the smaller amino acid side
chains [27]. In both subsets of mutations, the binding affinity of a drug is reduced either directly or
through the effects of receptor binding. This decreased affinity for drug would be displayed as a drugresistant phenotype.
StabilizationDestabilization Hypothesis
An alternative explanation for the behavior of the drug compensation mutants suggests that these
mutants allow for increased conformational flexibility in the capsid. This increased conformational
flexibility would compensate for the decrease in flexibility, which is manifested by decreased acid or
thermal lability, induced by the binding of drug. The increase in flexibility would allow a
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conformational transition to occur, presumably near the GH loop, that would be required for viral
uncoating. The conformational transition would lead to productive uncoating, even in the presence of
bound drug.
This hypothesis suggests that drug-resistant mutations would be of the type that destabilize protein
structures. Such mutations are typically from larger to smaller side chains or from highly branched to
less branched side chains [4345]. Four of five observed compensation mutants are of this type (Table
1). The remaining mutant, Val1153 rarrow.gif Ile, while a priori might not be predicted to destabilize
the capsid, has been shown experimentally to decrease viral thermostability when compared with native
HRV14 [28].
The advantage of the stabilization-destabilization hypothesis is that it allows for a single mechanism of
resistance for both compensation mutants occurring in the hydrophobic pocket as well as those that are
outside the pocket near the receptor binding site. Both of these sites are close to the GH loop of VP1 that
becomes disordered under acidic conditions. This stabilizationdestabilization hypothesis also explains
the behavior of acid-resistant mutants, which are predominately of the type which should stabilize a
protein to conformational changes. Wild-type viruses have a preferred stability profile, which allows
uncoating under the proper conditions. Mutations or addition of drug can perturb this profile, resulting in
decreased replication. A second perturbation might then compensate for the first, restoring virulence
(Figure 11). This behavior has been observed in HRV1A, in which mutants have been isolated that
require presence of drug to replicate. These mutations are more acid-labile
Figure 11
The effects of various rhinovirus manipulations. The solid line depicts
the native rhinovirus uncoating profile. Mutations and drugs can
effect this profile to make the virus more or less stable to
pH- or temperature-induced changes.
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than the wild-type virus. At the optimal drug concentration for growth, the pH-stability of these
mutations is equivalent to that of native HRV1A in the absence of drug. Increasing drug concentration
beyond that which is optimal for growth further increases the acid-stability of these mutant viruses so
that they are more acid-stable than native virus without drug (Daniel Pevear, personal communication).
C. Clinical Resistance and Virulence
The clinical importance of these mutations has not been clearly demonstrated. In at least one case a drugresistant mutant was able to grow in vitro in single-cycle growth experiments as well as wild-type
HRV14 [28]. Frequently, however, drug-resistant or acid-stable mutants will not grow as well in cell
culture as native virus [93].
The final analysis of the clinical importance of resistant mutants awaits the results of clinical trails. In
the one study published to date, HRV2 mutants of the compensation type (resistant to the capsid-binding
compound chalcone) were tested in healthy human volunteers [65]. The drug-resistant mutants caused
significantly fewer colds than the normal virus. Mutants that require the presence of drug to grow did
not cause any apparent disease. In this single case it appears that the mutant viruses were not as virulent
as the parent strain.
VI. Animal and Clinical Studies
The idea that an inhibitor that binds to a nonenzymatic, nonreceptor site of a virion could inhibit viral
replication in vivo would a priori be considered to be an unlikely scenario by most in the field of
structure-based design. If this idea were proposed knowing only the structure of the native virus
(especially HRV14, which has a closed-pocket conformation) skepticism would abound. This type of
project arose not from a structure-based approach, but from the tried-and-true screening approach. The
compounds were first shown to be effective in inhibiting viral replication in vitro [52]. This was
followed by an experiment showing that these compounds could inhibit at least one enterovirus in a
mouse model [94].
In this study, suckling mice were inoculated with an echovirus and if untreated they developed a fatal
paralytic illness. When treated either prophylactically or within a few days of the viral inoculation,
virtually all of the mice were protected. This is dramatic proof of the concept that these compounds can
inhibit picornaviral infection in vivo.
There are important differences between mice and men and between enteroviral paralytic illness and
rhinoviral upper respiratory tract infections.
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The differences between mice and men should be obvious to the casual reader. Enterovirus-induced
paralytic illnesses are systemic infections, and the virus must at some point move through the body and
be subject to circulating drug. Upper respiratory infections resulting from rhino-or enteroviruses tend to
be localized to the pharynx, which would require that drug titers remain high in this region of the body.
In spite of these difficulties, there have been some dramatic successes in clinical trials of antirhinovirals,
although clearly there are still obstacles to overcome. Two different classes of antipicornavirus
compounds have been shown to be successful at inhibiting at least some types of infection when
administered prophylactically. The WIN compound 54954, given orally, has been shown to inhibit
Coxsackie virus A21 in humans [6]. The Jaansen compound R77975 has been shown to inhibit HRV9
when administered intranasally 6 times daily [95,96], but R77975 has not been shown to inhibit
infection when used therapeutically (after symptoms occur). Further clinical trials with WIN 54954 were
suspended due to the developments of side effects. More recently VP 63843 has been shown to have
dramatically improved effect when compared with WIN 54954 in the prophylactic treatment of
Coxsackie virus A21 infection in humans and it will be tested therapeutically.
The inability of R77975 to show a therapeutic effect is more likely due to poor pharmacodynamics
rather than a fundamental inability for this compound to inhibit an established infection. The
pharmacodynamic problem was witnessed in the R77975 study, which showed that administration
intranasally 3 times a day did not yield prophylactic protection. Yet if administered 6 times daily the
prophylaxis occurs. Therefore if more potent and metabolically stable compounds are found, more
positive clinical results would be expected.
VII. Future Directions
The structures of the picornaviruses (native, with receptor bound, in the presence of acid, with a myriad
of compounds bound, and of acid- and drug-resistant mutants) have yielded valuable information about
possible molecular mechanisms for their uncoating. These same studies have suggested the mechanism
by which these uncoating inhibitors work. A by-product of this research is the hypothesis that these
compounds may mimic naturally occurring factors that occupy the VP1 pocket. The hunt for these
natural compounds and their significance is underway.
This understanding of the mechanism of action, as well as the structure-activity studies, have also
yielded valuable information for future development of antipicornaviral drugs. The determination of
compound's size, shape, and other physical requirements for activity will also be of assistance.
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From the animal and human experiments it is clear that the cure for some common colds is within reach.
These therapies are also quite likely to be efficacious in enteroviral diseases, for example in cardiogenic
coxsackie virus infection [97].
Acknowledgments
We would like to acknowledge that there have probably been hundreds of people who have contributed
to work reviewed here. We would like especially to thank the many people at ViroPharma, Sterling
Winthrop, Purdue University, and the University of Wisconsin at Madison with whom we have worked
over the years. We would also like to thank Dirksen Bussiere and Yvonne Martin for reading and editing
this manuscript prior to submission.
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20
The Integration of Structure-Based Design and Directed Combinatorial
Chemistry for New Pharmaceutical Discovery
Roger Bone and F. Raymond Salemme
3-Dimensional Pharmaceuticals, Inc., Exton, Pennsylvania
I. New Challenges For Drug Discovery
Rapid advances in cell and molecular biology, together with comprehensive genome sequencing efforts,
are providing detailed correlations between specific pathological conditions and discrete molecular
targets. The same tools of recombinant DNA technology that identify key gene targets also provide the
means for target biosynthesis in quantities sufficient for both the high-throughput screening of
compound libraries for leads and the structure-based refinement of leads using x-ray crystallography and
NMR spectroscopy.
The rapid expansion in genomics data makes it inevitable that targets will be identified whose functions
are so poorly understood that the most rapid and efficient way to establish their involvement in disease
will be through the development of prototype drugs. New approaches to drug discovery that are able to
integrate many different types of information are needed to seize this opportunity and drive an optimally
efficient discovery process.
In what follows, we describe a practical integration of structure-based design and combinatorial
chemistry aimed at enhancing the effectiveness of both approaches. Three-dimensional structures
provide the information required to most efficiently direct the design and optimization of new lead
compounds. Combinatorial chemistry technologies, which are based on high-throughput automated
methods of chemical synthesis, produce new classes of lead compounds and permit the rapid generation
of structureactivity relationships
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Figure 1
An integrated technology for drug discovery combines the
precision of structure-based design with the parallelism of
combinatorial synthesis. Chemi-informatics systems track
and integrate all data emerging from the discovery cycle.
(SAR). Chemi-informatics plays a key role in this integration by assuring that properties important in
drug development are both factored into compound design and cumulatively tracked throughout the
discovery process (Figure 1). The product of this approach is a permanently useful set of drug-design
parameters. The integration of these technologies promises to produce an increase in the efficiency of
drug discovery and may ultimately offer a means for reducing the aggregate failure rate of compounds
selected for development. One paradigm for achieving the integration of these technologies is described
below.
II. Structure-Based Design
Structure-based drug design has become a highly developed technology that is in active use in most
major pharmaceutical companies. Structure-based design is an iterative process in which lead
compounds identified by screening, de novo design, or mechanism-based features are systematically
elaborated to improve potency and specificity [1,2]. The process involves successive rounds of structure
determination of leadtarget complexes, design of lead modifications using molecular modeling tools,
synthesis of new drug leads, and measurement of the chemical and biological properties of the modified
leads using screens for target
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function. Iterative refinement and optimization of drug leads is an effective strategy for generating
potent preclinical candidates. Structure-based design can also be used to design new chemical classes of
compounds that present similar substituents to the target using a template or scaffold which is
chemically distinct from previously characterized leads [3,4].
Structure determination typically relies on x-ray crystallography or high-field nuclear magnetic
resonance to directly visualize the 3-dimensional structure of a molecular target and the structures of
complexes of the target with drug leads. Alternately, many targets fall into identifiable classes that
frequently enable the development of homology models of the 3-dimensional target structure or a
mechanism-based strategy for drug-lead generation. Ongoing genome sequencing efforts have led to the
identification of hundreds of potential therapeutic targets, many of which represent possible sources of
crossover pharmacology. Homology modeling is a key feature of an integrated drug discovery effort
because it allows this genomics information to be utilized early in the development of target ligands or
in the engineering of ligand specificity.
Although structure-based design is an effective technology, current limitations center on the inability to
quantitatively predict how specific modifications of the lead will actually affect ligand binding affinity
[5,6]. This reflects the complexity of the drug-binding process and our inability to accurately predict the
conformational response of macromolecular structures to ligand binding. In addition, we have only
limited ability to accurately calculate molecular energy parameters or to accurately estimate the effects
of factors such as polarizability, solvation, and entropy that may have an important influence on drugbinding energetics. Although computational methods will continue to improve, most design work (and
algorithms) still relies heavily on heuristic rules (Figure 2) that have been developed through experience
and that guide the structural and medicinal chemists in the systematic modification of lead compounds
[7]. As a practical consequence, many cycles of serial lead modification are required in order to produce
molecules of suitable potency and specificity to be considered preclinical drug candidates.
Structural information can increase the efficiency with which pharmacokinetic or toxicological liabilities
in lead compounds are eliminated by suggesting where compounds can be modified so as to alter drug
properties without affecting target potency. Structural data can also be used to direct de novo design of
alternate and distinct chemical classes of lead compounds, each of which might be expected to have a
different pharmacological profile [3,4]. New chemical compound classes can also be designed from
existing lead compounds by recombining substituents and core regions (scaffolds) from existing lead
compounds. Chemically distinct lead series can then be optimized in parallel so that when a preclinical
candidate is found to have inadequate drug properties, a backup is immediately available for preclinical
evaluation. As
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Figure 2
Some heuristic rules frequently used in structure-based drug
design. The positions of bound water molecules are key
indicators for lead modification sites.
outlined below, both the scaffold modification and structural recombination strategies are key
components of an integrated drug discovery technology that combines structure-based design and
combinatorial chemistry.
III. Combinatorial Chemistry
Combinatorial chemical technology enables the parallel synthesis of organic compounds through the
systematic addition of defined chemical components using highly reliable chemical reactions and robotic
instrumentation [811]. Large libraries of compounds result from the combination of all possible
reactions that can be done at one site with all the possible reactions that can be done at a second, third,
or greater number of sites. Combinatorial chemical methods can potentially generate tens to hundreds of
millions of new chemical compounds as mixtures, attached to a solid support, or as individual
compounds. The first combinatorial libraries with millions of members were oligopeptide and
oligonucleotide libraries for which reliable and versatile chemical reactions already existed [10,12,13].
However, these libraries were not generally useful as a source of leads for small-molecule
pharmaceuticals due to the relatively high molecular weight of the resulting leads and other limitations
of oligonucleotides and oligopeptides as drugs. More recently, drug-like libraries have been produced
that offer much greater utility as a source of drug leads (Figure 3). However, practical limitations of the
versatility and reliability of the chemical reactions used to make these libraries have resulted in
somewhat smaller sets of compounds [1418].
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Figure 3
Generation of combinatorial libraries.
Traditional combinatorial libraries based on
polypeptides or polynucleotides were built up
by oligomer condensation and achieved a
high level of diversity through conformational
flexibility. Small-molecule libraries of drug-like
molecules can be built up using a variety of
strategies that focus on maximizing chemical
diversity while (generally) minimizing
conformational flexibility.
From the drug discovery perspective, large combinatorial libraries have the same utility as conventional
pharmaceutical or natural-product compound libraries, i.e., as a source of leads for new drugs. The
design of large combinatorial libraries is driven by the requirement that individual reactions be highly
reliable and versatile, while producing libraries with the highest possible degree of chemical diversity.
Individual steps must be optimized, the compatibility of building blocks must be examined thoroughly
and the synthesis must be automated. As a consequence, a significant investment in time and resources
must be made before a library can actually be produced. Designed and used in this way, large
combinatorial libraries provide a new source of screening leads. However, libraries with maximum
diversity, which may be used for blind lead discovery, do not address the principle rate-limiting step
in drug discovery, the elaboration of a suitable SAR around the lead compound after an active lead has
been discovered.
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While initial combinatorial chemical strategies have focused on the exhaustive synthesis of all members
of a given library, it is inevitable that advances in automated chemistry and equipment will soon make it
possible to synthesize a vastly greater number of compounds than it will be practical to
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screen. For example, for a simple library created using amine and acid condensations onto a given amino
acid scaffold, over a million compounds can be produced from commercially available reagents. If this
simple library is expanded to incorporate the hundreds of commercially available or easily synthesized
amino acids scaffolds, the number of compounds that can possibly be made increases to greater than
108. Although biological, chemical, and physical assays can be automated so that hundreds of thousands
to millions of compounds can be screened annually, the process is expensive and the reliability of the
screening process decreases when measurements are made on mixtures of multiple compounds. Various
estimates of the number of drug-like molecules with molecular weight (MW)<1000 range from 1050 to
10130, making it clear that there is no way to exhaustively screen all of the compounds that can be made.
What is needed instead are strategies for focusing the process of combinatorial chemistry to permit the
rapid refinement of drug properties.
IV. Integrated Structure-Based Design And Combinatorial Chemistry
The limitations and strengths of structure-based design and combinatorial chemistry approaches are
complementary. On a basic level, structure-based drug design suffers from the inability to predict the
energetic effects of even the most conservative modifications of a lead compound. The result is that
many compounds must be individually synthesized to achieve discovery program objectives.
Combinatorial chemistry addresses this limitation by providing the methodology to synthesize many
compounds in parallel so that an extensive array of structure-activity relationships can be developed.
Predictions of the effects of modifying a particular substituent can then be based on empirical SAR data
rather than ab initio or semi-empirical computations.
Combinatorial chemical approaches can potentially produce millions of compounds, more than are
desired or useful, which tax target-screening efforts and compound tracking. Furthermore, the
approaches that allow such large numbers of compounds to be screened, using mixture [12,17], solid
phase screening [19], or single point assays [16,18], generally result in a reduction in data quality, which
makes the development of structure-activity relationships very difficult. Structure-based design methods
address this problem by making the search for new leads and development of SAR more directed and by
scaling back the numbers of compounds that are synthesized. Instead of producing all possible members
of every library, efforts focus on the design of templates that can bias libraries towards a particular target
class and the selection of the library members with the greatest probability of interacting with the target.
Thus the integrated application of structure-based design and combinatorial chemical technologies can
produce synergistic improvements in the effi-
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Figure 4
The information flow in a drug discovery process that
connects elements of structure-based design and
combinatorial chemistry for
drug discovery.
Document
ciency of drug discovery (Figure 4). The process begins with the knowledge-based design of a library
template or scaffold and involves the synthesis of small subsets of library members. As with structurebased design approaches, the process is an iterative one in which SAR data and structural data guide not
only the iterative selection of library members for testing, but also the design of later generation library
scaffolds. The integrated process is described in detail in the next few paragraphs.
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Figure 5
Generation of a virtual combinatorial library by finding
substituents of a custom scaffold that can be
accommodated in the binding site of a molecular target
or meet other 3D structural criteria. Once the virtual
library is synthesized in the computer, individual
members can be selected using structural or additional
criteria and synthesized using automated equipment.
The first step in the process is the generation of a chemical template or scaffold that can be derivatized
at multiple sites using reliable chemical reactions to produce a large combinatorial library (Figure 5).
This custom chemical template is designed based on the structure of the target using the same heuristic
set of rules used for traditional structure-based drug design. Useful 3-dimensional pharmacophore
models are best derived from crystallographic or nuclear magnetic resonance structures of the target, but
can also be derived from homology models based on the structures of related targets or 3-dimensional
quantitative structure-activity relationships (3D QSAR) derived from a previously discovered series of
active compounds [20]. In addition, the mechanism of action of the target or any other information that
exists regarding the target or the target class can be used in the design to maximize the chances of
finding hits [21,22]. The combinatorial libraries are designed so that a few thousand to millions of
discrete molecules can be produced by reaction of the custom-designed template with appropriate
proprietary and commercially available chemical building blocks.
The next step in the process involves the implementation of the automated synthesis and generation of
the library. Significant lead time is anticipated before a library can be produced because even the most
reliable chemical reactions require optimization if they are to be carried out by a robot, particularly if
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the reactions are to be implemented with the template attached to a solid support. When the synthesis is
optimized and fully automated, thousands to millions of compounds are accessible.
One of the key features of this process is that all of the compounds that can potentially be made by
elaboration of a custom scaffold are first made in virtual form in a computer. Each of the chemical
reactions required to create the library is encoded in a program that then systematically combines the
template and the building blocks to create a 2-dimensional representation of each member of the library.
Next, 3-dimensional representations are created and molecular-property descriptors are calculated for
each member of the library. Molecular property descriptors encompass molecular connectivity, dipole
moments, calculated partition coefficients, and many other calculable molecular properties. The virtual
library of compounds can then be computationally screened and the library members ranked according
to their ability to interact with the target receptor or 3-dimensional pharmacophore model [2325]. The
compounds can also be ranked by their inability to interact with any number of alternative targets whose
inhibition is undesirable, or their ability to meet any range of desired chemical or physical properties
that may be important in drug pharmacology. Alternately or additionally, compounds can be ranked
according to their ability to span or sample the physical-chemical property space to produce the most
diverse set of compounds for initial screening [26,27].
Small sublibraries of the large virtual library that best satisfy the selection criteria are chemically
synthesized using automated methods and then the biological and/or chemical properties of each
compound are measured using automated assays. The SAR data that emerge from the assays are stored
in a central database and used in the selection process to drive additional rounds of sublibrary selection,
synthesis, and assay. Multiple mathematical models are developed to correlate the computed structure
and properties of each synthesized library member with the biological, chemical, or physical properties
that are measured during each cycle of testing [28]. A key feature of this approach is that compounds
can be selected not only on the basis of which are predicted to perform the best in the target assay but
also on the basis of their ability to perform the best in the target assay but also on the basis of their
ability to distinguish between or validate the SAR models that are generated. The observed and
predicted properties of a given sublibrary are compared so that the set of assumptions upon which
property refinement is based is constantly updated. In principle, this process can become completely
automated so that leads are discovered and refined with very little manual intervention [28].
To achieve the greatest improvements in drug discovery efficiency, empirical data of various kinds must
be collected throughout the iterative refinement process. It is desirable to obtain more accurate
dissociation constants rather than IC50 or single-point percent-inhibition values. In addition, the 3dimensional structures of interesting targetinhibitor complexes are determined
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Figure 6
The combination of structure-based design and combinatorial
chemistry can facilitate the generation of recombined
compounds to rapidly produce potent compounds with
maximum chemical and structural diversity.
to provide information regarding how substituents are interacting with the target and when binding
modes change. This information may be essential if SAR models are to remain predictive over large
numbers of compounds.
The integrated drug discovery process utilizes as much information as is available to find and optimize
initial lead compounds. Because the automated synthesis of large libraries of compounds requires
reliable and versatile chemical reactions, initial libraries are designed to discover new chemical lead
classes or to develop SAR models. Both library and substituent designs evolve as hits are encountered
and the structures of targetinhibitor complexes are determined. Single modifications on both the
template and substituents may be made during the process based on the structures of targetlead
complexes. When sufficient SAR data has accumulated and the structures of target complexes with key
templates and substituents have been determined, potent compounds with the desired drug like
compositions are designed and synthesized using a structure-based recombination strategy (Figure 6).
The integration of these recent advances in drug discovery offers the possibility to substantially decrease
the time required to find initial leads and develop them into prototype drugs. Similarly, the interval
required to produce preclinical development compounds and backup candidates is also expected to
shrink. Perhaps most interesting is the potential of this integrated technology, which is ultimately based
on an abstract information model of the biological process, to dramatically increase the reliability of
successful drug development
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by factoring in and refining important drug properties concurrently with the optimization of drug affinity
and specificity for a specific molecular receptor. This objective is achieved by integrating assays and
computational models that relate to important drug development issues (e.g., oral absorption, optimal
pharmacokinetics, minimal toxicity, etc.) directly into the iterative design process.
V. Chemi-Informatics
The development of methods that can optimize the parallel refinement of drug potency and
pharmacological properties is a key objective in enhancing the efficiency and productivity of drug
discovery. To address this problem and to handle the dramatic increase in experimental data generated
using robotic synthesis and assay methodologies, advanced informatics systems are required to collect
and exploit data relating to the properties of the chemicals being produced. These systems will amplify
the synergy between structure-based design and combinatorial chemistry and provide a means for
reducing the aggregate failure rate of development candidates.
The poor success rate for preclinical development candidates (about 1 in 20) results from our limited
ability to predict such drug properties as intestinal absorption, excretion, metabolism, toxicity, efficacy,
and side effects. Rendering the prediction more difficult is the certainty that these drug properties are
composite properties that result from the operation of many biological processes. Recently, progress has
been made towards understanding the underlying molecular basis for some of the individual components
that contribute to the observed drug properties. For instance, the absorption of compounds into Caco-2
cells in culture may be predictive of intestinal absorption [2931]. Retention times of compounds during
artificial membrane chromatography has also been correlated with oral absorption [32]. Recently,
molecular transporters have been identified, cloned, and characterized that are responsible for the
absorption of dipeptides and the excretion of organic cations [33,34]. The enzymatic basis for the
metabolism of xenobiotics (cytochrome P450, glutathione transferase, etc.) has been known for some
time, at least in part. In fact, many of the individual components of the composite biological processes
can be developed into high-throughput assays, providing the opportunity to collect SAR data and
develop SAR models.
These observations, together with the integrated structure-based design and combinatorial chemical
technology described here, define a comprehensive new strategy for drug discovery that has the
potential to reduce the aggregate failure rate for development candidates (Figure 4). The creation of
virtual libraries of compounds that are readily accessible through the use of automated
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synthesis methods allows for the first time an extensive and systematic investigation of structure-activity
relationships related to drug properties. Small sublibraries can be selected, synthesized, and screened in
high-throughput assays with the goal of developing predictive SAR models for individual components
of the biological processes responsible for pharmacokinetic and toxicological properties of drug
candidates. Eventually, it will be possible to assemble the predictive SAR models for various component
processes to produce predictive models for bioavailability and toxicology. At this point it will be
possible to use these models to guide the selection of sublibraries directed against therapeutic targets so
that library members with the most drug like properties and the fewest liabilities are chosen. Steering
away from compounds with undesired properties will focus the selection process on molecules with an
improved probability of successful development. This strategy permits the simultaneous refinement of
multiple chemical properties in addition to target efficacy and will shorten the drug-discovery process.
Developing a system capable of collecting multivariate SAR data and exploiting the data to produce
predictive SAR models is a major systems integration task. However, recent advances in computers,
operating systems, and computational chemical tools now enables the implementation of a system that
can track compounds, store chemical property data in a comprehensive relational database, and operate
on virtual libraries in an iterative fashion to develop SAR models and refine chemical properties [28].
Tools for the production of virtual libraries have been developed by several groups and large virtual
libraries can be produced within a few days using high-power computer workstations. A variety of tools
also exist for selecting compounds based on criteria such as the ability to fit a target receptor [23],
similarity or diversity [26,27], or any number of other properties that might be important for interaction
with the target receptor. Statistical programs also exist that are up to the task of developing SAR models
[20]. To obtain SAR models that have utility in the drug-discovery process it will be necessary to collect
data on the properties of large numbers of compounds so that the models are predictive across diverse
chemical classes of lead molecules. This requires implementation of a comprehensive relational
database to collect and correlate SAR data.
The challenge is to integrate these tools into a system that can collect and operate on vast arrays of
chemical-property data in an intelligent fashion to direct and refine the properties of robotically
synthesizable compounds. One approach that has been used successfully is to create a hierarchical clientserver system with a powerful central selection algorithm (Figure 4) that chooses compounds from
virtual libraries based on their computed properties, supervises data analysis, and integrates results from
experimental measurements [28,35]. This system, which has been termed DirectedDiversity in the
author's laborato-
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ries, holds the promise of a major reduction in the time required to produce new preclinical compounds
with enhanced development potential.
VI. Conclusion
The preceding outlines a new drug-discovery paradigm that integrates structure-based design, directed
strategies for combinatorial chemical synthesis, and a comprehensive chemi-informatics system for
accumulating and analyzing information regarding chemical properties. Three-dimensional structures
provide the information required to most efficiently direct the design and optimization of new lead
compounds. High-throughput automated methods of chemical synthesis produce new classes of lead
compounds and provide for the rapid generation of structureactivity data. Chemical informatics
systems track chemical compounds, store chemical property data, develop predictive SAR models, and
provide a means for intelligently directing the drug-discovery process. By applying this approach not
only to the therapeutic target, but also to molecules involved in absorption, clearance, metabolism, or
toxicology it will be possible to develop predictive models for bioavailability and toxicology. Ultimately
this approach will greatly increase the cost effectiveness and efficiency of drug discovery by reducing
the aggregate failure rate for development candidates.
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21
Structure-Based Combinatorial Ligand Design
Amedeo Caflisch
University of Zrich, Zrich, Switzerland
Claus Ehrhardt
Novartis Pharma Inc., *Basel, Switzerland
I. Introduction
Structure-based ligand design is fascinating and challenging. Whenever it is possible to determine the
three-dimensional structure of a pharmacologically relevant enzyme or receptor, computational
approaches can be used to design novel high-affinity ligands. These methods can complement the broad
screening efforts, which represent traditional lead discovery.
In this chapter we focus on our approach to computer-aided ligand design. It is based on the docking of a
diverse set of molecular fragments into the active site of a macromolecular target and on the use of a
combinatorial strategy to connect them to form candidate ligands. The methodology is illustrated by an
application to human thrombin, a trypsin-like serine protease fulfilling a central role in both hemostasis
and thrombosis. The selective inhibition of thrombin is expected to prevent thrombotic diseases.
Ligand-design programs are being developed at an ever increasing rate and some are related to various
aspects of our ligand design approach. The LEGO software tool is based on the combination of multiple
fragment docking, automatic connection by small linker units (one to four atom chains), and searching
of three-dimensional databases for complementary molecules [1,2]. It has been implemented within the
MOLOC molecular modeling system [3], which allows the visualization of the functionality maps and
interactive model building of the growing ligands. Another related approach is that embodied in the
program LUDI [48]. It makes use of statistical data from small-molecule
* Formerly Sandoz Pharma Ltd.
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crystal structures to determine binding sites of molecular fragments, i.e., discrete positions on the
binding site surface suitable to form hydrogen bonds and/or to fill hydrophobic sites of the receptor.
Alternatively, it uses simple rules or the output of the program GRID [912] to generate the interaction
sites. Finally, the fragments fitted in the interaction sites are connected by linker groups. Other fragmentbased programs are GROUPBUILD [13]; GROW [14], HOOK [15], NEWLEAD [16], SPROUT [17],
and TORSION [18]. These and other strategies for computer-aided structure-based ligand design have
been reviewed by several contributors [13,19,20].
II. Docking Molecular Fragments
A. Multiple Copy Simultaneous Search
The present approach for ligand design is based on the combinatorial selection of molecular fragments
optimally docked on the protein binding site to form a population of diverse candidate ligands. The
multiple copy simultaneous search (MCSS) procedure combines the advantages of random distribution
and simultaneous minimization of a set of replicas of a chemical fragment to obtain maps of
energetically favorable positions and orientations (local energy minima) [21,22]. These maps, which
contain all possible low-energy minima of a fragment-protein complex, are called functionality maps. A
plethora of structural and thermodynamic data on inhibitor-enzyme complexes [2326] suggest that the
burial of nonpolar groups of the ligand in hydrophobic pockets of the protein is important for binding
affinity and that intermolecular electrostatic interactions determine selectivity. For this reason, and
because most of the known enzymes' binding sites have both hydrophilic and hydrophobic character,
very diverse functional groups are used in MCSS. Representative examples include charged (e.g.,
acetate, benzamidine, methylammonium, methylguanidinium, pyrrolidine); polar (e.g., methanol, 2propanone, N-methylacetamide); aromatic (e.g., benzene, pyrrole, imidazole, phenol); and aliphatic
(e.g., propane, isobutane, cyclopentane, cyclohexane) groups. Although most of these fragments are
rigid, MCSS can also generate the functionality maps of flexible medium-size fragments, e.g., the amino
acid side chains. Additional functional groups and more complex heterocyclic systems are currently
being introduced to increase the diversity of the resulting ligands and to better characterize the
specificity of the binding pockets (A. Caflisch and C. Ehrhardt, unpublished results).
As shown by a flowchart in Figure 1, the method is fully automated, although certain critical parameters
(e.g., number of replicas, radius of the sphere for random distribution, CHARMM parameters for the
minimization) can be adjusted by the user to optimize it for specific applications. Several thousand
replicas of a given group are randomly distributed inside a sphere
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Figure 1
Schematic representation of the MCSS procedure. Conditional
statements are enclosed by diamonds.
whose radius is chosen large enough to cover the entire region of interest. This can be a known binding
site or the entire protein, if one wants to explore alternative binding pockets. The initial random
distribution also can be performed inside a parallelepiped if the region of interest is elongated in one or
two directions. A minimal distance can be given as input to avoid bad contacts between functional group
atoms and protein atoms for the initial distribution.
Subsets of between 500 and 3000 randomly distributed replicas of the same group are simultaneously
minimized in the force field of the protein. The
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interactions between the group replicas are omitted. The polar-hydrogen approximation (PARAM19) of
the CHARMM force field is used [27]. In the application of the MCSS method to the sialic acid binding
site of the influenza coat protein hemagglutinin [21], HIV-1 aspartic proteinase [22], and thrombin
[20,28] the protein was kept fixed; hence, the forces on each replica consist of its internal forces and
those due to the protein, which has unique conformation and, therefore, generates a unique field. The
positions are compared every 1000 steps to eliminate replicas converging toward a common minimum.
Further details concerning the methodology are given in References 21 and 22, while a critical
assessment has been presented in Reference 20.
B. Simple Approximations of Solvation Effects
In previous applications of MCSS [21,22] the effects of the solvent were neglected, i.e., all proteinfragment interactions were calculated with a vacuum potential [27]. This choice was based on the
principle that fast methods are necessary to perform effective searches of the binding site and that good
candidate ligands subsequently can be ranked in terms of their binding free energy [20,28]. A possible
difficulty with this approach is that minimized positions may be missed or misplaced due to the lack of a
solvation correction during the MCSS minimization.
Electrostatic Shielding
In MCSS studies of thrombin [20,28], it was observed that minima of charged groups tend to cluster in
the vicinity of charged side chains on the thrombin surface and in the S1 (basic groups) or S1' pocket
(acidic groups). It is then necessary to estimate the electrostatic desolvation of both protein and fragment
to obtain a realistic ranking of the minima [28]. As a simple test of the importance of electrostatic
shielding, a distance-dependent dielectric function [29] was introduced instead of the unit dielectric
constant in the vacuum potential. The overall shape of the acetate map did not change, but three more
minima were found close to the Lys60F side chain in S1' [20]. It is difficult to find a physical meaning
in favor of the distance-dependent dielectric function. Nevertheless, it is a simple and useful
approximation, since it yields a smoother and more realistic potential surface than the vacuum
Coulombic interaction.
Hydrophobic Effect
Aliphatic and aromatic fragments do not bear any partial charges in the polar-hydrogen approximation.
Hence, MCSS determines their optimal position in the
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protein binding site exclusively by van der Waals interactions. Minima of nonpolar fragments may be
found in hydrophilic pockets because of the lack of an energy penalty for protein desolvation. A
representative example of a cluster of propane minima in a mainly hydrophilic region of the HIV-1
aspartic proteinase binding site is shown in Reference 20. In a simple attempt to approximate
desolvation of polar regions of the protein, the attractive contribution of the van der Waals interaction
energy was switched off between atoms of nonpolar MCSS fragments and all protein polar hydrogen,
nitrogen, oxygen, and sp2 carbon atoms. In addition, the van der Waals radius of nitrogen and oxygen
atoms was increased from the PARAM19 default value of 1.6 to 2.2 and the van der Waals radius
of the aliphatic carbons was reduced by 0.1 to avoid the too large van der Waals distance between
carbons often produced by PARAM19. The modified force field yields thrombin functionality maps of
propane, cyclopentane, cyclohexane, and benzene in agreement with structural data of known inhibitors
(see Section II. C). In addition, these nonpolar groups are prevented from occupying hydrophilic
pockets.
As a further test of the modified force field, the MCSS procedure was used to generate the propane
functionality map on the surface of the A peptide chain of the leucine zipper, i.e., residues 249281 from
the yeast transcriptional activator protein GCN4. Leucine zippers are composed of amphipathic
helices containing heptad repeats (abcdefg) in which hydrophobic residues are frequent at a and d. As
the x-ray structure indicates [30,31], the two amphipathic helices are held together by hydrophobic
interactions between residues in the a and d positions (Figure 2). The B peptide chain was removed and
MCSS was run separately with the original and the modified force field starting from the same random
distribution of 5000 propane replicas around helix A. The sixty most favorable minima obtained with the
modified force field are distributed in twelve clusters, seven of which match the Val and Leu side chains
of the B helix involved in the interhelical interactions (Figure 2). Of the remaining five clusters, labeled
A to E in Figure 2, B has minima in contact with Val and Ala side chains, D with Leu and Tyr side
chains, while A, C, and E with both a Val or Leu side chain and the alkyl part of Lys side chain. The
sixty most favorable minima obtained with the original force field form sixteen clusters (not shown).
Only four of these match Val and Leu side chains of the B helix, while eight clusters desolvate one (or
more) polar group on the hydrophilic (exposed) surface of the A helix.
The functionality maps of polar groups generated with a distance-dependent dielectric and those of
nonpolar groups obtained with the modified force field are closer to those obtained by using a
continuum dielectric model [3235] to postprocess the MCSS minima and estimate solvation effects
[28]. Hence, simple modifications of the force field may result in more accurate projections of the
binding free energy. Since these modifications are used during the
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Document
Figure 2
Stereo view of the sixty propane minima (thick lines) obtained with the modified force
field (see text) on the surface of the A peptide chain (medium lines) of the GCN4 leucine
zipper (PDB code 2ZTA). Although the B peptide chain was removed during the MCSS
procedure, its backbone and hydrophobic side chains are also drawn (thin lines) to show
how the propane minima match the aliphatic groups of chain B. Hydrophobic residues
are labeled at their C atom.Five clusters of propane minima that do not match the
hydrophobic side chain of the B helix involved in the interhelical interactions are
labeled from A (top right) to E (bottom center) and discussed in the text.
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minimization phase, a more realistic distribution of functional group minima is generated.

C. Thrombin Functionality Maps
Human thrombin is one of the best characterized enzymes from a structural point of view (Figure 3). It
binds a series of diverse inhibitors without major rearrangements in its conformation, as shown by a
number of x-ray crystallography studies [26,3639]. Its S3 and S2 precleavage subpockets have
hydrophobic character, whereas at the bottom of the S1 or recognition pocket the carboxy group of
Asp189 is a salt bridge partner for basic side chains (Figure 3). D-phenylalanyl-L-prolyl-L-arginine
chloromethane, PPACK, (Figure 4a), and N-((2-naphthylsulfonyl)glycyl)-DL-pamidinophenylalanylpiperidine, NAPAP, (Figure 4b) are the archetypal active-site inhibitors of
thrombin. The crystal structure of the thrombin-NAPAP complex is shown in Figure 3. The PPACK and
NAPAP inhibitors bind to the thrombin active site by occupying the S3 and S2 pockets with their
hydrophobic moieties and by positioning their basic group (guanidinium of PPACK, benzamidine of
NAPAP) into S1 to form a salt bridge with Asp189.
In continuation of a project aiming at the structure-based design of low molecular weight, active-site
directed inhibitors of human thrombin [40], MCSS was used to generate a series of functionality maps
of the thrombin S3 to S2' pockets [20,28]. A detailed description of the thrombin functionality maps and
the continuum approximation used to postprocess the MCSS minima is given in Reference [28]. From
the analysis of the results for the nonpolar groups it is evident that hydrophobic moieties prefer to bind
to the S3 and S2 pockets (Figure 5). The solvent exposed face of the Trp60D indole is another favorable
site, though the intermolecular van der Waals interactions are much smaller. Binding to the S2' region is
favored by interactions with the Leu40 side chain but implies a desolvation penalty because of the burial
of part of the Arg73 guanidinium and/or the Gln151 side chain. The latter might be an artifact of the
rigid protein structure used in the minimization, since the side chains of Arg73 and Gln151 are flexible
enough to displace their polar groups towards a more exposed region. Binding to the neighboring Leu41
side chain in S1' is highly unfavorable because of the concomitant desolvation of Lys60F.
For polar groups with zero net charge there are several hydrophylic groups on the thrombin main chain
that might be involved in strong hydrogen
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Figure 3
(a) Stereo view of the thrombin molecule in its complex with NAPAP. The side chains of
thrombin involved in the binding of NAPAP and the disulfide bridges are shown in
stick-and-ball representation with black sticks. NAPAP is shown in stick-and-ball
representation with white sticks. (b) Zoom image of the active site region. Figures made
with the program MOLSCRIPT [42].
bonds. These are 214CO, 216NH, 216CO, and 219NH in S1; 193NH and 195NH in the oxyanion hole;
41CO in S1'; 40CO in S2'; and 147NH and 148NH on the autolysis loop, whose exposure is dependent
on crystallization conditions and inhibitor type.
Two main conclusions can be drawn from the analysis of the minimized positions of the charged
functional groups. First, the minima with the lowest binding free energy have optimal hydrogen bonds
with the Asp189 side chain in the S1 pocket. Representative examples are the lowest energy minimum
of benzamidine (Figure 5) and the lowest energy minima of methylguanidinium and methylammonium
(Figure 4 of Reference 28). Since the Asp189 side chain
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Figure 4
Chemical structure of PPACK (a) and NAPAP (b).
is more buried than the side chain of Lys60F, the minima of positively charged groups interacting with
the former have a more favorable binding free energy than those of the negatively charged groups close
to the latter. This is due to reduced shielding of the chargecharge interaction and the smaller
desolvation of the carboxylate oxygens of Asp189 compared to the amino group in Lys60F [28].
Second, polar groups on the protein surface are not ideal partners for a charged functional group because
the high desolvation penalty for these groups might not be completely compensated for by the favorable
electrostatic interaction energy. This finding is analogous to the results for the polar functional groups,
i.e., their binding to a partially exposed charged side chain of the protein may result in an unfavorable
total binding free energy.
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Figure 5
Stereo view of the three lowest energy minima of benzene obtained with the
modified force field and the lowest energy minimum of benzamidine (thick lines for
heavy atoms and thin lines for polar hydrogens) in the thrombin active site (thin lines).
The inhibitor PPACK is also shown (medium lines), though it was removed during the
MCSS procedure. Some C atoms of thrombin are labeled.
III. Connecting Molecular Fragments

A. Computational Combinatorial Ligand Design
Overview
The recently developed program for computational combinatorial ligand design (CCLD) requires as
input atomic coordinates and partial charges of the protein atoms, as well as the coordinates of the
MCSS minima and the individual contributions to the free energy of binding [28]. An additional file
contains a number of control parameters and, for each functional group used for MCSS, a list of atoms
which can be used for connection (linkage atoms). The following procedures are performed during a
regular execution of CCLD (Figure 6): The MCSS minima are first sorted according to their
approximated binding free energies [28]; then a list of bonding fragment pairs and a list of overlapping
fragment pairs are generated (see below). This is followed by the combinatorial generation of putative
ligands (see below).
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Figure 6
Schematic representation of the CCLD program. Variable assignments are symbolized
by :=. Conditional statements are enclosed by diamonds. [From Reference 28.]
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Lists of Bonding Fragment Pairs and Overlapping Fragment Pairs

The user has to specify for each functional group type which atoms are to be used for connection to
other fragments. For each linkage atom CCLD generates a set of possible linkage points, i.e., points that
will be used to determine the position and orientation of the link. All possible pairs of minimized
positions are then analyzed and added to the list of bonding fragment pairs if they can be linked;
otherwise, if two fragments have bad contacts they are added to the list of overlapping fragment pairs. A
pair of bonding fragments may be connected by a linker unit, by a single covalent bond (1-bond), or by
fusing two overlapping atoms belonging to different fragments (0-bond). The linker units are small since
their function is to optimally connect two fragments without adding considerably to the molecular
weight. The following linker elements have been implemented so far: Keto and methylene (2-bond),
amide and ethylene (3-bond). The user is free to choose minimal and maximal values for the distance (d)
between linkage atoms for each connection type. In the application to thrombin the following values in
ngstroms were used: d<0.43, 0-bond; 1.2< d<1.8, 1-bond; 2.2 <d< 2.7, 2-bond; 3.6 <d<4.0, 3-bond.
The list of bonding fragment pairs and the list of overlapping fragment pairs are created only once
before entering the combinatorial search (Figure 6). The use of these lists results in a significant increase
in the speed with which ligands are generated.
Combinatorial Ligand Generation
Starting from the MCSS minimum with the most favorable binding free energy the ligand generation
algorithm proceeds in an iterative and exhaustive way by linking an additional fragment to the actual
construct. Such an elongation step is very fast since it is sufficient to check that the new fragment may
be connected to one of the fragments in the actual construct (by looking in the list of bonding fragment
pairs) and that the new fragment does not overlap with any of the fragments in the actual construct
(Figure 6). The combinatorial explosion problem is kept under control by pruning, which is performed
according to the average value of the free energy of binding of the fragments. Whenever the addition of
a fragment to the growing ligand results in an average value of the binding free energy higher than a
user specified threshold, the construct is reduced by deletion of the latest added fragment (Figure 6). A
ligand with energy below the threshold is saved if it is larger than a user specified minimal size and if it
is not a substructure of a ligand found previously.
A CCLD run requires from 23 minutes (for 200 to 300 fragments) to less than one hour CPU time (for
about 1000 fragments) on an SGI R4400 processor.
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B. Candidate Ligands of Thrombin

To test if CCLD is able to reproduce the known thrombin inhibitors, the functional groups of PPACK
and NAPAP were given as input molecular fragments in the orientation derived from the crystal
structures of the complexes. The CCLD program generated a set of candidate ligands that not only
contained the PPACK and NAPAP structures but also a number of interesting hybrid molecules
consisting of fragments from both inhibitors. A representative example is shown in Figure 7. This
putative ligand consists of the C-terminal part of NAPAP, whose piperidine ring is connected at the 3position to the PPACK D-Phe by an amide linker. The latter has its carbonyl oxygen involved in a
hydrogen bond with the Gly216 NH.
In another run, the MCSS minima of benzamidine (Figure 5), benzene (Figure 5), cyclopentane, and
cyclohexane [28] were used as starting molecular fragments. In a few seconds of CPU time of an SGI
Indigo2 (R4400 processor), CCLD generated a series of molecules showing the same interaction
patterns as those of known thrombin inhibitors, i.e., hydrophobic moieties in S3 and S2, hydrogen bonds
with the polar groups of Gly216, and benzamidine in S1. One of these putative ligands is shown in
Figures 8 and 9. It is involved in the same interactions as in the NAPAP-thrombin complex except for
the hydrogen bond with the CO of Gly216. Its cyclohexane ring in S3 is connected to the
Figure 7
PPACK-NAPAP hybrid ligand (thick lines) generated by CCLD starting
from the functional groups of PPACK and NAPAP (thin lines). The amide linker
connecting the piperidine in 3-position to the D-Phe was created by CCLD.
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Figure 8
Minimized structure of a putative ligand suggested by CCLD (thick lines).
The CCLD run used MCSS minima of benzamidine, benzene, cyclopentane, and
cyclohexane. The putative ligand was minimized in the thrombin active site, whose residues
within 8 of any atom of the ligand were allowed to move. The remaining residues of
thrombin were kept rigid. The NAPAP structure is also shown (thin lines) as a basis of
comparison.
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Figure 9
Stereo view of the minimized complex between the putative ligand shown in Figure 8
and thrombin (thin lines). Intermolecular hydrogen bonds are drawn by dashed lines.
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N-acylpyrrolidine ring in S2 by a single methylene linker. This is a novel design and the candidate
ligand appears to be more rigid than NAPAP, since it has a smaller number of rotatable bonds. Hence,
the penalty paid for the loss in entropy upon binding should be smaller for this CCLD hit than for
NAPAP.
In a study with a more diverse set of starting molecular fragments, ligands containing both a core
similar to known inhibitors and additional intermolecular hydrogen bonds and/or van der Waals
interactions were generated [28].
IV. Conclusion
A combinatorial approach for the computer-aided design of putative ligands of proteins or receptors of
known three-dimensional structure has been presented. Diversity of these candidate ligands is provided
by first docking a set of diverse molecular fragments. For aliphatic functional groups a modified force
field (switching off the attractive part of the van der Waals interaction with polar atoms of the protein)
was introduced to better approximate solvation effects, thereby avoiding the docking of apolar fragments
into hydrophilic cavities of the macromolecular target. The second part of the present ligand design
approach consists of a combinatorial strategy for the connection of optimally docked molecular
fragments by small linkage elements having optimal interactions with the target molecule. An
application was presented in which candidate inhibitors of thrombin were found.
It is important to note that the most difficult part of ligand design is the prediction of binding affinity. A
method to estimate relative binding constants has recently been applied to a series of similar inhibitors
of HIV-1 aspartic proteinase [41]. Our own efforts are concentrated on the development of an approach
that will predict relative binding affinities and will be general enough to be used for any enzyme or
receptor of known structure (A. Caflisch, D. Arosio, and C. Ehrhardt, work in progress).
One of the purposes of this chapter was to show that structure-based ligand design is a fascinating and
progressing research field. It is fascinating not only for its ultimate goal, i.e., the discovery of ethical
drugs, but also because it is based on, and thereby increases, our understanding of molecular interactions
and recognition phenomena on an atomic level. Another reason is its multidisciplinary character, which
requires skills in different branches of science, from theoretical physics and chemistry to computer
science and statistics. That structure-based computer-aided ligand design is a progressing field is evident
in many chapters of this book. This is mainly a consequence of the methodological developments and
the ever-increasing performance of computers.
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Acknowledgments
We thank J. Apostolakis and Professor A. Plckthun for helpful discussions. The calculations were
performed on an SGI Indigo2 and an eight-processor SGI Challenge (R4400 processors). The
CHARMM program within the version 4.0 of the QUANTA software package (Biosym-MSI Inc) was
used for some of the minimization performed in this work. The CCLD program is available from A.
Caflisch.
This work was supported by the Swiss National Science Foundation (Schweizerischer Nationalfonds
grant nr. 3100-043423.95) and by Novartis Pharma Inc.
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22
Peptidomimetic and Nonpeptide Drug Discovery: Impact of StructureBased Drug Design
Tomi K. Sawyer
Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan
I. Introduction
Peptidomimetic and nonpeptide drug discovery has evolved to become an extraordinarily intriguing area
of interdisciplinary research. It challenges synthetic, computational, and biophysical chemists,
biochemists, pharmacologists, and drug delivery scientists to collaboratively discover lead compounds
that exhibit sufficient potency, selectivity, metabolic stability, and in vivo pharmacological efficacy to
warrant further development as drug candidates. Over the past two decades a highly focused effort in
both industry and academia has advanced the rational transformation of first generation peptide lead
compounds to significantly modified analogs having minimal peptide-like chemical structure [118].
Such work is typically illustrated by systematic backbone and/or side chain modifications,
transformation into macrocycles, structure-conformation analysis (x-ray, NMR, CD), and computerassisted molecular modeling. These extensive structure-activity and structure-conformation studies have
enabled the creation of prototype peptidomimetic second-generation analogs from initial peptide
leads. Over recent years the emergence of 3D structural information on target proteins has significantly
impacted peptidomimetic drug discovery strategies. Particularly noteworthy has been the advancement
of the de novo design of chemically novel compounds that possess very limited peptide-like
substructure, but include structure-based functional group modifications which may make new
intermolecular interactions with the target protein (versus a first generation peptide lead compound).
Furthermore, the integration of such structure-based drug design efforts with high-throughput
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screening and synthetic chemical library generation is markedly reshaping peptide, peptidomimetic, and
nonpeptide drug discovery research. In this chapter a few examples of peptidomimetic and nonpeptide
drug discovery are detailed to highlight the scope of such work relative to a few specific targets (e.g.,
receptors, proteases, and signal transduction proteins) in which structure-based drug design has
contributed in significant ways.
A. Peptides: Molecular Diversity and -- Space
Peptides exhibit extraordinary molecular diversity by virtue of their varying primary structures (Figure
1). For many peptide hormones, neurotransmitters, and releasing factors the substructure of amino acids
that contribute to molecular recognition (binding) and biological activity (signal transduction) at their
target receptors have been determined [2]. Such work has led to the generation of pharmacophore
models of either agonist or antagonist analogs and, in some cases, the design of peptidomimetics. Yet,
for most peptide growth factors, cytokines, and large-sized (>50 amino acids) peptide hormones, the
identification of the amino acid substructure which accounts for molecular recognition and signal
transduction has been a difficult task, and proposals for pharmacophore models remain significant
challenges. In this regard the term pharmacophore is defined as the collection of relevant groups
(substructure) of a ligand which are arranged in three-dimensional space in a manner complementary to
the target protein and are responsible for the biological property of the ligand as a result of binding of
the ligand to its target protein [8b].
The three-dimensional structural properties of peptides (Figure 2) are defined in terms of torsion angles
(, , , ) between the backbone amine nitrogen (N), backbone carbonyl carbon (C'), backbone
methine carbon (C), and side chain hydrocarbon functionalization (eg., C, C, C, C of Lys) derived
from the amino acid sequence. A Ramachandran plot ( versus ) may define the preferred
combinations of torsion angles for ordered secondary structures (conformations), such as helix, turn,
turn, or sheet. With respect to the amide bond torsion angle () the trans geometry is more
energetically favored for most typical dipeptide substructures, however, when the C-terminal partner is
Pro or other N-alkylated (including cyclic) amino acids the cis geometry is possible and may further
stabilize -turn or -turn conformations. Molecular flexibility is directly related to covalent and/or
noncovalent bonding interactions within a particular peptide. Even modest chemical modifications by
N-methyl, C-methyl or C-methyl can have significant consequences on the resultant conformation
[6; also, see Phe analogs in Figure 2].
The N-C-C' scaffold may be further transformed by introduction of olefin substitution (e.g., CC rarrow.gif C=C or dehydroamino acid [19]) or insertion (e.g., C-C' rarrow.gif C-C=C-C' or
vinylogous amino acid [20]). Also the C carbon may be substituted to advance the design of so-called
chimeric amino acids
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Figure 1
Examples of native peptide hormones, neurotransmitters, and releasing factors
[9]. Finally, with respect to N-substituted amides it is also noteworthy to mention the intriguing
approach [21] of replacing the traditional peptide scaffold by achiral N-substituted glycine building
blocks. Overall, such N-C-C scaffold or C-C side chain modifications provide significant
opportunities for expanding peptide-based molecular diversity (i.e., so-called peptoid libraries) as well
as to extend our 3D structural knowledge of traditional -- space.
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Figure 2
Three-dimensional structural properties of peptides: backbone and side chain
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B. Peptidomimetic Drugs: Concepts, Strategies, and Technologies

Historically, the major focus of peptidomimetic design has evolved from receptor-targeted drug
discovery research and has not been directly impacted by an experimentally-determined 3D structure of
the target protein. Nevertheless, a hierarchial approach of peptide rarrow.gif peptidomimetic molecular
design and chemical modification has evolved over the past two decades, based on systematic
transformation of a peptide ligand and iterative analysis of the structure-activity and structureconformation relationships of second generation analogs (Figure 3). Such work has typically
integrated biophysical techniques (x-ray crystallography and/or NMR spectroscopy) and computerassisted molecular modeling with biological testing to advance peptidomimetic drug design.
A plethora of sophisticated synthetic chemistry approaches have entered into the arena of peptide-based
molecular design, including well-established applications of unusual amino acids and dipeptide
surrogates, among other types of chemical modifications. Such backbone or side chain modifications
may afford stability of the parent peptide to peptidases and have provided conceptual impetus for yet
more sophisticated molecular design and peptidomimetic chemistry studies [1-18]. For example, a few
of the known amide bond replacements (Figure 4) include: aminomethylene or [CH2NH], 1;
ketomethylene or [COCH2], 2; ethylene or [CH2CH2], 3; olefin or [CH=CH], 4; ether or
Figure 3
Hierarchial approach in peptide rarrow.gif peptidomimetic structure-based drug design
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[CH2O], 5; thioether or [CH2S], 6; tetrazole or [CN4], 7; thiazole or [thz], 8; retroamide or

[NHCO], 9; thioamide or [CSNH], 10; and ester or [CO2], 11. These amide bond surrogates
provide insight into the conformational and H-bonding properties that may be requisite for peptide
molecular recognition and/or biological activity at receptor targets. Furthermore, such backbone
replacements can impart metabolic stability towards peptidase cleavage relative to the parent peptide.
The discovery of yet other nonhydrolyzable amide bond isosteres has particularly impacted the design of
protease inhibitors, and these include: hydroxymethylene or [CH(OH)], 12; hydroxyethylene or
[CH(OH)CH2] and [CH2CH(OH)], 13 and 14, respectively; dihydroxyethylene or
([CH(OH)CH(OH)], 15, hydroxyethylamine or [CH(OH)CH2N], 16, dihydroxyethylene 17 and C2symmetric hydroxymethylene 18. In the specific case of aspartyl protease inhibitor design (see below)
such backbone modifications have been extremely effective, as they may represent transition state
mimics or bioisosteres of the hypothetical tetrahedral intermediate (e.g., [C(OH)2NH] for this class of
proteolytic enzymes.
Both peptide backbone and side chain modifications may provide prototypic leads for the design of
secondary structure mimicry [11, 2231] as typically suggested by the fact that substitution of D-amino
acids, N-Me-amino acids, C-Me-amino acids, and/or dehydroamino acids within a peptide lead may
induce or stabilize regiospecific -turn, -turn, -sheet, or -helix conformations. To date, a variety of
secondary structure mimetics have been designed and incorporated in peptides or peptidomimetics
(Figure 5). The -turn has been of particular interest to the area of receptor-targeted peptidomimetic
drug discovery. This secondary structural motif exists within a tetrapeptide sequence in which the first
and fourth C atoms are < 7 separated, and they are further characterized as to occur in a nonhelical
region of the peptide sequence and to possess a ten-membered intramolecular H-bond between the i and
i+4 amino acid residues. On of the initial approaches of significance to the design of -turn mimetics
was the monocyclic dipeptide-based template 19 [22] which employs side chain to backbone constraint
at the i+1 and i+2 sites. Over the past decade a variety of other monocyclic or bicyclic templates have
been developed as -turn mimetics, and specific examples include 20 [23], 21 [24], 22 [25], 23 [26] and
24 [27]. Most recently, the monocyclic -turn mimetic 25 has been described [28] and illustrates the
potential opportunity to design scaffolds that may incorporate each of the side chains (i, i+1, i+2 and i+3
positions), as well as five of the eight NH or C=O functionalities, within the parent tetrapeptide
sequence. tetrapeptide sequence modeled in type IIV -turn conformations. Similarly, the
benzodiazepine template 26 has shown [29, 30] utility as a -turn mimetic scaffold which also may be
multisubstituted to simulate side chain functionalization, particularly at the i and i+3 positions of the
corresponding tetrapeptide sequence modeled in type IVI -turn conformations. A recently reported
[31]
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Figure 4
Backbone amide bond surrogates: [CONH] replacements
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Figure 5
Secondary structure modifications using -turn -turn, and -sheet scaffolds.
-turn mimetic, 27, illustrates an innovative approach to incorporate a retroamide surrogate between the
i and i+1 amino acid residues with an ethylene bridge between the N' (i.e., nitrogen replacing the
carbonyl C') and N atoms of the i and i+2 positions, and this template allows the possibility for all three
side chains of the parent tripeptide sequence. Finally, the design of a -sheet mimetic, 28, provides an
attractive template to constrain the backbone of a peptide to that simulating an extended conformation
[32]. The -sheet is of particular interest to the area of protease-targeted peptidomimetic drug discovery.
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12640-0567a.gif
Figure 6
Contemporary drug discovery: integration of structure-based drug design,
synthetic chemical libraries, and high-throughput mass screening technologies
Finally, the convergence of structure-based drug design (biophysical and computational chemistry),
synthetic chemical libraries, and high-throughput screening technologies have established a new
paradigm for drug discovery (Figure 6). This powerful alliance of scientific disciplines is accelerating
the identification of lead compounds and/or the optimization of drug candidates. The impact to
academic, biotechnological and pharmaceutical research will be immense.
C. Receptor, Protease, and Signal Transduction-Protein Targets
Structure-based drug design and peptidomimetic drug discovery has emerged as a powerful approach in
many areas of pharmaceutical research, including receptor agonists and antagonists, protease inhibitors,
and, more recently, in the rapidly developing area of signal transduction, in which the protein targets
include catalytic and noncatalytic domains of a particular intracellular macro
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molecule. A noncomprehensive listing of such receptor, protease, and signal-transduction protein targets
is shown in Table 1.
With respect to receptor-targeted peptidomimetic drug discovery, the most noteworthy success has been
attained for G-protein-coupled receptor agonists and antagonists as well as, more recently, cell-adhesion
integrin receptor antagonists (see below). However, it is important to state that the impact of highthroughput screening in the discovery of nonpeptide ligands (typically antagonists) at G-protein-coupled
receptors has yielded extraordinary success. Although screening-based nonpeptide drug discovery will
not be extensively reviewed here, the possibility of common pharmacophores between peptide and
nonpeptide ligands may exist (limited cases) in relation to receptor binding. Nevertheless, in most cases
receptor mutagenesis studies suggest the existence of different binding pockets for peptide and
nonpeptide ligands, regardless of whether they both are functionally similar as related to agonism or
antagonism [33]. With respect to both protease-and signal-transduction protein-targeted peptidomimetic
drug discovery, the emergence of 3D structural information to provide high resolution molecular maps
of the catalytic (or noncatalytic) domain has provided incredible opportunities for structure-based drug
design.
II. Peptide Ligand Lead Discovery and Structure-Based Drug Design
As stated previously, peptidomimetic drug discovery was first advanced by molecular design concepts
and chemical modification strategies focused on
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Figure 7
Convergent pathways in peptide, peptidomimetic, and nonpeptide drug
discovery
using receptor-targeted peptide ligands or second generation agonist or antagonist analogs to develop
pharmacophore models (see Figure 3). However, this represents only a part of the sophisticated
convergent pathways that exist currently to advance both peptidomimetic and nonpeptide drug discovery
(Figure 7). In this scenario both native and foreign (biological or chemical origin) peptides may provide
the opportunity for ligand structure-based drug design. From pharmacophore models of key peptide
leads the iterative transformation to peptidomimetic second generation analogs may proceed through
either peptide scaffold-or nonpeptide template-based approaches. Examples of such work are detailed
below.
Independent of the hierarchial approach to peptide ligand structure-based design of peptidomimetics is
the work that encompasses screening-based nonpeptide lead discovery. Such nonpeptides may be of
chemical or biological origin (see below), and historically have been identified by targeted or random
screening approaches. Although they are not designed to be peptidomimetics in the chemical structure
sense, many appear to be biological mimics (e.g., receptor agonists or antagonists, protease inhibitors,
signal-transduction protein inhibitors, or antagonists) of a known native or foreign peptide. And, in few
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cases, a pharmacophore model of a nonpeptide lead (or series) may show similarity to that of a
pharmacophore model of a peptide ligand. Examples of such work are detailed below.
A. Peptidomimetics: Receptor Agonists and Antagonists
Specific examples that illustrate peptide scaffold-and nonpeptide template-directed drug design
strategies are shown in Figure 8 and include -opioid endorphin (END) agonist, 29 [34]; thyrotropinreleasing hormone (TRH) agonist, 30 [13]; fibrinogen (GPIIa/IIIb) antagonists, 31 [35] and 32 [36];
CCKA antagonist, 33 [37]; CCKB/gastrin antagonist, 34 [38]; endothelin antagonist, 35 [39]; growth
hormone secretagogue (GHRP), 36 [40]; somatostatin agonist (partial), 37 [41], substance-P (NK1)
antagonists, 38 [42]; neurokinin-A (NK2) antagonist, 39 [43]; and neurokinin-B (NK3) antagonist, 40
[44]. For the most part, the above compounds have been advanced as the result of extensive structureactivity studies and, typically, more focused structural studies (NMR) on a conformationally
constrained, linear or cyclic peptide lead or series. Such structure-conformation activity studies have led
to the development of pharmacophore models to guide iterative structure-based design strategies.
One example is that of integrin receptor gpIIb/IIIa antagonists that are structurally derived from the
tripeptide sequence Arg-Gly-Asp, which is common to gpIIb/IIIa protein ligands such as fibrinogen,
vitronectin, fibronectin, von Willebrand factor, osteopontin, thrombospondin, and the collagens [45]. As
shown in Figure 9, transformations of the linear peptide ligand Arg Gly-Asp-Phe by both peptide
scaffold (at the Arg-Gly backbone) modification and substitution of the Arg side chain by a benzamidine
moiety provided the peptidomimetic lead 31 that is active in vivo as an antiplatelet agent [35]. On the
other hand peptidomimetics such as 32 illustrate nonpeptide template-based design strategies derived
from iterative transformations of a cyclic peptide lead
in which a
-turn about the Asp residue was implicated in a pharmacophore model for the bioactive conformation
[36]. Specifically, the benzodiazepinone substructure of 32 may effectively replace this predicted -turn
conformation about the Asp, and the N-Me-Arg replacement with piperidine moiety was also compatible
to high-affinity receptor binding.
A second example is that involving the use of a glucopyranoside nonpeptide template by Hirschmann
and coworkers [41, 46] for systematic functionalization to create novel peptidomimetics for the
somatostain (SRIF) and substance-P (NK1) receptors. As illustrated in Figure 10, the cyclic hexapeptide
SRIF agonist provided a macrocyclic lead structure that was transformed to a glucopyranoside template
designed to substitute for a postulated turn about the Tyr-D-Trp-Lys-Thr substructure of the parent
peptide ligand. The prototype
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Figure 8
Receptor-targeted peptidomimetics exemplifying ligand structure based drug design
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Figure 9
Peptide scaffold- and nonpeptide template-based design strategies:
gpIIb/IIIa antagonists
peptidomimetic 37 was found to be a moderately potent SRIF-like agonist (partial) in cellular assays
[41]. This discovery extends previous studies on TRH (see peptidomimetic 30, Figure 8) which utilized
a cyclohexane ring system as a nonpeptide template to functionalize with the pyroglutamic acod and His
side chains as well as the C-terminal carboxamide group of the parent peptide ligand [13]. However, in
the comparative analysis of analogs of the SRIF-mimetic 37 it was also found that N-acetylation of the
Lys side chain moiety yielded a potent antagonist of substance-P (NK1 receptor). This indicated that
slightly different functionalization of the nonpeptidic glucopyranoside template was quite compatible
with NK1 receptor molecular recognition. Intuitively, a reversed design strategy to convert the latter
glucopyranoside-based NK1 ligand to a cyclic peptide was next investigated (Figure 10), and
successfully led to the discovery of a novel cyclic peptide ligand also exhibiting potent NK1 receptor
binding and antagonism [46].
The above examples of peptide scaffold- or nonpeptide template-based peptidomimetic agonists or
antagonists illustrate various strategies to elaborate bioactive conformation and/or pharmacophore
models of peptide ligands at their receptors. In many cases, receptor subtype selectivity has also been
achieved by systematic structural modifications of prototypic leads of peptidomimetics. Thus, although
the 3D structures of G-protein-coupled receptors (GPCRs) remain as elusive (except for models
constructed from homology-based low-
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Figure 10
Peptide scaffold- and nonpeptide template-based design strategies: somatostatin
agonist and substance-P (NK1) antagonists
resolution 3D structures of bacteriorhodopsin or rhodopsin, see below) the development of

pharmacophore models using the hierarchial approach in peptide rarrow.gif peptidomimetic structurebased drug design (see above, Figure 3) remains quite promising to creative science. Indeed, targetedreceptor screening of synthetic chemical libraries of highly modified peptide molecules (e.g., Nsubstituted Gly peptoids [21] is rapidly expanding our database of structurally diverse ligands. Such
structurally unique lead compounds will provide the opportunity for further pharmacophore modeling
strategies to be used in the discovery of novel agonists or antagonists at GPCR and other receptor types.
B. Peptidomimetics: Protease Inhibitors
Specific examples of peptidomimetics which illustrate peptide scaffold- and nonpeptide templatedirected drug-design strategies as applied to protease in-
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Figure 11
Protease-targeted peptidomimetics derived by ligand structure-based drug
design
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hibitors (Figure 11) include renin inhibitors, 41 [47] and 42 [48]; HIV protease inhibitors 43 [49] and 44
[50]; angiotensin-converting enzyme inhibitors 45 [51] and 46 [52]; collagenase inhibitor, 47 [53];
gelatinase inhibitor, 48 [54], stromelysin inhibitor, 49 [55]; elastase inhibitor, 50 [56]; thrombin
inhibitors, 51 [57] and 52 [58]; and interleukin-converting enzyme inhibitor, 53 [59]. In general, the
design of protease inhibitors has focused on both the natural substrate structure and the mechanism of
substrate cleavage to provide first-generation inhibitors. Also, these initial leads are typically peptide
scaffold-based to provide the possibility for -sheet conformation, which may permit extensive Hbonding between the backbone amide groups of the inhibitor and complementary H-bond donor or
acceptor groups of the enzyme active site. Furthermore, the traditional approach to designing protease
inhibitors includes the substitution of nonhydrolyzable amide surrogates (see below Figure 4) at the P1P1' cleavage site. Specificity to a particular protease may sometimes be extrapolated directly from the
primary structure of the substrate (e.g., human renin substrate specificity is conferred from the
Val-Ile-His13~ in which
angiotensinogen N-terminal octapeptide sequence ~His6-Pro-Phe-His-Leu
refers to the cleavage site). In many cases substitution of the scissile amide (substrate) by a transition
state bioisosteres or an electrophilic ketomethylene moiety have provided tight-binding first
generation pseudopeptide inhibitor leads.
One example of protease inhibitor design that illustrates the peptide scaffold-based approach is that for
HIV protease inhibitors. Albeit over the past several years HIV protease inhibitor research has become a
highly advanced example of iterative structure-based drug design (see below), the first discoveries of
pseudopeptide and peptidomimetic inhibitors of this aspartyl protease were not made with knowledge of
the 3D structure of the target enzyme. Specifically, the natural product pseudopeptide pepstatin (Figure
12), a typical inhibitor of the aspartyl protease family of enzymes, was determined to be weakly potent
against HIV-1 protease [60]. Relative to pepstatin, the central P1-P1' statine (i.e., Sta or
Leu[CH(OH)]Gly) moiety was further evaluated within the context of an optimized N- and Cterminal amino acid sequence using a chemical-library strategy [61]. As shown in Figure 12, a resultant
pseudo-tetrapeptide Ac-Trp-Val-Sta-D-Leu-NH2 was found to be a relatively potent HIV protease
inhibitor. In another approach, a designed renin inhibitor (55; Figure 12) was determined to be a highly
potent HIV protease inhibitor [62]. Further optimization studies led to the discovery [49] of the first
bonafide peptidomimetic inhibitor of HIV protease (Tba-Cha[CH(OH)CH2] Val-Ile-Amp; 43) of HIV
protease. This compound (43) provided the first evidence of cellular anti-HIV activity and, therefore,
supported proof-of-concept studies related to the therapeutic significance of targeting HIV-1 protease.
Replacement of the peptide scaffold by the pyrrolidinone-type -sheet mimetic 28 in a
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Figure 12
Peptide scaffold-based design strategies: HIV protease inhibitors
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chemically-related P1-P1' Phe[CH(OH)CH2] Phe-modified lead has been reported [63] to yield
effective peptidomimetic inhibitors of the HIV-1 protease (56; Figure 12). The pyrrolidinone-type lead
has shown enhanced cellular permeability relative to its peptide backbone-type counterparts. In a third
approach guided by HIV substrate-based design, the cleavage site dipeptide Phe-Pro was substituted by
the transition state bioisostere to provide the highly potent and selective HIV protease inhibitor 57, a
P1-P1' Phe[CH(OH)CH2N] Pro-modified heptapeptide [64]. As compared to this pseudopeptide, a
pioneering effort focused on peptide ligand structure-based design provided a second series of highly
potent, selective, and cellularly-active HIV protease inhibitors [50] as represented by the recently FDAapproved anti-HIV drug 44 (Saquinavir). The design of still more HIV protease inhibitors having novel
chemical structures (e.g., C2-symmetric scaffolding, P1-P1' transition state bioisostere cyclization, and
achiral nonpeptide template replacement) has progressed at an extraordinary pace (for reviews see
Reference 65), and in a majority of cases such work has been strongly impacted by knowledge of the 3D
structure of the target enzyme and/or inhibitor complexes of it (see below).
A second example of protease inhibitor design that properly illustrates the peptide scaffold-based
approach is that of thrombin inhibitors. Work with these compounds led to the identification of highly
potent, selective, and in vivo-effective lead compounds. A member of the serine protease family,
thrombin cleaves a number of substrates (e.g., fibrinogen) and activates its platelet receptor (a G-proteincoupled receptor) by proteolysis of the extracellular N-terminal domain which results in self-activation
(for a review see Reference 66). Initial lead inhibitors of thrombin were substrate-based, including the
fibrinogen P3-P1 Phe-Pro-Arg sequence [67] and simple Arg derivatives such as Tos-Arg-OMe [68].
Also, the natural product cyclothreonide-A, a macrocyclic peptide containing a Pro-Arg ketoamide
sequence, provided an inhibitory peptide ligand lead [69]. As shown collectively in Figure 13,
compounds 52 and 5860 provided the opportunity to try different strategies to advance the design of
thrombin inhibitors. Particularly noteworthy from these early peptidomimetic lead discovery studies was
the design effort [70] that led to the highly potent thrombin inhibitor 52 (Agatroban), a sulfonamidemodified Arg derivative, which incorporated an unusual cyclic amino acid substituent C-terminal to the
P1 moiety as opposed to reactive electrophilic groups (e.g., ketone, aldehyde, or boronic acid).
Interestingly, replacement of the Arg side chain moiety within a structurally similar analog 60 by a
amidinobenzyl group was shown [71] to be optimal when the stereochemistry at the P1 -carbon has a Dconfiguration suggesting that the mode of binding may be different for 52 versus 60. In this regard, xray crystallographic analysis of thrombin-inhibitor complexes (see below) have provided insight in the
interpretation of the structure-activity relationships of the aforementioned lead compounds.
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Figure 13
Peptide scaffold-based design strategies: thrombin and tripeptidyl peptidase II
inhibitors
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The recent discovery of peptidomimetic inhibitors of the serine protease TTP-II (tripeptidyl peptidase-II)
further illustrates the peptide scaffold-based design approach [72]. Specifically, relative to a known TTPII cleavage site on the endogenous neuropeptide CCK-8 (i.e., Asp1-Tyr[SO H]-Met arrowd.gif Gly-Trp3
Met-Asp-Phe8-NH
) the design of a highly potent inhibitor 61 (Figure 13) was successfully achieved by
iterative structure-based optimization of the P3-P1 sequence. Noteworthy of the relatively simple
structure of the TTP-II inhibitor 61 was that it contains no functional group C-terminal to the P1 carbon. Such absence of an electrophilic moiety, transition state bioisostere, or other type of
nonhydrolyzable amide substituent is rather unique relative to most examples of substrate-based
protease inhibitors.
2
C. Peptidomimetics: Signal-Transduction Protein Inhibitors and Antagonists

Beyond receptors and proteases exists the rapidly emerging area of signal-transduction protein-targeted
drug discovery research. To date, a multitude of catalytic and noncatalytic proteins have been identified
which are critical components of intracellular signal-transduction pathways. These signal-transduction
proteins provide the molecular basis for communication from extracellular effectors (e.g., hormones,
neurotransmitters, growth factors, and cytokines) to stimulate cells in specific and regulated manner.
Signal-transduction pathways often involve protein-protein interactions, including examples of enzymesubstrate (e.g., kinases, phosphatases, transferases, and isomerases) as well nonenzymatic complex
formation (e.g., adapter proteins, exchange factors, and transcription factors). As compared to receptoror protease-targeted peptidomimetic drug discovery, there are significantly fewer examples reported in
the field of signal-transduction research. Thus, in several cases peptide ligands (as prototype
peptidomimetic leads) will be described to illustrate opportunities for structure-based drug design.
Specific examples which illustrate peptide scaffold- and nonpeptide template-directed drug-design
strategies are shown in Figure 14 and include: Ras farnesyl transferase inhibitors, 62 [73], 63 [74], 64
[75], and 65 [76], Src SH2 domain antagonists, 66 [77], 67 [78], and 68 [79]; and the protein tyrosine
phosphatase PTP1B inhibitor 69 [80].
An example of the signal-transduction protein-targeted inhibitor design which illustrates both peptide
scaffold- and nonpeptide template-based approaches is that for the Ras farnesyl transferase inhibitor
discovery. Such compounds show potential as new therapeutic agents for Ras-related carcinogenesis
[81]. Substrate sequences for farnesyl transferase have the consensus ~Cys-AA1-AA2-Met motif (AA
refers to Val or Ile). Both substrate-based
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Figure 14
Signal-transduction protein-targeted peptidomimetics derived by structure-based drug design
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inhibitors [7375,82,83] and, more recently, a novel non-Cys containing peptide inhibitors [76,84] have
led to potent and cellularly active compounds. As illustrated in Figure 15, the collected substratebased inhibitor 70 was designed to covalently attach farnesyl to a peptide via a phosphinic acid linker
replacement for [82], and this compound has been determined to be both potent against the target
enzyme and cellularly effective. Relative to peptide substrate structure-based design efforts,
peptidomimetics incorporating [CH2NH]-substitutions (e.g., 62, [73]) or a benzodiazepinone
replacement of the central dipeptide moiety (e.g., 63, [74]) have yielded high affinity inhibitors. Another
series of very potent Ras farnesyl transferase inhibitors have been designed in which the central
dipeptide has been substituted by various isomeric and/or homologated derivatives of amino benzoic
acid (e.g., 64 [75]), including a particularly effective analog biphenyl derivative 71 [83]. The above
studies indicated that both conformationally flexible or constrained peptide scaffolds as well as
nonpeptide template replacements can be used to link the Cys and Met substructures. It is also
important to point out that although compounds such as 6264 have free sulfhydryl groups (Cys) there
is no evidence that they become farnesylated, and therefore the binding mode and effect on catalytic
function of the target enzyme are unique relative to their peptide substrate counterparts. Recently, a
novel peptide inhibitor series, as exemplified by Cbz-His-Try(O-benzyl)-Ser(O-benzyl)-Trp-D-Ala-NH2,
has been discovered [76]. These inhibitors do not contain a Cys residue and structure-based design
efforts have successfully led to a series of peptidomimetics (e.g., 65) having only one chiral center.
Interestingly, this novel inhibitor series has been determined to competitively inhibit farnesyl
pyrophosphate binding rather than the binding of peptide substrate to the target enzyme. Another Hissubstituted peptidomimetic inhibitor of Ras farnesyl transferase has been recently reported [84] as
exemplified by 73, which was designed relative to a peptide substrate-based parent analog (72, Figure
15). Although the 3D structure of Ras farnesyl transferase is not known, biochemical studies suggest
that a divalent metal ion (e.g., Zn2+) may coordinate with the above inhibitor sulfhydryl or imidazole
groups at their corresponding binding sites on the target enzyme.
Another example of the signal-transduction protein-targeted drug design that illustrates peptide scaffoldbased approaches is that for Src SH2 domain antagonist discovery. Such compounds show promise as
new therapeutic agents for Src-related carcinogenesis, osteoporosis, and immune diseases [85]. The Src
SH2 domain is a prototype example of a superfamily of intracellular signal-transduction proteins that
possess structurally homologous SH2 domains that specifically recognize cognate phosphoproteins in a
sequence-dependent manner relative to a critical phosphotyrosine (pTyr) residue (i.e., ~pTyr-AA1-AA2AA3-AA4~ Furthermore, recent x-ray crystallographic studies of a several SH2 protein targets (e.g.,
phosphopeptide complexes) is greatly impacting the
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Figure 15
Peptide scaffold- and nonpeptide template-based design strategies: farnesyl transferase
inhibitors
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opportunity for iterative structure-based drug design in this field or research (see below [86]). Relative
to Src SH2 domain antagonist lead discovery, peptide library studies [87] have shown the ~pTyr-GluGlu-Ile~ as a preferred consensus sequence. Peptide scaffold-based approaches to replace the internal
dipeptide, Glu-Glu, by both flexible and rigid linkers have been explored [88] but were unsuccessful in
yielding potent analogs. As shown in Figure 16, prototype peptidomimetics 66 [77] and 67 [78] illustrate
a successful approach in which stereoinversion at the second residue (P+2 relative to the pTyr) to the Dconfiguration and side chain substitution to hydrophobic functionalities (e.g., cyclohexyl and naphthyl)
which provides accessibility to the known hydrophobic binding pocket for the P+3 Ile side chain.
Indeed, such compounds showed binding affinities essentially identical to that of the N- and Cterminally extended phosphopeptides containing the pTyr-Glu-Glu-Ile sequence. Substitution of the
pTyr residue of 66 by the difluoromethyl-phosphonate modified analog F2Pmp provides a more
metabolically stable derivative 74 (Figure 16) and a prototype lead to advance the design of cellularly
active second-generation compounds. More recently, structure-based drug design studies of compound
65 have led to the discovery of potent and Src SH2-selective peptidomimetic lead compounds [89], and
this is further detailed below as related x-ray crystallographic structures of Src SH2-phosphopeptide
complexes.
III. Nonpeptide Ligand Lead Discovery and Structure-Based Drug Design
As previously illustrated in Figure 7, the convergent pathways to design drugs which act as mimics
(agonists), antagonists, or inhibitors of native peptide (protein) ligands at their target receptors,
proteases, signal-transduction proteins, and so forth, include foreign nonpeptides. The origin of such
nonpeptides may be either biological (e.g., natural product) or chemical (synthetic compound collection
or libraries) that have been subject to biochemical screening to identify leads for further molecular
design and structure-activity studies. Over recent years the success of screening-derived nonpeptide lead
discovery and iterative transformation to drug candidates has been quite impressive, and many aspects
of this area of research have been reviewed [12, 15]. Nevertheless, it is intriguing to explore the
potential relationship between peptide ligands (including peptidomimetic derivatives) and such
screening-derived nonpeptide ligands as related to pharmacophore modeling and structure-based drugdesign studies.
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Figure 16
Peptide scaffold-based design strategies: Src SH2 domain antagonists
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A. Molecular Diversity and Screening-Based Identification of Nonpeptide Ligands

Precedence for the success of nonpeptide drug discovery can be traced to the identification of morphine
(75; Figure 17) as nonpeptide natural product agonist at -opioid peptide receptors [90]. To date, the
robust momentum of nonpeptide drug discovery continues to be accelerated by sophisticated
biochemical screening technologies. The scope of molecular diversity as well as therapeutic targets for
such screening-based nonpeptide ligand lead compounds includes the following examples (Figure 17):
substance P (NK1 antagonist, 76 [91]; angiotensin AT1 antagonist, 77 [92]; growth hormone-releasing
peptide (GHRP) agonist, 78 [93]; cholecystokinin CCKA antagonist, 79 [94]; CCKB/gastrin antagonist,
80 [95]; CCKA agonist, 81 [96]; endothelin antagonist, 82 [97]; gonadotropin-releasing hormone
(GnRH) antagonist, 83 [98]; vasopressin V1 antagonist, 84 [99]; gastrin-releasing peptide antagonist, 85
[100]; glucagon antagonist, 86 [101], neurotensin antagonist, 87 [102]; angiotensin AT1 agonist, 88
[103]; oxytocin antagonist, 89 [104]; and HIV protease inhibitor, 90 [105]. A significant number of
screening-derived nonpeptide leads have been identified for G-protein-coupled receptors (GPCRs), and
in a majority of cases these compounds have been determined to be competitive antagonists. Albeit the
3D structures of this receptor superfamily have not been directly determined, homology model-building
and site-directed mutagenesis studies are impacting structure-activity analysis of agonist and antagonist
ligands (peptide, peptidomimetic, and nonpeptide) for several GPCR targets (see below).
B. Nonpeptides: Exploring Pharmacophore Relationships to Peptide Ligands
Relative to a number of GPCR targets, there exists significant opportunity to compare chemical
structures and 3D pharmacophore models of both peptide and nonpeptide ligands. Such comparative
analyses can explore the possibility of similar 3D substructural elements that may account for their
molecular recognition at the binding site(s) of the receptor. The fact that a vast number of screeningderived nonpeptide leads are multifunctionalized 5- to 7-membered ring heterocycles (e.g., alkaloid and
benzodiazepine) and contain conformationally rigid substructural elements (e.g., biphenyl, spiro-bicyclic
rings, and N-substituted amide or amine linkages) suggests the likelihood that such compounds are
binding with highly favorable entropic driving forces as compared to the more conformationally flexible
peptide ligands. In this regard, efforts to rigidify peptide-based scaffolding or replace it by nonpeptide
templates has been the underlying theme of peptidomimetic design strategies, and concepts for the latter
approach date back to the proposed used of cycloaliphatic ring systems that might be
multifunctionalized to create topographi-
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Figure 17
Nonpeptide drug discovery: examples from screening-based approaches
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cally-designed peptidomimetics (or, as also defined, peptoids [106]). Nevertheless, screening-based

nonpeptide drug discovery has advanced a treasure of structure-function information to provide insight
into both structure-based design and molecular recognition [12,15,107]. In a few (limited) cases, there
exists a likely possibility of similar pharmacophoric features or substructural elements between
nonpeptides and their peptide-ligand counterparts (Figure 18).
Historically, drug discovery research on opioid GPCR receptor targets (e.g., , , ) has provided insight
to explore the pharmacophores of both agonist and antagonists derived from endogenous peptides (e.g.,
endorphin, endorphin, and dynorphin) versus nonpeptides (e.g., the -receptor selective agonist
morphine and its N-allyl-substituted antagonist derivative naloxone). Relative to the N-terminal Tyr
moiety (side chain and -amino functionalities) of the endogenous opioid peptides [108], the N-methyltyramine substructure of morphine represents a likely common pharmacophore for agonist ligand
binding to the -receptor (Figure 18). In the case of angiotensin II receptor antagonist drug discovery, it
has been proposed [109] that a common pharmacophore may exist relative to the C-terminal His-ProPhe-OH sequence of angiotensin II and nonpeptide 91 (Figure 18). In fact, these studies provided design
insight leading to the discovery of the drug candidate 77 (Lorsartan). A third example in which
correlation between peptide and nonpeptide pharmacophore models becomes apparent is that of
neuropeptide-Y (NPY) versus the benextramine-based derivative 92 [110] or arpromidine-based
derivative 93 [111] as illustrated in Figure 18. In both cases, the C-terminal Arg-Gln-Arg-Tyr-NH2
sequence of NPY was modeled relative to the nonpeptide structures such that the guanido functionalities
were superimposed upon the corresponding basic (i.e., guanido or imidazole) substructural elements of
either 92 or 93.
In some cases, the availability of x-ray crystallographic information of both the peptide and nonpeptide
ligands may provide insight into the pharmacophore modeling studies. An example of this exists for
oxytocin antagonist structure-based drug design studies [112]. As shown in Figure 19, pharmacophore
models of both a cyclic hexapeptide oxytocin antagonists and conformationally-constrained,
tolylpiperazine camphorsulfonamide nonpeptide antagonist (89) suggest the likelihood of common
substructural elements that were key for molecular recognition at the oxytocin receptor, and led to the
design of a highly potent derivative 94. Nevertheless, such comparative pharmacophore mapping
studies are very simplistic since the 3D structures of the target receptors are not known. Furthermore,
site-directed mutagenesis studies
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Figure 18
Comparative substructural elements of peptide and nonpeptide ligands: -opioid receptor
agonists, angiotensin, and NPY receptor antagonists
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Figure 19
Comparative substructural elements of peptide and nonpeptide ligands: examples
of oxytocin receptor antagonists and HIV protease inhibitors
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often suggest that peptide and nonpeptide ligands have different modes of binding to their receptors (see
below). It is also important to point out that the discovery of nonpeptide agonists will likely provide
important structural information to advance our understanding of ligand binding and activation of
receptors as well as insight for pharmacophore modeling. In this regard, nonpeptide agonists 78 (growth
hormone-releasing peptide receptor) and 81 (cholecystokinin receptor CCKA subtype) are noteworthy
exceptions to the rule that nonpeptide screening-based leads are antagonists (see above, Figure 17).
Finally, beyond receptor targets the advantages of high-throughput screening of chemical files, natural
products, and synthetic libraries are increasing for proteases as well as other enzyme and noncatalytic
targets. An recent example of such efforts is the nonpeptide HIV protease inhibitor 90 [105], which was
originally identified from screening a chemical file. Through iterative structure-based drug design
studies, including x-ray crystallographic analysis of both ligand and inhibitor-enzyme complexes, the
pyrone template has led to the discovery of highly potent, selective, and cellularly active lead
compounds (see below). In retrospect, the original concept of superimposing the key substructural
elements of the nonpeptide ligand 90 to a known peptidomimetic inhibitor of HIV protease is illustrated
in Figure 19. A more detailed account of this effort and successful elaboration of the nonpeptide lead
structure is described below.
IV. Protein Target 3D Structural Models and Structure-Based Drug Design
A significant impact in both peptidomimetic and nonpeptide drug discovery has emerged over recent
years as the result of the determination of the 3D structures of protein targets by x-ray crystallography or
NMR spectroscopy [113119]. In addition, computational methodologies such as QSAR, 3D
QSAR/CoMFA, homology-modeling, ligand docking, molecular dynamics, and mechanics, and solventaccessible surface visualization have greatly impacted such research. Furthermore, programs such as
GRID, GROW, GrowMol, LEGEND, BUILDER, LUDI, FOUNDATION-SPLICE, and CONCERTS
have enabled 2D and 3D database searching and de novo design [115,120,121]. Overall, the iterative
cycle of structure-based drug design (Figure 20) has evolved to be an engine of invention for several
examples of peptidomimetic and nonpeptide drug discovery.
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Figure 20
Iterative cycle(s) for structure-based drug design
A. Receptor Targets
Amongst the known superfamilies of cell membrane anchored receptors, significant research has been
focused on GPCR targets (Table 1). The GPCR superfamily of receptors consist of seven
transmembrane-spanning (TM) helices. Sequence homology among them varies from 2570% (for
reviews see References 33, 122132). The initial development of 3D structural models of GPCR targets
has been developed from homology-building methodologies based on a low-resolution structure of
bacteriorhodopsin [133]. Representative examples of recent studies that provide insight to
pharmacophore modeling and structure-based drug design of peptide, peptidomimetic, and nonpeptide
ligands are discussed below.
3 G-Protein-Coupled Receptors
Recent studies have explored several GPCR targets with respect to ligand-receptor binding interactions
using site-directed mutagenesis to explore agonist
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versus antagonist ligands, and such work provides insight to pharmacophore modeling. Specific
examples of such work as focused on peptide, peptidomimetic, and/or nonpeptide ligands, and working
3D structural models of their GPCR targets include angiotensin II AT1 and AT2 subtypes [134],
neurokinin NK1 and NK2 subtypes [135], cholecystokinin/gastrin CCKA and CCKB subtypes [136],
opioid -, -, and -subtypes [137], vasopressin V1A subtype [138], bradykinin B2 subtype [139],
neurotensin [140] and -melanotropin MC1 subtype [141]. From such work it has been inferred that
different binding-site interactions may exist for peptide versus nonpeptide ligands as based on their
differential sensitivities to site-directed mutants of the native GPCR. The recent development of 3D
structural models of the neurotensin [140] and -melanotropin MC1 subtype [141] GPCRs provide
interesting case studies. Both examples provide the correlation of significant structure-activity databases
and experimentally determined (NMR) structures of key peptide analogs with predicted molecular
contacts at their respective target receptors. As illustrated in Figure 21, the proposed peptide agonist
binding interactions for neurotensin and -melanotropin analogs at their human GPCR targets may be
used to further guide the molecular design and synthesis of second-generation peptidomimetic
derivatives.
In the first example, the neurotensin C-terminal octapeptide was subject to conformational searching
(~Arg-Pro-Tyr~ sequences from the Brookhaven Protein Databank), manual docking to the homologybuilt neurotensin GPCR receptor model, and constrained molecular dynamics simulation to provide a 3D
structure of the ligand-receptor complex [140]. A compact structure of the peptide in its complexed
conformation was consistent with a Type-1 -turn as previously determined by structural and structureactivity studies. Key molecular contacts predicted from this neurotensin GPCR model include
hydrophobic interactions with the C-terminal Ile and Leu side chains, -cation interactions with each
Arg residue side chain, and a cluster of aromatic-aromatic interactions with the Tyr side chain. No
electrostatic interactions were predicted, and the primary contact residues on the neurotensin GPCR
model were those comprising the third extracellular loop.
In the second example, the -melanotropin (MC1) GPCR model was constructed [141] by homologybuilding methods relative to both bacteriorhodopsin and rhodopsin fingerprint maps, and the MSH
superagonist peptides [Nle4, D-Phe7]-MSH and Ac-cyclo[Nle4, Asp5, D-Phe7, Lys10]-MSH410-NH2 were
modeled in conformations derived from previous experimental studies (i.e., a Type-II -turn at the
common tetrapeptide sequence ~His-D-Phe-Arg-Trp~). Of the alternative binding modes that were
described for the above the two MSH peptide ligands, one predicts the possibility of a network of
aromatic-aromatic and hydrophobic interactions between the MC1 receptor and the D-Phe and Trp side
chains of the MSH ligand (Figure 21). In addition, this MC1 receptor model predicts multiple
electrostatic and -cation interactions between
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Figure 21
GPCR 3D structural models for neurotensin and -melanotropin agonists
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the MC1 receptor and the Arg side chain of the peptide ligand. The primary contact residues of this
particular MC1 receptor model were all transmembrane domain derived and lie within 47.5 angstrom
(centroid to centroid). In conclusion, the development and refinement of 3D structural models of GPCR
targets, iterative site-directed mutagenesis studies, and systematic testing of key agonists and/or
antagonists will undoubtedly make a significant impact in the structure-based drug design of
peptidomimetic and nonpeptide ligands at these receptors. Such work may be expected to synergize well
with ligand-based pharmacophore modeling strategies which have become quite sophisticated in recent
years as the results of advanced computational chemistry methodologies.
B. Protease Targets
Protease-inhibitor drug discovery illustrates significant success in both mechanistic and 3D structurebased drug discovery for each of the representative classes (i.e., aspartyl, serinyl, metallo, and cysteinyl)
as exemplified in Table 2 (for a review see Reference 142a). In retrospect, pioneering achievements in
the design of peptidomimetic inhibitors of angiotensin-converting enzyme (for reviews see References
142b,142c) to have led to 45 (Captopril [51]) and 46 (Enalapril [52]). Such work has provided great
impetus to the area of proteasetargeted drug discovery. Over the past two decades a pervasive effort
integrating substrate-based inhibitor design, x-ray crystallography or NMR spectroscopy of inhibitorprotease complexes, high-throughput mass screening, and combinatorial chemical technologies has
evolved to further advance this area of research.
Aspartyl Proteases
The aspartyl proteases include pepsin, renin, cathepsin-D, chymosin, and gastricsin as well as microbial
enzymes (e.g., penicillopepsin, rhizopuspepsin, and endothiapepsin) and retroviral proteases (e.g., HIV1 protease). The first high-resolution x-ray crystallographic structures of this protease family were
determined for penicillopepsin [143], rhizopuspepsin [144], endothiapepsin [145], pepsinogen [146],
and pepsin [147]. Based on homology-building strategies, 3D structural models of renin were
subsequently constructed [148] to first guide the structure-based design of peptidomimetic inhibitors (for
a review see Reference 149). Furthermore, in several cases the x-ray crystallographic structures of renin
inhibitors were determined [150] as ligand-enzyme complexes with rhizopuspepsin, endothiapepsin, or
pepsin. Eventually, the x-ray crystallographic structures of renin (apo/complexes) were achieved to
provide high-resolution molecular maps of the target enzyme [151]. As illustrated in Figure 22, substratebased inhibitors such as the highly potent peptidomimetic 95 [151a] show well-defined hydrophobic
pockets for the P3-P1' side chains as well
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as hydrogen-bonding to the backbone of the inhibitor that exists in -sheet type extended conformation.
Structure-based design strategies of renin inhibitors have focused on systematic transformation of its
substrate (angiotensinogen). Noteworthy examples which illustrate topographical designed include
inhibitors 96 [152], 97 [153], 98 [154], and 99 [155], of which the latter macrocyclic inhibitor is quite
novel in terms of having two D-aromatic amino acids and lacking a transition state bioisostere
replacement at the P1-P1' site.
In contrast to renin, the discovery of HIV protease inhibitors provides a high degree of synchronization
of x-ray crystallography studies with iterative structure-based drug design efforts as well as the
identification of nonpeptide ligands from mass screening (e.g., coumarins and pyrones) or 3D
computational searching (e.g., haloperidol) to advance what has become a milestone achievement in
rational drug design [113]; and for reviews on HIV protease see Reference 156 and the first chapter of
this book). Representative of the scope of the many contributions to the discovery of both
peptidomimetic and nonpeptide inhibitors of HIV protease (Figure 23) are the de novo designed C2symmetric
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Figure 22
Protease 3D structural models: renin-inhibitor complex and drug design
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Figure 23
Protease structure-based drug design: HIV protease-targeted peptidomimetics and nonpeptides
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inhibitors 100 [157] and 101 [158]; the nonsymmetric peptidomimetics 44 [50] and 102106 [159163,
respectively]; and a series of nonpeptides derived originally from either 3-D computational searching
107 [164] or high-throughput sceening 108110 [165168], respectively. Of these compounds, FDA
approval has been recently granted to 102 (Indinivar), 103 (Ritonavir), and 44 (Saquinavir).
Currently, it is believed that there exists well over 150 x-ray crystallographic structures of HIV proteaseinhibitor complexes, not including mutated forms of the target enzyme that have also been determined to
be important to develop inhibitors which will be effective in so-called HIV resistant strains. The initial
series of x-ray crystallographic structures of HIV protease included the apoprotein [169] and enzymeinhibitor complexes derived from substrate-based analogues having P1-P1' substitutions by
N1e[CH2NH]N1e [170], Leu[CH(OH)CH2]Val [171], Phe[CH(OH)CH2N]Pro [172], and
Leu[CH(OH)]Gly or statine [173]. As illustrated in Figure 24, the first reported HIV protease-inhibitor
complex [170] with the pseudopeptide 111 provided a high-resolution map of the active site of the
enzyme as formed in a C2-symmetric fashion by the homodimer, and the flaps of each monomeric
subunit (i.e., residues 3557) were shown to make intermolecular interactions with the backbone of the
inhibitor by both direct hydrogen-bonding and through a structural water molecule (W301). Relative to
the C2-symmetry of the target enzyme, the discovery of C2-symmetric inhibitors was successfully
achieved by the design of Phe[CH(OH)]gPhe- and Phe[CH(OH)CH(OH)]gPhe-modified
peptidomimetics (gPhe refers to gem-diamino-Phe in which the C-CO2H moiety is replaced by CNH2) as exemplified by 101 [157] Among the plethora of other structure-based drug design strategies
focused on HIV protease inhibitor discovery it is also noteworthy to highlight the nonpeptide leads 100
and 108111 as they displace a key structural water (i.e., W301) and, as opposed to all previously
discovered substrate-based inhibitors, are capable of direct hydrogen-bonding interactions to the HIV
protease flap regions (Figure 24). These discoveries provide impetus for molecular design strategies to
consider tightly bound water molecules as possible secondary ligands in either de novo design or
iterative structure-based design of novel peptidomimetic or nonpeptide lead compounds. Previous
studies [174] on biotinstrepavidin and an x-ray crystallographic structure of the complex showed that
structural water molecule displacement (relative to the apoprotein) by key functional groups of the
ligand (biotin) was possible. It is noted, however, that HIV protease is unique from other members of the
aspartyl protease family with respect to the role of structural water W301 role in substrate/ inhibitor
binding. The catalytic water, which is critical to the mechanism of substrate cleavage for all aspartyl
proteases, has been a key feature in the design of a plethora of so-called transition state modified
inhibitors that incorporate a tetrahedral
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Figure 24
Protease 3D structural models: HIV protease-inhibitor complex and drug design
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hydroxymethyl substituent within various types of nonhydrolyzable surrogates of the scissile amide
bond (e.g., [CH(OH)], [CH(OH)CH2], and [CH(OH) CH2N]; see above). Nevertheless, there exist
examples of highly potent inhibitors of renin e.g., the [CH2NH]-modified 41 [47] and macrocylic
peptide 99 [155] and HIV protease e.g., the pyrone-based series 108111 [165168] respectively) that
do not possess a tetrahedral CH(OH) moiety per se.
Serinyl Proteases
The serinyl proteases include trypsin, chymotrypsin-A, elastase, thrombin, kallikrein, cathepsins-A, G,
and R, Factor VII, Factors IXa-XIIa, and tissue plasminogen activator. High-resolution x-ray
crystallographic structures of this protease family have been determined for thrombin (for a review see
[175]; also refer to Table II [176182]), Factor Xa [183], trypsin [184], kallikrein-A [185], and elastase
[186190]. As illustrated in Figure 25, a substrate-based inhibitor of thrombin having a boronic acid,
B(OH)2, substitution for the scissile amide was determine by x-ray crystallography to form a covalent
bond to the active site Ser-195 residue [176]. The N-terminal Ac-D-Phe-Pro moiety of this inhibitor
binds in a -sheet type extended conformation that involves hydrogen-bonding contacts to the enzyme
and well-defined hydrophobic and aromatic-aromatic (edge-to-face) stacking interactions. The inhibitor
Arg side chain binds in an extended conformation and the guanidino moiety forms bidentate hydrogenbonding interactions with an Asp189 residue at the base of the S1 specificity pocket as well as
additional hydrogen-bonds to the enzyme, one of which is mediated through a structural water. Relative
to the substrate-based peptidomimetic inhibitors of thrombin having C-terminal electrophilic groups
(e.g., aldehyde, ketone, and boronic acid), the discovery and structure-based design of nonpeptide
inhibitors not having P1 electrophilic functionalization has also been extremely successful as represented
by 52 [70], 60 [71], and 113 [182]. As shown in Figure 25, the design of a highly potent and selective
amidinopiperidine-based thrombin inhibitor 113 was derived from analysis of the x-ray crystallographic
structures of thrombin complexed with inhibitors 52 and 60. The latter two compounds showed different
trajectories of their P1 side chains (i.e., guanidinoalkyl and amidinophenyl, respectively) into the S1
pocket to account for the observed opposite chirality preferences at the C-position of the P1 amino acid
residues. Also, the C-terminal cycloalkyl moieties of both 52 and 60 were observed to bind to the socalled inhibitor P-pocket (i.e., the P2 substrate pocket), thus explaining that these compounds were not
binding in a substrate-like conformation such as the peptidomimetic inhibitors Ac-D-Phe-Pro-boroArgOH as described above. Thus, the design of the novel amidinopiperidine-based inhibitor 113 illustrates a
transposition of the P-pocket binding group to an N-substituted Gly--Asp scaffold.
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Figure 25
Protease 3D structural models: thrombin-inhibitor complex and drug design
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For other serinyl proteases, such as Factor Xa, kallikrein and elastase, the availability of x-ray
crystallographic structure of the enzymes (apo/complexes) provides further examples in which structurebased drug design is being advanced (Table 2). In particular, elastase-targeted drug discovery is
highlighted here as it illustrates substrate-based peptidomimetic inhibitor design strategies that have
focused on key P2P3 side chain and backbone hydrogen-bonding interactions with the enzyme (for
reviews see Reference [191]). Several x-ray crystallographic structures have been determined for
pancreatic elastase [187,188,189b,190,191] and leukocyte elastase [186,187,189a], including complexes
with peptide substrate-based inhibitors having P1 electrophilic functionalities such as benzoxazole, [188]
trifluoromethyl ketone [56,187,189], and -,-difluoro--ketoamide [190]. Recently, the design of
peptidomimetic inhibitors incorporating nonpeptidyl P2-P3 replacements has resulted in the discovery of
highly potent compounds [56,187]. Specifically, a lead series of highly potent trifluoromethylketonebased inhibitors of human leukocyte elastase which incorporate a N-carboxymethyl-3-amino-6-arylpyridone template (50, 116; see Figure 26) was developed and shown by x-ray crystallography to
provide backbone hydrogen-bonding and a novel trajectory of a P2 group from the pyridone ring to the
enzyme. Further modification of the pyridone ring to give the bicyclic pyridopyrimidine derivative 117
was predicted from molecular modeling studies to provide additional hydrogen-bonding to the enzyme
as well as another site on the bicyclic heteroaromatic ring system for tethering various hydrophobic or
hydrophilic groups. Finally, a series of novel dipeptide-based inhibitors (e.g., trifluoromethylacetyl-LeuPhe-p-isoproylanilide and a peptidomimetic derivative) are particularly intriguing because they bind
backwards as based on analysis of the x-ray crystallography structures of their complexes with
pancreatic elastase [191]. In this binding mode the trifluoromethylacetyl moiety is proximate to the
active site Ser residue and the ligand backbone and side chain substructures make hydrogen bonding and
hydrophobic contacts with the enzyme, respectively.
Cysteinyl Proteases
The cysteinyl proteases include papain; calpains I and II; cathepsins B, H, and L; proline endopeptidase;
and interleukin-converting enzyme (ICE) and its homologs. The most well-studied cysteinyl protease is
likely papain, and the first x-ray crystallographic structures of papain [193] and a peptide
chloromethylketone inhibitor-papain complex [194] provided the first high resolution molecular maps of
the active site. Pioneering studies in the discovery of papain substrate peptide-based inhibitors having P1
electrophilic moieties such as aldehydes [195], ketones (e.g., fluoromethylketone, which has been
determined [196] to exhibit selectivity for cysteinyl proteases versus serinyl proteases), semicarbazones,
and nitriles are noteworthy since 13C-NMR spectro-
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Figure 26
Protease 3D structural models: elastase-inhibitor complex and drug design
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scopic studies provided direct evidence that the active site Cys25 of papain forms a reversible covalent
bond with such electrophiles (see review Reference 142). The x-ray crystallographic structures of
cathepsin-B [197] and picornaviral 3C protease [198] have also been determined, but only in the
apoprotein form. Recently, high-resolution x-ray crystallographic structures of ICE-inhibitor complexes
have been determined [199,200]. This enzyme is structurally unique relative to other cysteinyl proteases
(for a review see Reference 201) in that it has a heterodimeric architecture in which two subunits form
the catalytically active enzyme site (actually, two p10/p20 heterodimers apparently create a tetrameric
form of the competent protease). As shown in Figure 27, an x-ray crystallographic structure of ICE
complexed with a substrate peptide-based chloromethylketone inhibitor (118 [200]) that is irreversibly
bonded to the active site Cys285 shows the P1 Asp specificity pocket to be comprised of two Arg
residues that lie at the base of the S1 binding pocket. Relative to other side chain binding pockets, a
hydrophobic channel type S4 site exists for the P4 Tyr of the inhibitor, whereas the P3-P2 Val-Ala side
chains are well exposed to solvent. With respect to the peptide backbone of the inhibitor, hydrogen
bonding interactions between the P3 Val (both NH and CO) and the P1 Asp (NH) and the P10 monomer
are predicted.
The x-ray crystallographic structure of ICE complexed with the inhibitor Ac-Tyr-Val-Ala-Asp-aldehyde
(119 [202]), supports structure-activity studies [202] that employed a systematic analysis of N-methylamino acid substitutions in which hydrogen bonding interactions between inhibitor and enzyme tolerated
only N-Me-Ala replacement at the P2 site. Furthermore, C-terminal modification of P1 Asp by
irreversible alkylating groups, such as the aryloxymethyl ketone analog 120 [203], have led to the first
reported peptidomimetic inhibitor of ICE (121 [204]). Noteworthy in the structure of this
peptidomimetic inhibitor is that a pyridone template provided an effective P2-P3 replacement, similar to
that for the elastase inhibitor design (see above). It is likely that the backbone hydrogen bonding
network between 121 and ICE is conserved as compared to the x-ray crystallographic structure of the
substrate peptide-based inhibitor 118 complexed to the target enzyme. Finally, the x-ray crystallographic
structure of the ICE homolog referred to as apopain, or CPP32, as a complex with a peptide-aldehyde
inhibitor has been recently determined [205] and provides additional insight as the specificity of
substrate recognition at both the S1 (P1 Asp) and S4 (P4 Asp) subsites. Thus, the opportunity for iterative
structure-based drug design exists to advance novel peptidomimetic and nonpeptide inhibitors of ICE
and/or its homologs.
Metalloproteases
The metalloproteases include both exopeptidases (e.g., angiotensin-converting enzyme, aminopeptidaseM, and carboxypeptidase-A) and endopeptidases (e.g.,
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Figure 27
Protease 3D structural models: ICE-inhibitor complex and drug design
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(e.g., thermolysin, endopeptidase 24.11 or NEP, collagenase, gelatinase and stromelysin). Historically,
the most well-studied metalloprotease is thermolysin (for a review see [206]), and the first x-ray
crystallographic structures of thermolysin [207] and several structurally distinct peptide inhibitors (see
Table 2 [208212]) provided the first high-resolution molecular maps of the active site and insight into
the mechanistic roles of the metal ion for substrate hydrolysis. Specifically, the binding interactions of
P1P1' transition state amide bond isosteres (e.g., [P=O(OH)NH]) as well as metal-chelating
functionalities (e.g., hydroxamates, carboxylates, and sulfhydryls), introduced as N-substitutions on
P1'P2' peptide scaffolds [208212] have been determined. Moreover, the structural and mechanistic
information derived from studies on thermolysin have provided insight into the design of inhibitors of
the therapeutically relevant target, angiotensin-converting enzyme or ACE (for reviews see Reference
142). This has been particularly significant since the 3D structure of ACE has not yet been determined.
However, it is noted that x-ray crystallographic structures of carboxypeptidase, a related metalloprotease
of the exopeptidase group, for both the apoprotein and a Gly[P=O(OH)NH]Phe-modified inhibitor
complex have been reported [213]. As illustrated in Figure 28, the x-ray crystallographic structure of the
Phe[P=O(OH)NH]Leu-modified peptidomimetic inhibitor 122 complexed with thermolysin show the
Zn2+ coordination and P1'-P2' (or P1-P2' collected product) mode of binding. Relative to this structure
the predicted molecular interactions between ACE and its inhibitors (e.g. Captopril [51], Enalaprilat
[52], and Fosfinoprilat [214] as well as an emerging class of dual specific inhibitors of ACE and NEP
(e.g., 123 [215]) may be envisaged.
The ability to design specific inhibitors of several matrix metalloproteases (MMPs) is rapidly
developing (for reviews see Reference 216). Such MMP targets include fibroblast collagenase (MMP-1),
gelatinase-A (MMP-2), and stromelysin (MMP-3). Both x-ray crystallography and NMR spectroscopy
have provided 3D structural information for several MMPs as well as MMP-inhibitor complexes (see
Table 2; [217226]). In the example of collagenase, first generation substrate-based inhibitor design
strategies have focused on modifying the P1P1' cleavage site (e.g., Gly-Phe, Ala-Tyr, and Ala-Phe) by
N-terminal functionalities capable of Zn2+ coordination (e.g., sulfhydryl, carboxyl, phosphonoalkyl, and
hydroxamate [227229]). As shown in Figure 29, an x-ray crystallographic structure of the Nhydroxamate-modified peptide 124 complexed to fibroblast collagenase provides a molecular map of
both its hydrogen-bonding interactions to the enzyme active site and binding to bound Zn2+ [219].
Potent MMP-1 inhibitors have been designed that tether the P2' side chain to the inhibitor's C-terminus
as macrocylic rings [53,230]. In the example of gelatinase-A, potent inhibitors have been designed (e.g.,
48, Figure 11 [54]) by N-terminal hydroxamate and P1' extended aromatic side chain modifications
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Protease 3D structural models: thermolysin-inhibitor complex ACE inhibitor drug design
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of a P1'P3' peptide scaffold [54]. To date, no 3D structural information is available for MMP-2 with
respect to either the apoprotein catalytic domain or inhibitor complexes thereof. Finally, in the example
of stromelysin-1, potent inhibitors have been designed (e.g., 49, Figure 11 [55]) by N-terminal
carboxyalkylamino functionalization that includes a P1 substituent. An x-ray crystallographic structure
of a related MMP-3 inhibitor 125 shows (Figure 29) the hydrogen-bonding interactions at the active site
and carboxylate coordination with the Zn2+ [223]. Finally, a recently determined x-ray crystallographic
structure of an Phe[P=O(OH)CH2]Ala-modified peptide inhibitor 126 complexed with astacin (Figure
29) shows the extensive hydrogen-bonding network between inhibitor, enzyme, Zn2+, and a structural
water [226]. It is expected that iterative structure-based design of inhibitors of the MMP family will
enable the discovery of novel compounds with superior binding affinities and/or selectivities.
C. Signal-Transduction Protein Targets
Beyond proteases the opportunity for structure-based drug design is being realized in the rapidly
developing area of signal-transduction research (e.g., intracellular protein and nucleic acid targets). Both
x-ray crystallography and/or NMR spectroscopy have significantly contributed to a wide-scope database
of 3D structural information for various catalytic and noncatalytic signal-transduction protein targets
(see Table 3). These include tyrosine kinases (e.g., growth factor receptor kinases and Src family
kinases; for reviews see Reference 231), serine/threonine and dual specificity kinases (e.g., mitogenactivated protein kinases and CDK2 and cAMP-dependent protein kinases; for reviews see Reference
232), phosphotyrosine phosphatases (e.g., PTP1B and Syp; for reviews see Reference 233),
phosphoserine/phosphothreonine and dual specificity phosphatases (e.g., VH1 and CDC25; for
reviews see Reference 234), noncatalytic adapter proteins (e.g., Crk, Grb2, Shc, and IRS-1; for
reviews see Reference 85), transferases (e.g., Ras farnesyl transferase; for reviews see Reference 81),
proline cis-trans isomerases (e.g., FKBP-12 and cyclophilin A; for reviews see Reference 235), and GTPbinding proteins (e.g., p21 Ras and -/ heterotrimeric G-protein for GPCR superfamily; for reviews
see References 236 and 237, respectively). The diversity of targets, mechanistic relationships (e.g.,
enzymesubstrate or regulatory proteinprotein interaction), and potential therapeutic opportunities has
created great impetus for focused research in the area of signal transduction (for reviews see References
265267).
Src Homology-2 and Homology-3 Domains
The identification of noncatalytic regulatory domains referred to as Src homology (SH) domains has
been rapidly advanced over recent years as a critical link
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Figure 29
Protease 3D structural models: matrix metallo protease-inhibitor complexes
and drug design
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Table 3 Some Known 3D Structures of Signal-Transduction Proteins (Apo/Complexes)
Protein Target
Apo/Complex
Resolution
Reference
Src homology domains

Abl SH2
(Apo)
NMR
238
Src SH2
(Apo)
2.5
239
phosphopeptide
2.7
239
phosphopeptide
NMR
240
PLC (C) SH2
phosphopeptide
NMR
241
Shc SH2
phosphopeptide
NMR
242
Lck SH2
phosphopeptide
2.2
243a
p85 (N) SH2
phosphopeptide
2.0
244
Syp SH2
phosphopeptide
2.0
245
Grb2 SH2
phosphopeptide
2.1
246
Syk SH2
phosphopeptide
NMR
247
Zap70 SH2-SH2
phosphopeptide (tandem)
1.9
248
Src SH3
(Apo)
NMR
249a
peptide
NMR
249b
Abl SH3
peptide
2.0
250
Crk SH3
peptide
1.5
251
Grb2 SH3
peptide
NMR
252
Grb2 SH3-SH2-SH3
(Apo)
3.1
253
Insulin receptor Tyr kinase
(Apo)
2.1
254
cAMP-dep. protein kinase
(Apo)
3.9
255
Tyr and Ser/Thr kinases
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(Ser/Thr)
peptide
2.9
255
(Apo)
2.4
256a
protein (p27Kip1 inhibitory domain)
2.3
256b
(Apo)
2.3
257
BH-PTP (Tyr)
(Apo)
2.2
258a
PTP1B (Tyr)
(Apo)
2.8
258b
peptide (C215S mutant enzyme)
2.6
258c
VH1 (Tyr and Ser/Thr)
(Apo)
2.1
259
PP-1 (Ser/Thr)
(Apo)
2.1
260
(Apo)
1.95
261
peptide
1.8
261
peptide
NMR
262
Cyclophilin
peptide (cyclosporin-A)
2.8
263
FKBP-12
FK-506 (macrolide antibiotic)
1.7
264
Cell cycle-dep. protein

kinase (CDK2)
Mitogen-activated protein kinase
(MAPK)
Tyr and Ser/Thr phosphatases
pTyr binding domains

IRS-1 PTB
IRS-1 PTB
Pro cis-trans isomerases
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in deconvoluting both enzyme-substrate and regulatory protein-protein interactions for a number of

signal-transduction pathways (for reviews see Reference 268). This emerging superfamily of proteins
includes SH2 and SH3 domains, the so-called choreographers of multiple signalling pathways, and
include very intriguing new therapeutic targets [85]. The SH2 domains have been determined to bind
cognate phosphotyrosine (pTyr) containing proteins in a sequence-dependent manner relative to the
amino acids contiguous to the C-terminal side of the pTyr residue (e.g., for Src SH2 a preferred
sequence is ~pTyr-Glu-Glu-Ile~ versus ~pTyr-Tyr-Asn-Tyr for Grb2 [87,269]. The SH3 domains have
been determined to specifically bind Pro-rich sequences of cognate proteins. Interestingly, as a result of
the pseudosymmetrical nature of the SH3 domains there is the possibility of binding both N rarrow.gif
C and C rarrow.gif N directions (e.g., for Src SH3 preferred sequences are ~Arg-Ala-Leu-Pro-Pro-LeuPro-Arg-Tyr and Ala-Phe-Ala-Pro-Pro-Leu-Pro-Arg-Arg, wherein Arg binds to a site 3 pocket [249b]).
With respect to SH2 domain structure-based drug design, the first x-ray crystallographic structures of
pTyr-containing peptide ligands complexed with Src SH2 domain [239] have been utilized to design the
first peptidomimetic antagonists [89]. As illustrated in Figure 30, a molecular map of the tetrapeptide
sequence ~pTyr-Glu-Glu-Ile~ complexed with Src SH2 [239] shows the pTyr binding pocket and a
second binding site for the P+3 Ile residue. As previously described, a prototypic peptidomimetic AcpTyr-Glu-D-Hcy-NH2 (66) was first discovered by a peptide scaffold design strategy (see Figure 16;
[77]) that took into account the x-ray crystallographic structure of Src SH2-phosphopeptide (Glu-ProGln-pTyr-Glu-Glu-Ile-Pro-Ile-Tyr-Leu, 127) complex. Further structure-based drug design
modifications have led to the discovery a series of potent peptidomimetics having novel C-terminal
functionalization (e.g., transposed side chain of the P+1 Glu or conformational constraint using a
pyrrolidine ring; see Figure 30) as represented by 128130 [89,270]. Studies focused on Src SH2 [88]
have shown that the phosphate ester of pTyr is particularly critical for molecular recognition, and that
significant loss in binding occurs by replacement with sulfate, carboxylate, nitrosyl, hydroxy, and
amino. However, backbone modifications of pTyr which replace its acylated amino functionality with
aromatic rings designed to form -cation type interactions with the Arg-A2 were effective substitutions
[271].
Recently, high-resolution 3D structures have been described for the noncatalytic adapter protein Grb2
with respect to the apoprotein (SH3-SH2-SH3 [153]) as well as the individual SH2 and SH3 domains
[246 and 252, respectively]. In the case of the SH2 domain of Grb2 an x-ray crystallographic structure
of a phosphopeptide complex provided insight to the molecular basis of the specificity of Grb2 SH2
binding of ~pTyr-Xxx-Asn-Yyy~ sequences. As illustrated in Figure 31, the binding interactions of LysPro-Phe-pTyr-Val-Asn-
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Figure 30
SH2 domain 3D structural models: pp60Src SH2 domain antagonists
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Figure 31
SH2 domain 3D structural models: Grb2 SH2-SH3 domain antagonists
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Val (131) showed that the phophopeptide adopts a -turn conformation about the P-P+3 residues and that
the P+2 Asn side chain carboxamide moiety is extensively hydrogen bonded to the protein. In contrast to
the well-defined binding pocket for the P+3 Ile of 127 to bind Src SH2, the P+3 Val of 131 engages in
limited surface hydrophobic interactions because the Trp121 residue of Grb2 SH2 sterically blocks the
phosphopeptide from attaining a similar binding mode. In the case of the SH3 domain, the binding of a
cognate Pro-rich peptide sequence ~Pro-Pro-Pro-Val-Pro-Pro-Arg-Arg~ shows distinct pockets which
recognize the Pro, Val, and Arg residues as illustrated in Figure 31. The peptide adopts a left-handed
polyproline type-II helical conformation which projects the three aforementioned residues to their
complementary binding pockets in the Grb2 SH3 domain.
Tyrosine Phosphatases and Phosphotyrosine Binding Domains
Two other types of signal-transduction proteins that recognize phosphotyrosine-containing sequences
are tyrosine phosphatases (e.g., PTP1B, Syp, and CD45) and proteins that contain a noncatalytic motif
referred to as a phosphotyrosine binding (PTB) domain. Although tyrosine phosphatases and PTB
domains are structurally quite different from each other they both are similar with respect to the binding
of pTyr-containing sequences with preference to the amino acids N-terminal to the pTyr residue. In
other words, the tyrosine phosphatase PTP1 binds EGF receptor (pTyr992)-based phosphopeptide
substrates Asp-Ala-Asp-Glu-pTyr-Leu-NH2 and Asp-Ala-Asp-Glu-pTyr-Leu-Ile-Pro-Gln-Gln-Gly
equally [272], and the substitution of pTyr by nonhydrolyzable F2Pmp in the hexapeptide derivative has
been reported [273] to give a highly potent inhibitor (see compound 69, Figure 14). In the case of PTB
domains, ~Asn-Pro-Xxx-pTyr~ (where Xxx is variable) has been determined [274] as the cognate
sequence for several PTB-domain-containing proteins such as Shc and the insulin receptor substrate-1
(IRS-1). The preference for amino acids N-terminal to the pTyr residue in binding to either tyrosine
phosphatases or PTB domains is, therefore, opposite of that known for SH2 domains [275].
Among the tyrosine phosphatase superfamily, PTP1B was the first to be discovered and structurallydetermined by x-ray crystallography, as the apoprotein catalytic domain [258b]. The first x-ray
crystallographic structure of PTP1B complexed with a phosphopeptide has also been very recently
determined [258c] using a catalytically inactive Cys215 rarrow.gif Ser PTP1B mutant and the
phosphopeptide Asp-Ala-Asp-Glu-pTyr-Leu-NH2 (133). As illustrated in Figure 32, the molecular
interactions between the tyrosine phosphatase and the phosphopeptide are dominated by electrostatic
(i.e., the pTyr, P-1 Glu, and P-2 Asp residues) and hydrogen bonding contacts to key amide functionalities
of the backbone of 133. The P+1 Leu side chain forms hydrophobic contacts with
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Figure 32
PTP and PTB 3D structural models: PTP1B inhibitor and PTB antagonists
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several PTP1B residues at the surface proximate to the well-defined pTyr binding pocket. Such 3D
structural information provides insight for the design of peptidomimetic inhibitors of PTP1B.
In the case of the IRS-1 PTB domain, x-ray crystallographic studies of PTB complexed with a pTyrcontaining peptide (134) complex have shown (Figure 32) that the phosphopeptide forms a type-I -turn
within the Asn-Pro-Ala-pTyr sequence, and the peptide backbone is extensively hydrogen bonded to the
PTB domain from the P+1-P-7 residues of 134 [261]. The pTyr binding pocket provides both electrostatic
and multiple hydrogen bonding contacts to the phosphate ester moiety, and hydrophobic interactions
exist for the P-1-P-3 side chains of the peptide. As in the case of PTP1B, such 3D structural information
provides the opportunity for structure-based drug design to discover potent inhibitors which may be used
for further exploration in cellular studies.
V. Future Perspectives
The impact of structure-based drug design on both peptidomimetic and nonpeptide drug discovery has
been significant over the past few years. The integration of sophisticated computational chemical
technologies, structural biology (x-ray crystallography and NMR spectroscopy), molecular diversity and
high-through-put screening, and targeted biological testing are expected to provide invaluable guidance
to drug discovery. These technological tools are contributing to an emerging 3D structure-activity
database that is the essence of rational drug design. Certainly, this intriguing area of peptidomimetic
and nonpeptide drug discovery and design is providing tremendous insight to our understanding of
molecular recognition and biochemical mechanisms. With particular regard to molecular diversity, the
generation of new leads from combinatorial chemistry focused on synthetic peptide, peptidomimetic, or
nonpeptide-type libraries will provide new opportunities (and challenges) for structure-based drug
design strategies (for reviews see Reference 276). Novel scaffolds and templates will continue to be
advanced and iteratively modified using randomized or targeted substructural replacements, and
such work is exemplified in this review. Of particular significance to the field of peptidomimetic and
nonpeptide drug discovery will be the rational use of D-amino acids, N-alkyl or C-alkyl amino acids,
N-substituted Gly, Xxx[Z]Yyy (dipeptide isosteres), benzodiazepines, and amino-benzoic acids in
structure-based drug design. Without question, structure-based drug design will be a decisive factor in
drug discovery efforts ranging from de novo design to iterative structural optimization of
peptidomimetic and nonpeptide lead compounds.
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Acknowledgments
I wish to acknowledge my colleagues at Parke-Davis for their critical review of this manuscript as well
as for their collaborative contributions to structure-based drug design research in the several areas,
including HIV protease inhibitor discovery, Ras farnesyl transferase inhibitor discovery, Src homology2 domain antagonist discovery, interleukin-converting enzyme inhibitor discovery, and melanocortin
receptor modeling development to probe MSH agonist and antagonists binding. I especially thank Mark
Plummer, Elizabeth Lunney, Charles Stankovic, Kim Para, Aurash Shahripour, Vara Prasad, Carrie
Haskell-Luevano, Daniel Ortwine, Daniele Leonard, Wayne Cody, and Christine Humblet in this regard.
I also very much thank Pandi Veerapandian for the opportunity to contribute a chapter to this book, and
for his critical review of this manuscript and excellent editorial suggestions.
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Index
A
Acquired immunodeficiency virus (AIDS), 41, 56, 62, 65, 70
Activate platelets, 247
Active site, 45, 48, 50, 54, 55, 56, 59, 61, 62, 63, 64, 65
Activity, 41, 48, 50, 55, 56, 64, 65, 67, 68, 69, 70
Acyclovir diphosphate, 154, 160, 164
Addison's disease, 195
Alcohol dehydrogenase (ADH) 202-203
Aldo-keto reductase superfamily, 240
Aldose reductase (ALR2)
catalytic mechanism, 233-234
inhibitors, 231-232
mutations, 234
NADPH cofactor binding, 232-233
relation to diabetic complications, 229-231
structure
active site, 233-235
NADPH-bound form, 231-233
ternary complex with zopolrestat, 235-239
-ketoamide transition state, 282
Document
ALR2 (see Aldose reductase)

Amantidine,
462
Amide bond
replacements, 563-565
1-amidinopiperidine, 256, 257
Amino acids
C alpha, 128
N-methyl, 128
side chain constraints, 124-128
Aminoalcohols 326
Amphipathic alpha-helices (see Leucine zipper)
Angiotensin, 321
converting enzyme, 120
Animal models
thrombosis of, 267
Anthopleurins, 297, 300, 301, 302, 312, 313, 314
Antibody-neuraminidase complexes, 470
Anticoagulation, 247
Anticoagulant protein, 257
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Anti-influenza drugs, 462, 477-480

Antirhinoviral agents
capsid-binding compounds, 497-500
clinical trials, 517-518
drug sensitivity groups, 503
resistance, 514-517
structure-activity relationships, 502-514
WIN compounds, 498-500
Antithrombin III, 248
Antitrypanosomal agents
eflornithine, 365-367
melarsoprol, 365-366
pentamidine, 365
suramin, 365-366
Antiviral
agent, 41, 67
Apparent mineralocorticoid excess (AME), 191
Argatroban, 251, 255
Arginine
boronate esters, 250
guanidinium, 251
Arrhythmias, 296, 298
Aspartic proteinases
Document
inhibitor binding, 323, 332

inhibitors, 323
mechanism, 328
specificity, 333
structure, 322
transition state analogs, 323
Atherosclerosis 395, 398
Autoimmune
disease, 395,398
atherosclerosis, 395, 398
insulin-dependant diabetes, 395, 398
myasthenia gravis, 398
rheumatoid arthritis, 395, 398
systemic lupus erythematosus, 398
disorders, 152
Available chemicals database (ACD), 381
B
Bacteriorhodopsin, 131
BCX-34, 166, 167
Benzodiazepinone peptidomimetic, 571
-barrels, 248
-turn, 122, 124, 126, 127
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Bicyclic peptidomimetic, 251

Bidentate hydrogen bond, 252
Binding
cleft, 45, 48, 61, 65
pocket, 56, 58, 60
Bivalent inhibitors, 257
Blood clot formation, 247
Boroarginine, 261
Boronate esters, 251
Boronic acid analog, 250
Bovine trypsin inhibitor mutants
phage display, 287
positional requirements for Fxa inhibition, 286-288
random mutagenesis, 287
site specific mutagenesis, 285
structure of complexes, 285
C
Calcium regulators, 296
cAMP generators, 296
Cancer, 395
Carbonyl reductase, 199
Cardiotonic activity, 301, 304, 305, 306, 314
Catalytic
Document
site, 52, 55, 61

triad, 247, 275
Catechol O-methyltransferase
active site, 349-350, 355-356
catalysis, 345, 350-351
inhibition mechanism, 356-358
inhibitors, 351-353
kinetics, 346
physiological role, 344-345
structure
AdoMet (see S-Adenosyl-L-methionine)
crystal structure, 347-350
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[Catechol O-methyltransferase] drug complexes, 354-355

Mg++ ion, 349-350
S-adenosyl-L-methionine, 349
sequence, 345, 347
substrates, 345,346
Cation-p site, 277, 282
Ceriamide, 473
Charge calculation, 381
CHARMm, 133
Chemical
library, 142
Chemi-informatics, 526, 535, 537
databases, 536
selection algorithm, 536
systems integration, 536
virtual library production, 536
Chemotherapy, 66
Chimeric receptor, 126
Chymotrypsin serine protease family, 248
Circular dichroism (CD), 438, 444
Coagulation
cascade, 247, 265
Document
factors, 247
Combination therapy, 62, 68, 69
Combinatorial
approach, 550-552
chemistry, 141, 525, 537
combinatorial explosion, 552
drug lead source, 528, 529
focusing, 530
parallel synthesis, 528, 532
refinement, 530
robotic instrumentation, 528, 532
scaffolds, 530, 532
structure-activity relationship (SAR), 529
pruning, 552
Compound selection, 531
chemi-informatics, 536
drug properties, 535
receptor fit, 536
similarity, 536
structure-activity relationship (SAR) models, 533, 536
virtual libraries, 533, 536
Computer programs
BIOGRAF, 381
Document
CHARMM, 542-544
computational combinatorial ligand design (CCLD), 550-552
DELPHI, 383
DOCK, 379-383
MCSS, 542-544
Conformational
change, 59, 60, 61, 70
degrees of freedom, 252
protein, 235-237
Congestive heart failure, 295, 296, 298, 301, 314
Cortiso, 193
Cortisone, 193
Cross linking, 137-138
Crystallography
cocktail soak approach, 377-379
Crystal structure, 45, 54, 56, 59, 61, 64
aldose reductase, 231-239
other aldo-keto reductases, 240-241
glyceraldehyde-3-phosphate dehydrogenase, 374-375
phosphoglycerate kinase, 376-377, 388
triosephosphate isomerase, 371-373
Cutaneous T-cell lymphoma, 167
Cyclic template, 252
Document
Cyclotheonamide A (CtA), 254
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Page 638
Cytokines, 395-398, 412, 420

alpha-beta, 396
atherosclerosis, 398
autoimmune disease and, 395, 98
beta sandwich 396
beta strands, 402
beta-trefoils, 396
4-helix bundle, 396
growth hormone, 398
immune system, 398
immunomodulation, 398
insulin-dependant diabetes, 398
myasthenia gravis, 398
network, 396
nuclear magnetic resonance (NMR) and, 396
production of, 395
prolactin, 398
receptors, 396
rheumatoid arthritis, 398
signalling by, 396
structures, 396
Document
tumour necrosis factor, 398

x-ray crystallography and, 396
D
ddI, 152
9-deazaguanine, 161, 163, 164, 165, 167
Diabetic complications, 229-231
Digoxin,295, 296, 297, 300
Dihydropteridine reductase, 197, 202
Diversity
directed, 536
of candidate ligands, 542,555
dNTP binding, 56, 61
Drug, 43, 45, 53, 55, 56, 60, 62, 63, 65, 66, 67, 68, 69, 70
binding affinity prediction, 555
combinatorial, 550-552
design, 55, 66, 70, 402
docking, 241-242, 379-383, 542-544
fragment approach, 541
inhibition, 70
lead
discovery, 377-384
optimization, 384-387
linked-fragment approach, 378
Document
resistance, 56, 66, 68, 70

scoring, 382-383
solvation effects, 544-547
Drug properties, 527, 535
absorption, 535
excretion, 535
metabolism, 535
refinement, 535, 536, 537
structure-based design, 527
DTic (tetrahydroisoquinnoline carboxylic acid), 124
DUP714, 250
E
Energy
minimization, 132-133
with CHARMM, 542-544
F
Fab, 45, 49, 51, 56, 58, 59, 69
Factor Xa
active site blocked (DEGR-Xa), 267, 269
active site substrate sequences, 271
modeled inhibitor complexes
amidinoaryls, 279
Document
antistasin peptides, 281

cyclotheonamide, 284
dansyl-Glu-Glu-Arg-CMK, 280
DX-9065a, 276
SEL-2711, 283
natural inhibitors of
AcAP's, 273-274
antistasin, 266, 268, 270, 271-272
ecotin, 268, 270, 274, 288
tick anticoagulant peptide (TAP), 266, 268, 270, 272-273
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Page 639
[Factor Xa] TFPI, 270-271, 285, 287

structure and function, 267-269, 274
synthetic inhibitors of
amidinoaryls, 277-279
antistasin peptides, 280-282
bisamidines, 277
BPTI mutants, 285-288
cyclotheonamide, 282
dansyl-Glu-Glu-Arg-CMK, 280
DX-9065a, 275-277
peptidyl argininals, 288
PPACK, 275
SEL-2711, 282
Factor XIII, 247
Factor XIIa, 119
Feline immunodeficiency virus (FIV), 441
Fibrin, 247
Fibrinogen, 247
Fibrinopeptide A, 250, 261
Flavonoid, 473
Fluoroketone analogs, 327
Fragment approach (see Computer programs, MCSS, CCLD)
Document
fragment-based programs, 541-542

Fourier maps, 154, 161, 166
G
Geminal diol analogs, 327
Genomic data, 525, 527
GG167 (see Neu5Ac2en,4-guanidino)
Glucopyranoside
peptidomimetic, 571
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
catalysis, 372, 374
crystal structure, 374-375
human, 374-375
inhibitors, 381-382, 384-387
Leishmania mexicana, 375-376, 385
sequence, 374
Glycol analogues 326
Glucocorticoid 193
G-protein coupled receptors (GPCR), 592-594
GRID maps, 475
H
Heart attack, 247
Hemagglutinin, 459, 460, 462, 463, 464, 477
Hematophageous organisms
Document
Ancylostoma caninum, 273

Haementeria officinalis, 266
Ornithidorous moubata, 266
Hemopexin domain, 172-174
Hemostasis, 247
Heparin, 248 cofactor II, 248
High throughput screening, 530
Hirudin, 251
HOE 140, 127-128
Homology
model building, 275-276,278
models, 527
scaffold development, 532
structure-based design, 527
sequence, 176
structure, 176-177
Hormone therapy, 206
Human immunodeficiency virus (HIV), 441
protease
cleavage sites, 7
flexibility, 7-8
inhibitors, 586-587 AG1284, 22-27
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Page 640
[Human immunodeficiency virus]

cyclic ureas, 21-22
hydroxycoumarins, 27-28
indinavir, 9, 15-17, 28-29
inversion of binding mode, 24
nelfinavir, 17-21, 29-30
nonpeptidic, 17-28
peptidic, 9-10
peptidomimetic, 10-19
ritonavir, 13-16, 28-29
saquinavir, 10-13, 28-29
symmetric binding, 13-15, 21-22
mutations, 28-32
resistance
primary mutations, 29-30
secondary mutations, 30-32
sequential passage, 28
vitality factor, 32
structure
active site, 5-7
three dimensional, 3-5
Wat 301, 6, 7, 10, 13, 16, 17, 21, 22, 24, 27
Document
integrase
amino acid sequence, 90-91
amino terminal domain, 92, 102
biochemical properties, 85-88
biophysical properties, 88, 92
carboxyl terminal domain, 89, 92
structure of, 102-103
catalytic core domain, 88-89
biophysical properties of, 92-93
mutation of hydrophobic residues of, 102-103
structure of, 93-102
comparison to toher
polynucleotidyl
transferases, 96-100
conserved acidic residues in
active site, 95-96
dimer, 100-102
domain structure, 88-92
inhibitors, 103-109
3'-azido-3'-deoxy-thymidine (AZT), 107-108
common pharmacophore of, 104-107
curcumin, 107, 111
design of, 110-112
Document
overview, 103-104, 108-109

rationale for, 85
Human prothrombin fragment F1, 261
Hydrophobic collapse, 252
Hydroxamate, 172, 182-184
Hydroxysteroid dehydrogenases, 191
7, 197, 199
11, 191-194, 203
17, 193-199, 205-207
20, 197, 199
Hypertension, 193
I
Inflammation, 395
Influenza virus
antigenic variation, 462, 468-470
classification, 459
drug resistance, 478, 479
inhibition, 478
replication cycle, 461
vaccines, 463
Inhibition, 50, 54, 60, 61, 65, 69, 70
mechanism, 65
Inhibitors, 41, 43, 45, 48, 50, 55, 56, 58, 59, 60, 61, 62, 63, 65, 66, 67, 68, 69, 70
Document
design, 358-359
Document
Page 641
[Inhibitors]
2-((3,4-dihydroxy-2-nitrophenyl)vinyl)phenyl-ketone, 352-353, 356
entacapone, 352-353, 360
first generation inhibitors, 351-352
tolcapone, 352-353, 360
Insulin-dependent diabetes 395,398
Interactions
electrostatic, 544
hydrophobic, 545
van der Waals, 545
Interferon
, 435, 439, 440
activity, 442
cytotoxicity, 439, 443
receptor, 441
, 435
structural studies, 443, 444
clinical uses, 436
definition, 435
, 435
activity, 448, 449
antibody studies, 445
Document
receptor, 446, 447, 450

intron A, 436
roferon, 436
side effects, 436
sub types, 435, 436
synthetic peptide studies, 448
signal transduction, 451
structural studies, 449-451
synthetic peptide studies, 446, 448
, 435, 439, 440,
activity, 441-443
antibody studies, 442
expression, 442, 443
receptor, 441
signal transduction, 443
structural studies, 443, 44
synthetic peptide studies, 441, 442, 444
, 435, 440
Interleukin-1
accessory protein, 398, 401
affinity for receptors, 401
, 398, 399, 401, 402, 406, 420, 421, 423, 427, 428
alternatively spliced, 399
Document
antagonistic activity, 416

auto-antibodies and, 401
autoimmune disease and, 401
barrel, 402, 404
, 398, 399, 401, 402, 420, 421, 423, 427, 428
barrel, 406, 409
bulge, 415-416
converting enzyme (ICE), 399, 402, 412
hairpins, 405
strands and, 402
beta-trefoil fold, 402, 403, 404, 407
binding, 412-416
affinity, 421
epitope, 410
pocket, 412
catalytic
activity of, 412
diad, 412
cysteine protease, 399, 412
enzyme mechanism, 412
epitopes of, 405, 413-416, 418
expression of, 399
fibroblast growth factors, 402
Document
gene duplication, 398

glycosylation of, 399, 409
GTPase proteins and, 401
Document
Page 642
[Interleukin-1]
homology with CED-3 protein, 412
hydrophobic patch, 406
hydrophobic residues, 404
immunoglobulin superfamily and, 399
Kunitz family, 402
leukemia, 395
location of, 399
low molecular weight antagonists, 421-427
monoclonal antibodies and, 421
monocyte phagocytes by, 399
mutational studies of, 407, 409
N-terminal extension
nuclear magnetic resonance (NMR), 402, 404
overexpression of, 419
peptide fragments, 416
peptomimetics, 412
precursor, 399
form, 399
receptor, 398, 402, 420
antagonist, 398, 399, 401, 407, 420, 421
binding, 404-405, 406, 407-410, 412-416
Document
recombinant, 421
sequence identity of, 398
signal transduction and, 401
site-directed mutagenesis, 412-416
structure of, 401-412, 419
synthesis of, 395, 419
substrate, 399
specificity, 412
subunits of, 412
therapeutic strategies and, 412
tumor necrosis factor and, (TNF) 421
type I, 399, 401, 420
type 2, 399, 401, 420
x-ray crystallography, 401-412
Ion-channel modulators, 296, 297, 301
Ischemia, 296, 297
K
Kampo drug, 473
Ketomethylene pseudo peptide bond, 260
Kinins, 119
turn propensity of, 123
bradykinin, 119
Document
Kininogen, 119
high molecular weight, 119
low molecular weight, 119
Kinin receptors, 120
agonist binding site, 131-133
antagonists of, 124
antagonist site, 137-138
B1 subtype, 120
B2 subtype, 119, 120
chimeras and, 138, 139
mutagenesis of, 133-134
Kallidin, 119
molecular dynamics of, 123
pharmacology of, 121
solution conformation of, 121
Kunitz domains, 271, 273, 282, 285
Kyte-Doolittle, 131
L
Lactam amide, 222-223
Leucine zipper
propane functional map, 545-546
stereo view, 546
Document
yeast transcriptional activator protein GCN4, 545-546

Leydig cells, 193
Licorice, 193, 195-196
Ligand docking, 133
Lock-and-key, 159
M
Malignant
tissue, 214
transformation, 214
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Page 643
Matrix-metalloproteinase (MMP), 171-186

fibroblast collagenase, 171-173, 176, 179, 183-184
gelatinase, 171-173, 184
matrilysin, 171-175, 182-184
neutrophil collagenase, 171-175, 183-184
stromelysin, 171-173, 184
Medicinal leech, 257
Metal requirement, 88, 89
Mineralocorticoid receptor, 193
Molecular
fragments
aliphatic, 542
aromatic, 542
charged, 542
polar, 542
modelling, 182-183
MuA transposase
structural comparison to integrase, 97-100
Mutation, 62, 67, 68, 69
Myristic acid, 214
Myocardial infarction, 247
N
Document
NAD, 199, 201

NADP, 199, 201
NADPH, 199-201
NAPAP, 255, 261
Napthalenesulfonyl, 257
Neuraminidase
active site, 470-472
carbohydrate structure, 467
dimensions, 465
enzyme function, 464, 473
inhibitors, 472-473, 476
molecular weight, 465
morphology, 464
topology, 466
Neu5Ac2en, 472, 473, 474, 477, 478, 480
4-guanidino, 475, 476, 477, 479
4-amino, 475, 476
Nonnecleoside ingibitor binding pocket (NNIBP), 56, 58, 59, 60, 61, 62, 63, 66, 67
Nonnucleoside reverse transcriptase inhibitors (NNRTI), 41, 45, 48, 49, 50, 56, 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70
Nonnucleoside, 41, 45, 56
inhibitor, 45, 56
inhibitor binding pocket, 56
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RT inhibitor, 41, 56
Nonpeptide
angiotensin agonist, 586-587
antagonist
angiotensin, 586-587
cholecystokinin, 586-587
endothelin, 586-587
gastrin-releasing hormone, 586-587
glucagon, 586-587
neurokinin, 586-587
neuropeptide Y, 588-589
neurotensin, 586-587
oxytocin, 586-587
drug discovery, 559, 570, 586-587
vasopressin, 586-587
NPC 17731, 124
NPC 18325, 137
NPC 567, 124
Nuclear magnetic resonance (NMR), 402, 404, 407
Nucleic acid, 48, 51, 54, 64, 65, 69
Nucleophile, 221
Nucleoside, 41, 50, 52, 53, 56
reverse transcriptase (RT) inhibitor, 41, 50, 52, 53, 54, 55, 56, 65
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Page 644
Nucleotide, 51, 53, 54, 55, 69

binding domain, 200
O
Octahydroindole carboxylic acid (Oic), 124
Oxyanion hole, 248, 250
P
Parkinson's disease
disease, 359
therapy, 359-360
Peptides
synthetic
structure-function studies in, 437, 438, 440-442, 444, 446, 448
Peptidomimetic
drug discovery, 591-598, 613
(see also Protease targets, Receptor targets, Signal transduction protein targets, Structure-based
drug design)
Peptidomimetics, 251
PGK (see
Phosphoglycerate
kinase)
Pharmacophore, 225, 560
peptide and nonpeptide models, 593-594
Phenanthroline
copper complexes as integrase inhibitors, 107
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Phosphoglycerate kinase (PGK)

catalysis, 376-377
crystal structure, 376-377, 388
human, 377
sequence, 377
Trypanosoma brucei, 376-377
Phosphorylysis, 151
Phosphostatine analogs, 327
Phosphotransfer, 222
Platelet aggregation, 247
Polymerase, 41, 45, 48, 50, 52, 54, 55, 56, 59, 61, 62, 63, 64, 65, 67, 69
active site, 45, 48, 50, 54, 55, 56, 59, 61, 62, 63, 64
catalytic site, 52, 55, 61
Polymerization, 41, 48, 50, 55, 61, 67, 70
mechanism, 56
Polynucleotidyl transferases, 96-100
Positive
inotropes, 295, 296, 297, 298, 300, 309, 310, 314
inotropy, 295, 297, 298, 305, 314
PPACK, 250, 261
Primer grip, 48, 61
Proliferative diseases
asthma, 214
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atherosclerosis, 214
fibrosis, 214
osteo arthritis, 214
psoriasis, 214
restenosis, 214
rheumatoid arthritis, 214
septic shock, 214
Protein C, 247
Protease targets
angiotensin-converting enzyme (ACE) inhibitors, 576, 607, 610
aspartyl proteases, 595-596
classes, 567-568
cysteinyl proteases, 605
inhibitors
elastase, 576, 597, 606
gelatinase, 576
human immunodeficiency virus (HIV) protease, 576-578, 595-596, 598, 600-602
interleukin-converting enzyme (ICE), 576, 597, 607-608
stromelysin, 576, 596, 609-612
thermolysin, 597, 610
thrombin, 576, 578-579, 596, 604
metalloproteases, 597-598, 607, 609-612
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Page 645
[Protease targets]
serinyl proteases, 603
x-ray structures
apoprotein and complexes, 595-598
Protein data bank, 173
Protein
kinase
activation loop, 218, 219
calmodulin dependent, 219
caseine (CK-1), 218, 220, 222, 224
catalytic
core, 214, 219-221
loop, 217, 221
cyclin dependent (CDK-2), 214, 215, 218-219
EGFR, 223
insulin receptor (IRK), 214-216, 218-220, 222
MAP, 214, 218-219
phosphorylase, 218-220, 222, 224
protein A (cAPK) 214-216, 218-220, 222-225
protein C, 224
phosphorylation, 217, 218

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Structure-based Drug Design

Structure-based Drug Design

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Structure-based Drug Design

14 Structural Aspects in the Inhibitor

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TFtransframe, PRprotease, RTreverse transcriptase, RHRNAse H, INintegrase. The location of the

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