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Vol.25 No.4 April 2004

Principles: Receptor theory in pharmacology

Terry Kenakin
Assay Development Compound Proling, GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, NC 27709, USA

Pharmacological receptor theory is discussed with special reference to advances made during the past 25 years. Thus, the operational model has supplanted analysis of drug receptor interaction in functional systems whereas the extended ternary complex model is used routinely to simulate quantitatively G-protein-coupled receptor (GPCR) behavior. Six new behaviors for GPCRs, centered on spontaneous production of receptor active states, ligand-selective receptor active states, oligomerization with other proteins (receptor and nonreceptor) and allosteric mechanisms, have been characterized and each holds the potential for new drug discovery for therapeutic benet. Pharmacological receptor models (the rst being an operational black box) preceded accurate knowledge of receptors by many years. In these cartoon-like schemes, drugs bound to receptors (dened as specic recognition units) to yield quanta of excitation to cells. Even this imprecise scheme enabled the construction of rudimentary receptor theory because the receptor was dened as the minimal unit needed to characterize the pharmacology of drugs; the cell simply furnished the herald for the drug receptor events. Under these circumstances, null methods (using equal functional responses) allowed the cancellation of cellular effects for quantication of drug receptor activity. Instead of being concerned with diverse responses to an agonist such as isoproterenol, including cardiac inotropy, lusitropy, chronotropy, vascular relaxation, pancreatic, lacrimal and salivary gland secretion, bronchiole, uterine and urinary bladder muscle relaxation, decreased stomach motility, skeletal muscle tremor and melatonin synthesis, the problem could be reduced to the interaction of isoproterenol with b-adrenoceptors. For models to be useful, they must describe data and predict effects. To do this, a mathematical formalism is required. A.J. Clark [1,2] (see [3] for history preceding Clark) was the rst to apply the mathematical approaches used in enzyme kinetics systematically to the effects of chemicals on tissues. The general aimhas been to determine the extent to which the effects produced by drugs on cells can be interpreted as processes following known laws of physical chemistry. (Clark, 1937) [1]
Corresponding author: Terry Kenakin (Terry.P.Kenakin@gsk.com).

Clark is credited with being the originator of receptor theory. It might not be appreciated today how important his application of simple chemical laws to systems of innitely greater complexity was to receptor pharmacology [2]. It is interesting to note the scientic atmosphere in which Clark published these ideas. The prevailing concepts in the years 1895 1930 were rooted in homeopathic theories (based on loose ideas around surface tension of the cell membrane) such as the Arndt Schulz law and the WeberFechner Law. Other vagaries, with little physicochemical basis, such as the law of phasic variation, essentially stating that certain phenomena occur frequently, were widely applied [4]. In this context the inuence of Clarks thinking (in the form of his two classic books on receptor theory [1,2]) cannot be over-estimated. Occupation theory An important difference between the effects of drugs on enzymes versus tissues is the quantication and prediction of agonist response. Empirical proportionality constants such as intrinsic activity [5] were used to make the equations better describe the experimental data obtained from tissues. Progress was made when a powerful framework was constructed around the theoretical quantities stimulus, stimulus response functions and efcacy and published in an article by R.P. Stephenson [6]. In general, classical theory evolved chronologically through concepts described by Clark and other researchers such as Ariens [5,7], Colquhoun [8], Ehlert [9], Furchgott [10,11], Gaddum [12], MacKay [13], Paton [14], Rang [15], Schild [16,17], Stephenson [5], Van Rossum [18] and Waud [19]. In terms of occupation theory, a response to ligand A is described by the function:   A 1Rt Response f Eqn 1 A KA where 1 is intrinsic efcacy (inherent ability of the drug to induce a physiological response), [Rt] is the receptor density, f is a function that relates the initial strength of activation (termed stimulus) to tissue response, and KA is the equilibrium dissociation constant of the drug receptor complex. With this model, drugs can be characterized with system-independent parameters (afnity and relative efcacy) in test systems and the effects applied to therapeutically relevant systems. New concepts arrived during the next 25 years to bring receptor theory to its present state. These involved the

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isolation and biochemical study of receptors, application of allosteric theories, use of recombinant receptor systems and presentation of the operational model of receptor function. The operational model One theoretical shortcoming of occupation theory is the ad hoc nature of the efcacy term. In 1989, Black and Leff [20] introduced a revolutionary idea that obviated the need for an empirical constant to account for efcacy; this formed the basis for the operational model. The ingenious premise on which this model is built is the fact that the efcacy term emerges from an experimentally observed behavior of pharmacological systems, namely the saturable relationship between receptor stimulation and the observed response. The equation describing drug action through the operational model is given as: Response A t Emax At 1 KA Eqn 2

drugs [21]. This model has gained widespread acceptance in the pharmacological community and is the model of choice for describing functional receptor pharmacology. The biochemical study of GPCRs and the ternary complex model With the advent of biochemical binding techniques came the capability of characterizing some of the players on the pharmacological receptor stage. Beneting most from this increased technology was the study of G-protein coupled receptors (GPCRs). The current model(s) of GPCR function uses the concept of allosterism, which was rst described for ion channels [22,23] and enzymes [24] and subsequently applied to receptors [7,25,26]. These ideas culminated in the ternary complex model for GPCRs, which was rst published by De Lean and colleagues [27]. This model describes a receptor that, when activated by an agonist, moves laterally in the cell membrane to physically couple to a trimeric G protein. This introduced a novel concept in receptor models, namely a synoptic (affording a general view of a whole) nature in which the sensitivity of the system (and potency of agonists) was subject to the availability of an external protein (i.e. a G protein). Supporting this model were data showing that physical complexes between receptors and G proteins could be isolated after addition of agonist to receptor systems [28,29]. Opening Pandoras (molecular) toolbox and extending models Before the widespread use of recombinant receptor systems, pharmacologists were conned in terms of the systems they had at their disposal. Most receptor work was performed on relatively few isolated tissues (e.g. guineapig ileum, rat atria and rat trachea). As put succinctly by Sir William Paton: The guinea pig longitudinal muscle is a great gift to the pharmacologist. It has low spontaneous activity; nicely graded responses (not too many tight junctions); is highly sensitive to a very wide range of stimulants; is tough, if properly handled, and capable of hours of reproducible behavior. (W.D.M. Paton, 1986) [30].

(see Box 1 for denitions of terms). Changes in the value of t lead to changes in the responsiveness of the system to agonists; that is, t controls both the potency of the agonist and the maximal response of the agonist (a reection of agonist efcacy). The tissue-specic component of t is the concentration of agonist-bound receptor that produces half the maximal tissue response. Thus, in highly coupled tissues, this value will be small (i.e. a small amount of receptor complex will produce a large response). However, the nature of the agonist also matters because the more efcacious the agonist, the smaller the amount of receptoragonist complex that is required to produce a response. Therefore, agonists will have a unique value of t for a given tissue. This allows comparisons between agonists; that is, the relationship between t values in one tissue will carry over to their relationship in other tissues. Figure 1 shows the relative agonist response to two agonists with a xed ratio of efcacy. With the operational model the relative agonist responses of both agonists can be predicted in any receptor system if the sensitivity of the system to one of the agonists is known. The operational model can negate the effects of volume control whereby differences in receptor density and efciency of receptor coupling cause differences in organ sensitivities to

Box 1. The operational model of drug action

A basic premise of the operational model is the experimentally observed fact that the relationship between receptor occupancy and response very often is hyperbolic in nature. Thus, the ligand-occupied receptor (AR) activates the cellular stimulus response cascade with a general equilibrium dissociation constant denoted by KE (the concentration of the AR complex that produces 50% of the maximal response). Response AR Emax AR KE Eqn I where [Rt] is the receptor density and KA is the equilibrium dissociation constant of the agonist receptor complex. The constant used to characterize the propensity of a given system and a given agonist to yield a response is the ratio Rt =KE ; this ratio is denoted by t. Substituting for t yields the working equation for the operational model: Response A t Emax At 1 KA Eqn III

where Emax is the maximal response that can be obtained from the system. The more efcient the process is from the production of AR to response, the smaller is the value of KE : Substituting mass action for the production of AR yields the equation for the operational model: Response A Rt Emax ARt KE KA KE Eqn II

To t experimental data that produce a dose response curve with Hill coefcients that are different from unity (i.e. as for a complex cellular stimulusresponse coupling mechanism showing cooperativity) the following equation is used: Response

tn An Emax A KA n tn An

Eqn IV

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(a) Fraction of maximal response

1.2 1.0 0.8 0.6 0.4 0.2 0.0 4 3 2 1 0 Log [agonist] = 300 100 30 10 3 1 0.3 0.1 1 2 3

(b)

1 / 2 = 10
1.2 1.0 0.8 0.6 0.4 0.2 0 1 2 3 0.0 4 3 2 1 0 1 2 3 1.2 1.0 0.8 0.6 0.4 0.2 0.0 4 3 2 1 0 1 2 3 1.2 1.0 0.8 0.6 0.4 0.2 0.0 4 3 2 1 0 1 2 3 1.2 1.0 0.8 0.6 0.4 0.2 0.0 4 3 2 1 0 1 2 3

Fraction of maximal response

1.2 1.0 0.8 0.6 0.4 0.2

1 = 0.3

1 = 1

1 = 10

1 = 30

1 = 300

0.0 4 3 2 1

Log ([A] /KA)

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Figure 1. Doseresponse curves calculated using the operational model. (a) The response is calculated according to Eqn 2 in the main text, with varying values for t (see Box 1 for the denition of t). It can be seen that the magnitude of t controls both the potency and the observed maximal response to the agonist. (b) The operational model preserves activity relationships between agonists (A) across all physiological systems. The curves show responses to two agonists differing in intrinsic efcacy by a factor of ten. The panel furthest to the left shows responses in the least efciently receptor-coupled tissue. The value of t for the most efcacious agonist (t1, solid line) is 3 whereas the value for t of the less efcacious agonist is 0.3 (t2, dashed line). Progression of panels from left to right represents tissues of increasing efciency of receptor coupling and/or receptor density. The tissue component of KE (general equilibrium dissociation constant) has been changed to affect differences in the receptor coupling efciency of the tissues (hence the sensitivity to the agonists). Abbreviation: KA ; equilibrium dissociation constant of the agonistreceptor complex.

This uniformity allowed quantitative experimentation that could be cross-checked in many laboratories; the resulting data laid the groundwork for modern receptor theory. However, with it came a lack of scope in terms of being able to study receptors at the extremes of their behaviors. With advances in technology and molecular biology, new observations could be made in old systems by using new approaches such as receptor point mutation, fusion proteins and stimulus-biased cell lines (enriched with selective G proteins through co-transfection and selective mating of GPCRs and G-protein subunits). These techniques have been coupled with the plethora of new technologies to observe receptor interaction with proteins. A classic example of the impact of recombinant systems is the nding of constitutive activity (elevated agonistindependent receptor activity) by Costa and Herz [31] and the denition of inverse agonism (ligand-induced reversal of constitutive activity). This drug activity was thought to be an oddity at the time, but was later seen to be rare only because the eyes to see it were not readily available to the pharmacological community at large. The increasing use of recombinant constitutively active receptor systems soon revealed that, as predicted by theory, inverse agonism
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is a common phenomenon (i.e. 85% of competitive antagonists are inverse agonists [32]). The observation of constitutive GPCR activity revealed a receptor behavior that could not be described by the original ternary complex model. Thus, the now commonly used extended ternary complex (ETC) model for GPCRs was devised by Sammama and colleagues [33] (Figure 2 and Box 2). The ETC model uses an important concept from twostate theory: the production of species bias with selective afnity. This suggests a mechanism for ligand efcacy: the selective afnity for different receptor species. It also gives efcacy a vector quality because it can be negative [a , 1, where a is the differential afnity of the ligand for the activated receptor (Ra)] and positive (a . 1). The ETC model has been referred to as a two-state model, probably because of the two unliganded receptor species: inactive receptor (Ri) and Ra. This is an unfortunate misnomer because the model actually describes innite receptor states when the receptor is ligand bound (the multiplier g confers a different afnity of the receptor for G proteins when the receptor is ligand bound). Thus, every value of g denes a new ligand-bound receptor state. This is important to note in view of experimental evidence that

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(a) Extended ternary complex model G L ARi ARa Kg ARaG

Box 2. Ternary complex models for GPCRs

The extended ternary complex (ETC) model (Figure 2a in the main text) describes a receptor that can exist in two states, active (Ra) and inactive (Ri), named for their ability to activate G proteins (G). These conformations coexist according to an allosteric constant unique for the receptor type (denoted L Ra =Ri : Ligands have afnity for Ri denoted by Ka (equilibrium association constant) and a differential afnity for Ra of aKa. Similarly, Ra that is not bound to ligand has an afnity for G proteins of Kg ; ligands can confer a different afnity of the receptor for G protein denoted gKg. The ETC model describes response production (elevated concentrations of Ra and ARa) as a fraction of the total receptor species (denoted by r) as:

Ka L
Ri

Ka

Ka

Kg
Ra G R aG

(b) Cubic ternary complex model ARiG Kg K ARi L RiG ARa L K L Kg K ARaG

LG=KG 1 agA=KA A=KA 1 aL1 gG=KG L1 G=KG 1

Eqn I

where KA and KG are equilibrium dissociation constants (reciprocals of association constants). Figure I shows the effects of changing a on the dose response curves of a system with existing constitutive activity (note the elevated basal activity in the absence of ligand). Formally identical effects are observed with changes in g values. The cubic ternary complex model (Figure 2b in the main text) allows interaction between the Ri and G proteins.

RaG
1.2

Kg Ra
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Fraction of maximal response

Ka

Kg

1.0 0.8 0.6 0.4 0.2 0.0 5 3 1 Log ([A]/KA) 1 3 = 30 10

Ri

1 0.3 0.1 0.03 0.01

Figure 2. Ternary complex models for G-protein-coupled receptors (GPCRs). (a) Extended ternary complex model. Receptor states (Ri) and (Ra) coexist according to the allosteric constant L. G protein (G) enters the system and binds to the activated receptor state Ra to produce the physiological response. Ligand A binds to either receptor state and also to Ra when it is bound to the G protein. The propensity of the system to produce constitutive activity (spontaneous formation of the active state RaG species) is dened by the allosteric constant L {L [Ra]/[Ri]}. The afnity of ligands for the receptors is given by Ka whereas the efcacy is described by two terms, a and g. The term a is the differential afnity of the ligand for Ra and the term g is the differential afnity of the ligand-bound ARa for G proteins. (b) Cubic ternary complex model. The inactive receptor species Ri and ARi are allowed to interact with G proteins (but not signal) in this variant model. b refers to the differential afnity of the receptor active state (over the inactive state) for the G protein.

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ligands confer unique conformations to receptors that differ from the constitutively active receptor state [34]. In thermodynamic terms, there must be a provision for the inactive receptor to also interact with G proteins; this is allowed in a more complete but more complex model for GPCRs, named the cubic ternary complex (CTC) model (Box 2) [35]. Recent evidence indicates that antagonists form GTP-sensitive, non-signaling ternary complexes with receptors (e.g. opioid peptide receptors [36] and histamine H2 receptors [37]) and that unliganded wild-type receptors (e.g. pheromone receptors Ste2p and Ste3p [38], and cannabinoid CB1 receptors [39]) and receptors bound to inverse agonists {SR141716A (see Chemical names) for CB1 receptors [40], and tiodidine for H2 receptors [37]} can sequester G proteins (in the form of antagonist-bound, non-signaling ternary complexes) from other systems to
Chemical names SR141716A: N-(piperidin-1-yl)-5-(4-chlorophenyl)-4-methyl-1Hpyrazole-3-carboxamide HCl
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Figure I. Response according to the extended ternary complex model for G-protein-coupled receptors (GPCRs). Response (ordinate axis) given as the concentration of the response-producing species [RaG] [ARaG] as a fraction of the total receptor number according to Eqn I. Curves were calculated for agonists of xed value for g (g 5) and varying magnitudes for a in a system with constitutive receptor activity L 0:01:

reduce constitutive activity. These data suggest that the CTC model applies for some receptor systems. In the worst-case scenario, recombinant systems can simply show uncharacteristic behavior of receptors or receptors under extreme conditions (i.e. the data take on a Pandora aspect whereby the resulting information is misleading and dissimulating). From this standpoint, such data reect what a receptor can do, and not necessarily what it does under normal physiological circumstances. However, pathological processes change synoptic receptor systems to set-points that might not have been imagined in normal in vitro pharmacological test systems. Therefore, such extremes can be therapeutically relevant. Beyond linkage models Linkage models such as the ETC and CTC models are extremely useful for deriving methods to quantify drug

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(a) No ligand present

(b) Add ligand

(c)

1.2 0.25 0.20 0.15 0.10 0.05 0.00

S4 S10 S7

1.0 Frequency 0.20 0.15 0.10 0.05 0.00

S10 S7 S4

[R] [AR]

0.8 0.6 0.4 0.2 0.0 1 10 20 30 40 50 Micro-conformations 60

S1

1 3

S1

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Figure 3. Probabilistic view of receptor conformations. This model assumes that a receptor, not bound by ligand, possesses a particular state distribution in conformational space and that ligands and/or G proteins change the resting distribution of the receptor states (micro-conformations) following binding. (a) The frequency of occurrence of 100 random receptor micro-conformations depicted as the height of the individual histograms. (b) Frequency of those same conformations in the presence of a saturating concentration of a ligand that stabilizes each conformation by a factor unique to the particular ligand and each conformation. (c) Gaussian distributions for the ensemble of receptor conformations not bound by a ligand (labeled R) and bound by a ligand (labeled AR). Some conformations are enriched by ligand binding at the expense of others.

effect. However, they are constrained in that they must specically pre-dene the species present in thermodynamic space. If there are more species than are dened, then the models fall short. A completely different approach is taken by a probabilistic model of GPCR behavior [41,42]. This model assumes that a receptor, not bound by ligand, possesses a particular state distribution in conformational space and that ligands and/or G proteins change the resting distribution of the receptor states following binding (i.e. some conformational states are enriched by the presence of the ligand and some are depleted) (Figure 3). The pharmacological activity of a ligand is dened by the quantity and type of receptor conformations that the ligand stabilizes in conformational space. For example, ligands that shift the distribution of conformations towards those that interact with and activate G proteins are agonists. The probability model is much more versatile than linkage theory models because it can predict receptor behavior beyond a single dimensional response (i.e. G-protein activation). GPCRs are known to have a wide range of behaviors (beyond activation of G proteins), such as the ability to form homodimers, heterodimers and higher-order oligomers and the ability to internalize, desensitize and interact with other membrane proteins such receptor activity-modifying proteins (RAMPs) and receptor component protein (RCP). The probability model can describe ligand efcacies as the formation of different ligand-stabilized ensembles of receptor conformations. Thus, the coincidence of ligand-formed ensembles with physiologically relevant conformations denes the pharmacological activity of the ligand [43]. A renaissance in receptor theory? The past 25 years have amounted to a virtual revolution in pharmacology with respect to receptor theory. In some cases, experiments necessitated modication of a theory, as in the discovery of constitutive activity by Costa and Herz [30] and the resulting ETC model [32]. In other cases,
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theory preceded experiment as, for example, in the prediction of protean agonism (positive agonism in nonconstitutive systems and inverse agonism in constitutive systems as a result of a . 1 and g , 1 in the ETC model [44]) and experimental verication thereafter (b2-adrenoceptors [45], a2-adrenoceptors [46] and bradykinin receptors [47]). Similarly, the observation of non-signaling antagonist ternary complexes and sequestration of G proteins by receptors suggest an experimental verication for the CTC model in some systems. GPCRs are now seen as interactive informationprocessing units beyond switches for G proteins; some of these receptor behaviors might have therapeutic application. Figure 4 shows six GPCR receptor behaviors that have been discovered during the past few decades and might lead to new therapeutic horizons. The discovery of constitutive activity has led to the denition of a new drug class (inverse agonists). It is not clear to what extent inverse agonism is therapeutically relevant. However, for some receptors that are known to be exceptionally constitutively active, inverse agonists might be the ligand of choice. For example, appetite control through ghrelin receptor antagonism [48] or histamine H3 receptor blockade [49] might require inverse agonism. Also, in some tumors, receptor overexpression might make inverse agonists useful modulators of cancer growth [50]. There is considerable evidence that drugs can form ligand-specic receptor active states and thus select certain metabolic pathways (Figure 4) [34]. This can yield more-selective agonists (i.e. biased agonists [51]) such as [Gly1,Arg19]hPTH1 28, a parathyroid hormone (PTH) receptor agonist that activates cAMP but lacks the activation of phospholipase C that is normally elicited concomitantly by the natural hormone PTH [52]. This concept extends beyond G-protein signaling, and is demonstrated by, for example, the opioid receptor agonists methadone and 1-a-acetyl methadone, which exhibit reduced propensity to produce desensitization [53].

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(f) Receptor dimerization

25-year receptor revolution

(a) Spontaneously occurring active states New mechanisms of signaling (e) Allosterism Ri (inactive) Ra (active state 1)

Constitutive activity, inverse agonism

(b) G-protein pleiotropy

Drug-like molecules for peptide receptors

G Ra (active state 2)

Organ-selective and ligand-selective agonists

(d) Auxiliary coupling proteins (i.e. RAMPs, RCP) G Tissue-directed recombinant screening Phenotypic versus genotypic organ selectivity

(c) Agonist-selective active states

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Figure 4. Discoveries of G-protein-coupled receptor (GPCR) behavior through biochemical research conducted during the past 25 years. See the main text for specic details and examples. Abbreviations: RAMPs, receptor activity-modifying proteins; RCP, receptor component protein.

Combinations of gene products (Figure 4) to yield drug phenotypes in cells (phantom genes [21]) have led to cell-specic drug sensitivity. For example, RAMPs [54] or RCPs can completely change the agonist and antagonist recognition properties of calcitonin and adrenomedullin receptors. This severely undermines the historical notion that the receptor is the minimal unit for drug recognition and activity. Increased functional screening of drugs is leading to a concomitantly increased availability of allosteric drugs (Figure 4) [55]. These ligands challenge conventional classication (e.g. the muscarinic acetylcholine receptor agonist effects of alcuronium are insensitive to the muscarinic receptor antagonist quinuclidinyl benzoate [56]). The advent of allosterism in receptor function will allow the investigation of probe-specic effects that are not possible with previous orthosteric analogs of natural ligands. For example, although blockade of the chemokine receptor CXCR4 is an important therapy for X4 HIV entry [57], knockout studies suggest that blockade of chemotaxis through this receptor can also lead to deleterious effects [58]. However, there are allosteric ligands (i.e. peptide L5H) that block HIV entry but do not affect CXCR4 function, which suggests a new approach to this problem [59]. Finally, another type of phantom gene prole can be observed with receptor dimerization (Figure 4), as in the unique opioid receptor prole produced by the heterodimerization of kappa and delta opioid receptors [60].
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The next 25 years? Considering the advancements made over the past 25 years, it is difcult to envisage GPCR-based therapeutics in the year 2029. However, in view of the emergence of GPCRs as complex information-processing units that are capable of differential cryptography, it might be supposed that new chemical scalpels will glean therapeutically useful behaviors. The advancements made during the past 25 years reinforce the belief that receptor theory is an indispensable tool in the drug discovery process.
References
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