Professional Documents
Culture Documents
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PROTOCOL LIST
Experimental design
Procedure for acquisition of transmission spectra 246
Post-processing of spectra 252
Application of band narrowing techniques 256
Raman experiments
Obtaining Raman spectroscopic data on protein–ligand interactions 273
Acquisition of spectra 278
Preparation of a silver sol by sodium citrate reduction of silver nitrate 290
Electrospray ionization
Nano-flow with a capillary ESI source 319
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xix
Chapter 2
Molecular modelling
Romano T. Kroemer
Department of Chemistry, Queen Mary and Westfield College, University of
London, Mile End Road, London E1 4NS, UK.
1 Introduction
Molecular modelling or computational chemistry have come a long way. From
the era when they were restricted to a small number of scientists using very
specialized and user-unfriendly software/hardware to the present where we are
confronted with a vast amount of well integrated programs and relatively cheap
hardware. The recent explosion of the Internet has added significantly to the
amount of software available, as it is often accessible directly via the net. Now-
adays, many of these programs can be used also by someone without program-
ming expertise, and it is not necessary to have studied theoretical chemistry for
years beforehand. However, the program in question should not be treated as a
black box either and the user must have an understanding of the underlying
theory and concepts.
This chapter is intended to give a flavour of the molecular modelling tech-
niques used in the context of protein–ligand interactions. It is beyond the scope
of this chapter to describe all programs/techniques in detail, and in many cases
they will be only mentioned briefly. However, the aim is to provide the reader
with an overview of some basic techniques in the area of protein–ligand model-
ling and to give some example applications using selected programs/techniques.
The selection of these examples is rather arbitrary and reflects the author’s ex-
perience and it is not intended to indicate any degree of superiority over other
programs with the same or similar functionality. Reference to other programs
will be given wherever possible.
In the context of modelling protein–ligand interactions one can think of a
variety of issues, which are outlined in Figure 1. The first point is that for many
applications one needs a protein structure to begin with. This structure can
come from experimental determination (usually X-ray or NMR). In cases where
there is no structure available, one can resort to the techniques of protein struc-
ture prediction. Usually, with the (protein) receptor structure in their hand, one
can start to analyse the structure for potential binding sites and/or the character-
istics of this site. In cases where the structure(s) of the protein(s) are available,
but not the geometry of the complex, elucidating the mechanisms by which
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ROMANO T. KROEMER
proteins or small molecule ligands dock is often required: here one tries to fit
two molecules together in energetically favourable conformations, by applica-
tion of computational procedures.
A further related problem has attracted considerable attention: The design of
a new ligand from scratch (de novo) with the aim of interfering with a specific
biological process. In some cases it is necessary to perform precise calculations
or predictions of the binding energies, with the aim to distinguish between
several possible ligands. Last but not least there are computational methods for
the statistical analysis of binding affinities for a set of ligand molecules. Normally
these latter procedures are applied when the receptor structure is not available,
but the binding affinities of several different ligands are known.
Before considering how we address these topics in a practical sense it is worth
pointing out that the boundaries between many of the topics (see the headings
of Figure 1) are rather diffuse and that there is sometimes considerable overlap
between them.
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ROMANO T. KROEMER
structure. This technique is based on the assumption that the tertiary structures
of two proteins will be similar if their sequences are related, and it is the
approach most likely to give accurate results.
At first sequences of proteins with known structure have to be identified. This
can be achieved by sequence database searching (18, 19). Knowledge of the
function or the family of the target protein might prove useful for the identi-
fication of homologues. The next task is the alignment of the target sequence
with those of the known structure. This is a very important step in the pro-
cedure, as serious errors at this stage are very difficult to correct later. If the per-
centage identities between the compared sequences are high ( 45%) the correct
sequence alignment is straightforward. When identity is low ( 25%) alignment
becomes difficult. However, knowledge of the family fold (e.g. a four-helix
bundle as in helical cytokines, associated with the presence of a hydrophobic
core) may allow for a good alignment even in these cases (20).
Two widely used approaches to comparative modelling are discussed
below.
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MOLECULAR MODELLING
Protocol 1
Fragment-based modelling using SYBYL COMPOSER
Equipment
• SYBYL software including the composer • Unix workstation
module
Method
1 Select the sequence (‘Select Sequence’).
2 Identify homologous, structurally known proteins and align their sequences with
the target sequence (‘Find Homologs’).
3 Assign seed residues for the start of an iterative 3D-alignment (fitting) procedure of
the known structures (‘Identify Seeds’).a
4 Perform the 3D-alignment, i.e. superposition of the known structures, and extract
the structurally conserved regions (‘Align Structures’).
5 Generate the structurally conserved core of the target protein (‘Build SCRs’).
6 Add the remaining parts of the target protein via database search (‘Add Loops’).
a
Alternatively one can perform steps 1 and 2 with other software, i.e. searching in other data-
bases and performing sequence alignments with different methods. In this case the user has
to provide three files for step 3: A ‘.pir’ file with the sequence of the target protein, a ‘.homol’
file indicating the homologous sequences, and a ‘.homlog’ file containing the alignment of
these sequences with the target sequence. The best strategy in this case would be to initiate
first a standard COMPOSER run, to modify the files that have been generated by the program
accordingly, and to re-start the procedure with these files.
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ROMANO T. KROEMER
104
MOLECULAR MODELLING
ring of polar interactions. 61% of the complexes analysed, however, had inter-
faces composed of a mixture of hydrophobic patches and polar interactions.
The studies mentioned above indicate that it might be of interest to analyse
and display the properties of a binding site in more detail, prior to any docking
or design studies. A commonly used technique is to calculate a surface repre-
sentation of the protein and to map certain properties such as hydrophobicity
or electrostatic potential onto it (see, e.g. 46–50). Calculation and graphical ex-
amination of the electrostatic potential around a molecule can provide other
useful information (51). The location of charged and polar groups in a protein
can have significant influence on the shape of the potential. As an example, this
has been demonstrated convincingly for the enzyme trypsin (Plate 5). In this
case the electrostatic potential around the molecule was calculated using the
finite difference Poisson–Boltzmann method (52). The potential contours reveal
how two proteins having both net positive charges are able to associate in a
complex.
Another procedure for analysing binding sites in proteins is to predict favour-
able positions for probes or small molecules (53, 54). One of the most popular
tools for this is the GRID program by Peter Goodford (55). Here the binding site
is embedded in a regular grid. A probe (atom, group, or small molecule such as
water) is then placed at the lattice intersections and the interaction energy be-
tween the probe and the protein is calculated using an empirical energy func-
tion. The resulting energy map can then be analysed for favourable interactions.
The program MCSS combines the analysis of the protein binding site with the
calculation of energetically favourable orientations of small functional groups
(56).
In many cases the analyses we have described so far in this chapter lead on
directly to the following methods: docking and ab initio design.
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ROMANO T. KROEMER
Criteria for differentiating between the various docking methods include the
way the receptor site is described, the type of ligand (small molecule and/or pro-
tein), the docking algorithm, and the evaluation procedure (scoring function). A
selection of different docking programs/methods is given in Table 1.
As described in Section 2, there are three different routes for determining 3D
structures of proteins: X-ray crystallography, high-resolution NMR, and homology
modelling. The structures coming from these sources have to be sufficiently
accurate in order to be of use for the docking experiments. For X-ray structures
this implies that their resolution should be in the region of 2 Å or lower and the
thermal factors (‘R-factors’) should be less than 30% (68). NMR structure deter-
mination normally results in an ensemble of structures. One can use the average
of these structures, in which case the rmsd variation between the structures
should be 1.5 Å. An approach for using the entire ensemble of structures for
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MOLECULAR MODELLING
docking has been recently reported (69). Structures resulting from homology
modelling are likely to have rmsd values higher than 0.7 Å for the C atoms (70)
and larger errors can be expected for the side chains (71). Nevertheless, protein
models structures have been successfully used for docking studies (72, 73).
Once a suitable protein structure has been selected for the docking process,
one has to determine the region of interest for the docking procedure. Usually
this is the active site of an enzyme, or the binding site of a receptor. In cases
where the location of this site is not known, it may be identified by graphical
inspection or by using an automated method (41, 59, 74). Many docking pro-
grams do not use an atomistic description of the binding site but rely on altern-
ative representations. For example, the molecular surface of a protein binding
site can be represented as an array of spheres (Figure 3). The centres of these
spheres can then be used in the calculation for fitting a ligand atom in the dock-
ing process. Another approach would be to represent the binding site by a grid,
where the grid points carry information about the interaction energies between
probe atoms and the binding site (55).
The ligand(s) have to be considered next. This could involve pre-calculation of
different conformers, assignment of rotatable bonds, building of a database of
suitable fragments (in order to introduce flexibility), or generation of a grid-
representation (discretisation) of the molecule(s). Other tasks may include the
evaluation of partial charges for the ligand atoms.
After the preparation has been done, the examination of the docking process
can start. Again, depending on the program used, a variety of strategies are pos-
sible. The docking step can be performed by matching (fitting) ligand atoms to
pre-calculated site points, where the latter represent potentially favourable inter-
actions. By matching the ligand atoms to these points different orientations of
the ligand in the protein are generated. Grid or systematic searches fit the ligand
into the active site by rotating and translating the ligand in discrete steps.
Fragment-based methods can introduce flexibility by docking several ligand
fragments in favourable orientations and subsequently reconnecting them. The
fragment methods can be used for the evaluation of existing inhibitors or can
be applied to so-called ab initio ligand design. Last but not least, kinetic docking
methods can fit ligands to receptor sites by exploring the potential energy
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ROMANO T. KROEMER
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MOLECULAR MODELLING
Protocol 2
Docking using AUTODOCK version 3.0
Equipment
• UNIX workstation • AUTODOCK software
Method
1 Prepare a file (extension .pdbq) containing the co-ordinates of the receptor (in pdb-
format), including polar hydrogens and partial atomic charges.a
2 Assign atomic solvation parameters and create a .pdbqs file, which contains co-
ordinates, charges, and solvation parameters. (mol2topdbqs, addsol)b
3 Define ligand torsions and generate the ligand .pdbq file. (deftors)
4 Use the receptor .pdbqs and the ligand .pdbq files in order to create the grid para-
meter file (.gpf) and the docking parameter file (.dpf). (mkgpf3, mkdpf3)
5 Calculate the grid maps. (autogrid3)
6 Perform the docking. (autodock3)
7 Create a pdb formatted file containing all docked conformations. (get-docked)
8 View the results using a molecular modelling program.
a
A .pdbq file has standard PDB format, with the exception that it contains partial atomic charges
in columns 71–76 of the file.
b
Steps 1 and 2 can be performed simultaneously as follows: Use SYBYL to add hydrogens (‘Bio-
polymer’, ‘essential_only’) and partial charges to the protein structure of interest. Save the struc-
ture as a file with the extension .mol2, use the AUTODOCK mol2topdbqs program to create the
.pdbqs file.
The way that different ligands are predicted to fit into a receptor provides
then a rationale for selecting certain compounds for synthesis. The geometries
of docked complexes can also be used as starting points for further studies, such
as ligand design (c.f. next section) or 3D QSAR (79).
The scanning of entire databases in order to identify putative ligands for a
known receptor has been reported as well (73, 80) and will become more import-
ant with the advent of large compound libraries generated by combinatorial
chemistry.
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ROMANO T. KROEMER
differences between the programs are related to the main stages of an ab initio
design procedure as follows:
In the following the reader will find a quick run-down of a number of differ-
ent procedures available. For further information he is referred to the preceding
chapter of this volume (Chapter 1).
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Figure 4 Active site characterization using hydrogen-bond vectors and lipophilic interaction
points.
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The elements in this function for the estimation of the free energy of binding
(Gbind) are the number of hydrogen bonds between ligand and receptor (f refers
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MOLECULAR MODELLING
to the geometry of the H-bonds), the number of ionic interactions, the lipophilic
contact area (A), and the number of rotatable bonds in the ligand. Some pro-
grams incorporate mixed functions containing force field like terms and other
descriptors, such as rotatable bonds or number of lipophilic contacts (97).
Another example for a de novo ligand design program is the LEAPFROG module
in SYBYL (31). It combines a number of ideas incorporated also in the programs
considered above. LEAPFROG follows a deterministic approach, by repeatedly
making some structural changes, and then either keeping or discarding the re-
sults, depending on the evolution of the ligand during the run. As a start it can
process two different types of input: A receptor structure (from experiment or
modelling) or, alternatively, a pharmacophore model. After selection of the input
the program can operate in three different modes (c.f. Protocol 3): The OPTIMIZE
mode aims at improving existing ligand structures. In the DREAM mode novel
ligands are suggested. In the GUIDE mode the user can interfere interactively
with the design process. New ligand structures are evaluated mainly on their
binding energy relative to their immediate precursor using an approximation of the GRID
procedure (55). Synthetic difficulty can be included in the scoring scheme. The
user can decide on the trade-off between variety and quality of the output
ligand structures. Therefore, the user may decide to emphasize in a first run the
variety. Subsequently, distinctively different structures can be chosen from the
output in order to be optimized in a second run. At the end of a run the struc-
tures are saved in a SYBYL database and are referenced by a molecular spread-
sheet containing the LEAPFROG binding energies.
Protocol 3
Setting up an ab initio ligand design procedure using
LEAPFROG
Equipment
• SYBYL software including the LEAPFROG • Unix workstation
module
Method
1 Start LEAPFROG. In the dialog box choose an input structure (‘cavity molecule’).
2 Select the operating mode (‘Guide’, ‘Optimise’, ‘Dream’, c.f. text).
3 Select whether to run the program interactively or in background.
4 If necessary alter other input data such as the fragment database used (‘Data’).
5 Choose either to calculate or to read (from a previous run) the sitepoint/box
description.a
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ROMANO T. KROEMER
Protocol 5 continued
When using any of the design techniques described above the user must bear
in mind that they incorporate only crude approximations of the binding ener-
getics and events. They are, however, excellent tools for providing novel ideas
in inhibitor design. The most promising candidates have to be synthesized and
tested. The test results can then be fed back into the design procedure in order
to initiate a new, hopefully improved, design cycle.
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Each of these terms contains different contributions and grouping them together
one can write alternatively (109):
Gelec. contains both the electrostatic solute–solute and the solute–solvent inter-
actions. Gcav. refers to the cavitation free energy on binding and can be defined
as the free energy required to form a solute-sized cavity in the solvent when all
interactions (dispersion and electrostatic) are switched off. Gconf. is the loss of
side chain conformational entropy on binding (TS). The van der Waals energy
(GvdW) is often neglected, under the assumption that the van der Waals inter-
actions at the interface of the associated complex are equal to those with the
solvent molecules in dissociated form. The last term (Grt) represents the loss of
translational and rotational entropy on complex formation. A number of tech-
niques for calculation of all these terms has been reported (52, 112–121).
Figure 5 Thermodynamic cycle for complex formation. Gsolv. refers to the free energy of
solvation. Ggas is the free energy of association in the gas phase.
The third method for calculating absolute free energies of binding, referred
to as the linear interaction energy (LIE) model, is a hybrid method including
elements of the former two. The method was introduced by Åquist et al. (122)
and employs a linear response approximation to calculate absolute binding free
energies as follows:
G bind
E elec. E vdW [5]
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MOLECULAR MODELLING
combined (128, 129). Recent advances in 3D QSAR and related techniques have
been summarized in two volumes of a book series (130) and in a number of
papers (131).
8 Concluding remarks
Although many of the computational methods presented in this chapter incorp-
orate various approximations and simplifications, they work remarkably well,
as shown by the many examples where experimental results could be repro-
duced or predicted with good accuracy. The usefulness of these approaches has
also been demonstrated by some success stories in the development of new
drug molecules (see, e.g. 73, 132, 133). Also, it has become clear that application
of these procedures is most useful in combination with experiments, either to
obtain confirmation from the latter or to provide guidelines for new ones. In
many instances this has proven to be highly synergistic.
The future looks bright for molecular modelling in the area of protein–ligand
interactions. New hardware with novel architecture and faster processors and
improved generations of software will advance the field significantly. Recent
developments such as the advent of combinatorial chemistry will lead to novel
computational methods, for example combinatorial docking and virtual screen-
ing (134, 135).
Acknowledgements
R. T. K. gratefully acknowledges the help of Martin Parretti for the preparation
of Plate 5.
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