Allosteric modulation of G-protein coupled receptors

European Journal of Pharmaceutical Sciences 21 (2004) 407–420

Mini-review

Allosteric modulation of G-protein coupled receptors Anders A. Jensen a,∗ , Tracy A. Spalding b a

Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen, Denmark b ACADIA Pharmaceuticals Inc., San Diego, CA, USA Received 28 July 2003; received in revised form 17 November 2003; accepted 17 November 2003

Abstract The superfamily of G-protein coupled receptors (GPCRs) has more than 1000 members and is the largest family of proteins in the body. GPCRs mediate signalling of stimuli as diverse as light, ions, small molecules, peptides and proteins and are the targets for many pharmaceuticals. Most GPCR ligands are believed to activate (agonists) or inhibit (competitive antagonists) receptor signalling by binding the receptor at the same site as the endogenous agonist, the orthosteric site. In contrast, allosteric ligands modulate receptor function by binding to different regions in the receptor, allosteric sites. In recent years, combinatorial chemistry and high throughput screening have helped identify several allosteric GPCR modulators with novel structures, several of which already have become valuable pharmacological tools and may be candidates for clinical testing in the near future. This mini review outlines the current status and perspectives of allosteric modulation of GPCR function with emphasis on the pharmacology of endogenous and synthesised modulators, their receptor interactions and the therapeutic prospects of allosteric ligands compared to orthosteric ligands. © 2004 Published by Elsevier B.V. Keywords: G-protein coupled receptors (GPCRs); Allosteric modulation; Allosteric modulator; Potentiation; Inhibition

1. Introduction Cell surface receptors are membrane bound proteins that translate extracellular signals delivered as neurotransmitters or hormones into intracellular cascades and events such as phosphorylation, calcium mobilisation and opening of ion channels. The three major classes of cell surface receptors are ligand-gated ion channels, tyrosine kinase receptors and G-protein coupled receptors (GPCRs). These receptor classes constitute the majority of the therapeutic targets identified today, with drugs targeted to GPCRs accounting for $18,548 million in sales during 2000 (Bailey, 2001). GPCR ligands generally act by stimulating a response (agonists) or blocking the activity of an endogenous agonist (competitive antagonists). Inverse agonists also block the constitutive activity of a GPCR. Since most ligands appear unable to bind simultaneously with each other (e.g. in radioligand binding), and are similarly affected by mutations in the receptors, it is generally assumed that they act through

∗

Corresponding author. Tel.: +45-35-30-64-91; fax: +45-35-30-60-40. E-mail address: [email protected] (A.A. Jensen).

0928-0987/$ – see front matter © 2004 Published by Elsevier B.V. doi:10.1016/j.ejps.2003.11.007

the binding site of endogenous transmitter at the receptor, the orthosteric site. In recent years, there has been increasing therapeutic interest in ligands that bind to other sites on the receptor, allosteric sites. Many allosteric modulators have little or no agonist or inverse agonist activity themselves, but they affect the receptor’s response to endogenous agonists and other ligands. Classical examples of allosteric modulators include benzodiazepines and barbiturates, which enhance or inhibit GABAA receptor function, and memantine which negatively modulates NMDA receptors (Kohl and Dannhardt, 2001; Möhler et al., 2002). These receptors are ion channels. In contrast, the vast majority of clinically-used GPCR ligands are believed to act orthosterically. Recently, rapid developments in combinatorial chemistry and high throughput screening techniques have helped identify numerous allosteric ligands, several of which already have become valuable pharmacological tools and may be candidates for clinical testing in the near future. In this review, we discuss the pharmacological characteristics of allosteric modulators, the interactions of specific allosteric modulators with their respective GPCRs, and the potential therapeutic advantages of allosteric versus orthosteric ligands.

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(Gether, 2000). The “rhodopsin-like” GPCRs in family A constitute the largest subfamily and are activated by wide range of structurally different ligands. In family A GPCRs, the orthosteric ligands bind to a crevice formed by transmembrane regions (TMs) 3, 5, 6 and 7 (e.g. rhodopsin, ␤2 -adrenoceptor) or to extracellular regions of the receptor (e.g. glycoprotein receptors for FSH, LH, etc.). Family B is a smaller family of receptors activated by peptides like secretin and glucagon, which bind to both extracellular and transmembrane regions of the receptor. Family C contains the “metabotropic-like” receptors for glutamate, calcium, ␥-aminobutyric acid (GABA) and various taste molecules, which bind exclusively to the extracellular amino-terminal domain (ATD) of the receptor (Fig. 1) (Gether, 2000). The recently published X-ray crystal structures of the rhodopsin receptor and the ATD of the metabotropic glutamate receptor subtype 1 (mGluR1) have been of immense importance for understanding family A and C GPCR function (Kunishima

2. GPCR function and allosteric modulation The term “GPCR” originates from these receptors’ ability to associate with heterotrimeric G-proteins and through them stimulate or inhibit the formation of second messengers such as inositol-3,4,5-tris-phosphate and cyclic AMP and modulate the function of ion channels (Gether, 2000; Pierce et al., 2002). They are also capable of interacting directly with ion channels and other effector molecules (Pierce et al., 2002). The GPCRs do not share an overall amino acid sequence identity and as shown in Fig. 1, their topologies vary considerably. The only structural feature shared by all GPCRs is the presence of seven predicted hydrophobic transmembrane ␣-helical segments of 20–25 amino acid residues (Gether, 2000; Palczewski et al., 2000), thus GPCRs are also frequently termed “7TM receptors”. The GPCR superfamily has classically been divided into subfamilies A, B and C based on amino acid sequence homologies (Fig. 1) (A)

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Fig. 1. The GPCR superfamily. The three subfamilies of GPCRs are depicted with examples of their endogenous agonists. The binding modes of the orthosteric ligands for each receptor type are depicted by a green rectangle. The GPCR signals either by coupling to heterotrimeric G-proteins consisting of ␣ and ␤␥ subunits (which trigger a wide range of metabolic cascades and ion channel activities) or by direct association with effector molecules. AC, adenylyl cyclase; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; PLC, phospholipase C; IP3 , inositol-3,4,5-tris-phosphate; DAG, diacylglycerol.

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et al., 2000; Palczewski et al., 2000; Tsuchiya et al., 2002; Liang et al., 2003; Hulme et al., 2003). In the late 1980s and early 1990s, observations of constitutive activity (receptor activity in the absence of agonist) and inverse agonism (suppression of constitutive activity by antagonists) fundamentally changed the perception of the molecular mechanisms underlying GPCR function (Costa and Herz, 1989). The extended ternary complex model or “two-state” model was formulated to explain these observations (Samama et al., 1993). A simpler version of this model was published by Leff (1995). In this model the GPCR fluctuates between active and inactive conformations. An agonist is a molecule that binds to and stabilises an active receptor conformation, while an inverse agonist stabilises an inactive receptor conformation. Compounds with similar affinities for active and inactive receptor conformations are termed “neutral antagonists”. Several authors have added the concept of allosteric modulation to the two-state model for family A GPCR (Tucek and Proska, 1995; Hall, 2000; Christopoulos and Kenakin, 2002) and family C GPCR function (Parmentier et al., 2002). The allosteric two-state model for family A GPCR function

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described by Hall (2000) is depicted in Fig. 2. The left side of the cube represents the equilibrium between the inactive and active GPCR conformations, either of which can be stabilised by ligands binding to the orthosteric site. It is homologous to the two-state model described by Leff (1995). The constants K and αK describe the affinity of the orthosteric ligand for the inactive and active receptor conformations, respectively, and the intrinsic activity (efficacy) of the orthosteric ligand is governed by α (α > 1 for agonists, α < 1 for inverse agonists and α = 1 for neutral antagonists). The top of the cube is directly analogous, except that the ligand binds to the allosteric site of the receptor. The affinity and the intrinsic activity of the allosteric ligand are governed by M and β, respectively. The rest of the model describes what happens when the orthosteric and allosteric ligands bind to the receptor simultaneously. Both ligands can potentially affect the binding and the intrinsic activity of the other ligand (binding cooperativity and activation cooperativity). These effects are described by the constants γ and δ, which are attributes of each pair of ligands. As shown on the front of the cube, the binding affinity of each ligand for the inactive receptor

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Fig. 2. Dissecting the allosteric two-state model. The allosteric two-state model cube. The orthosteric ligand is depicted as a square, the allosteric ligand as a circle, and the inactive and active GPCR conformations are given in grey and black, respectively. L, isomerisation constant for inactive and active GPCR conformations; M, association constant of allosteric ligand to inactive conformation; K, association constant of othosteric ligand to inactive conformation; α, intrinsic activity of orthosteric ligand; β, intrinsic activity of allosteric ligand; γ, binding cooperativity between orthosteric and allosteric ligand; δ, activation cooperativity between orthosteric and allosteric ligand.

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is modulated by the factor γ when the receptor binds both ligands simultaneously. Ligand binding to the active state of the receptor is modulated by both γ and δ (back of cube). The bottom of the cube shows the effect of simultaneous binding on the intrinsic activity of the orthosteric ligand (α). This is modulated by the intrinsic activity of the allosteric ligand (β) and δ. γ also indirectly affects the intrinsic activity of the orthosteric agonist unless β and δ = 1 or the allosteric ligand is present at saturating concentrations. Analogously, the intrinsic activity of the allosteric ligand is modulated by α, γ and δ when the receptor also binds an orthosteric ligand (the right hand side of the cube). Fig. 3A–D shows the predicted effects of four simulated allosteric modulators on the binding and function of an orthosteric agonist, to illustrate the effect of changing γ and δ. The simulated modulators have no intrinsic agonist activity themselves (β = 1), and the system is assumed to have reached equilibrium. Modulators 1 and 3 are negatively co-operative with the orthosteric agonist, and modulators 2 and 4 are positively co-operative. Modulator 1 stabilises a receptor conformation with lower affinity for the orthosteric agonist than the unliganded receptor, but has no effect on its intrinsic activity (γ = 0.1, δ = 1). This decreases the apparent affinity of the receptor for the orthosteric ligand (Kapp ) in radioligand binding studies (Fig. 3A). In functional assays, this causes a decrease in the potency of the orthosteric agonist, but it does not affect its intrinsic activity (Fig. 3B). Although the receptor binds the orthosteric agonist less well when occupied by modulator 1, it can still bind both molecules simultaneously, and thus modulator 1 reduces but cannot completely inhibit binding and receptor activation by the orthosteric agonist (Fig. 3C and D). Modulator 2 stabilises a receptor conformation with a higher affinity for the orthosteric ligand than the unliganded receptor (γ = 10, δ = 1), causing an increase in Kapp (Fig. 3A). As with modulator 1, this is reflected in an increase in the potency of the orthosteric ligand in functional assays, but it has no effect on its intrinsic activity (Fig. 3B). Modulator 3 does not affect the affinity of the orthosteric agonist for the inactive receptor conformation, but it decreases its affinity for the active receptor conformation and displaces the equilibrium between the active and inactive receptor conformation towards the inactive conformation (γ = 1, δ = 0.1). Since binding to the active conformation of the receptor is a major component of agonist binding, this causes a decrease in Kapp (Fig. 3A) and in the potency of the orthosteric agonist in functional assays (Fig. 3B). Since modulator 3 decreases the proportion of receptor in the active conformation, it also decreases the intrinsic activity of the orthosteric agonist (Fig. 3B). Similarly, modulator 4 (γ = 1, δ = 10) increases Kapp and the potency and intrinsic activity of the orthosteric agonist. This model predicts that allosteric modulators with δ = 1 will affect the Kapp of the orthosteric agonist.

The nature and degree of cooperativity between allosteric and orthosteric ligands is dependent on both ligands and receptor subtype. For example, the allosteric modulator N-benzyl-brucine has displayed positive cooperativity with the antagonist [3 H]N-methylscopolamine ([3 H]NMS) and negative cooperativity with [3 H]acetylcholine at the muscarinic acetylcholine receptor M2 , but positive cooperativity with [3 H]acetylcholine and negative cooperativity with [3 H]NMS on M3 (Lazareno et al., 1998). More surprisingly, the muscarinic agonist pilocarpine acted as an antagonist in the presence of alcuronium, while another agonist, oxotremorine, retained its agonistic properties although its potency was reduced (Zahn et al., 2002). The model shown in Fig. 2 is too simple to describe allosteric interactions of family C GPCRs, where the orthosteric ligand binding site is located at the extracellular amino terminal domain and allosteric modulators generally bind to the seven transmembrane domain. For example, several allosteric modulators of mGluRs have been reported not to affect the EC50 or Kapp of orthosteric ligands (Litschig et al., 1999; Pagano et al., 2000; Carroll et al., 2001). The interactions of Family C GPCRs with allosteric modulators are thus better described by the model proposed by Parmentier et al. (2002). Allosteric modulators also affect the binding kinetics of orthosteric ligands when binding simultaneously, often decreasing or increasing the dissociation and association rates of radiolabeled ligands (Stockton et al., 1983; Tucek and Proska, 1995). This is illustrated in Fig. 3E. This property of allosteric ligands has been used extensively to characterise their interactions with receptors, orthosteric ligands and other allosteric ligands binding through alternative allosteric sites (Ellis and Seidenberg, 1992; Gnagey et al., 1999; Lazareno et al., 2002). Changes in binding kinetics can be extreme and prevent assays from reaching equilibrium, complicating modelling studies such as those described above (Tucek and Proska, 1995). Non-equilibrium approaches have been used to overcome this difficulty (e.g. Lazareno et al., 1998). Allosteric modulators have classically been defined as ligands having no intrinsic agonist or inverse agonist activity themselves (β = 1). However, this presumption has been challenged repeatedly in recent years. Allosteric ligands such as gallamine and PD81,723 have been demonstrated to possess intrinsic agonist activity at muscarinic and adenosine receptors, respectively (Jakubik et al., 1996; Kollias-Baker et al., 1997), and the allosteric ligands BAY36-7620 and MPEP have been shown to be inverse agonists of mGluR1 and mGluR5, respectively (Pagano et al., 2000; Carroll et al., 2001). The model in Fig. 2 predicts this, as an allosteric ligand can have agonist (β > 1) or inverse agonist (β < 1) activity in the absence of the orthosteric ligand. Alcuronium has displayed both agonism and inverse agonism on recombinant M2 muscarinic receptors (Jakubik et al., 1996; Zahn et al., 2002). This phenomenon, “protean agonism”, has been reported for other receptors

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Fig. 3. Simulated pharmacological profiles of allosteric modulators. Predicted effects of orthosteric agonist/allosteric modulator pairs having different binding cooperativity (γ) and activation cooperativity (δ) values on ligand binding and receptor activation. Modulator 1: γ = 0.1, δ = 1; 2: γ = 10, δ = 1; 3: γ = 1, δ = 0.1; 4: γ = 1, δ = 10; N: no modulator. (A) Predicted effects of 1 ␮M concentrations of four allosteric modulators (1–4) on the apparent affinity (Kapp ) of a labeled orthosteric agonist in binding assays. (B) Receptor activation under the same conditions as (A). (C) Predicted effect of the four allosteric modulators on the binding of a constant concentration (200 nM) of a labeled orthosteric ligand. (D) Receptor activation under the same conditions as (C). Curves were calculated using the following equations (after Hall, 2000), where [A] = concentration of the orthosteric ligand and [B] = concentration of the allosteric ligand (other parameters are defined in Fig. 2): 100 × (K[A] + αKL[A] + γKM[A][B] + αβγδKLM[A][B]) percentage receptor occupancy by orthosteric ligand = 1 + L + K[A] + αKL[A] + M[B] + βLM[B] + γKM[A][B] + αβγδKLM[A][B] 100 × (L + αKL[A] + βLM[B] + αβγδKLM[A][B]) percentage receptor activation = 1 + L + K[A] + αKL[A] + M[B] + βLM[B] + γKM[A][B] + αβγδKLM[A][B] Parameter values: α = 100; β = 1; K = 106 ; L = 0.05; M = 106 . (E) Predicted effect of allosteric modulators on the dissociation kinetics of an orthosteric ligand. Dashed line: no modulator. The dissociation rate for the orthosteric ligand from the free receptor (koff ) = 0.1 min−1 ; modulator 5: koff for the receptor/allosteric ligand complex is increased to 0.2 min−1 . Modulator 6: koff for the receptor/allosteric ligand complex is decreased to 0.05 min−1 . Curves were calculated using the following equation (Lazareno et al., 2002): percentage receptor occupancy by orthosteric ligand = 100 exp(−t(koff + [B]Kocc koffX )/(1 + [B]Kocc )), where t = time, koff and koffX are the dissociation rates of the orthosteric ligand from the free receptor and the receptor/allosteric ligand complex. Kocc is the affinity of allosteric ligand for the receptor/orthosteric ligand complex. In this simulation, Kocc = 106 and [B] = 1 ␮M.

(see Kenakin, 2003). Since the detection of intrinsic activity of a ligand is dependent on the assay sensitivity, this aspect of the definition of an allosteric modulator may be academic. Examples of allosteric ligands with pronounced intrinsic agonistic activity (allosteric agonists) are given in Section 3.4.

3. Allosteric modulators of GPCRs 3.1. Endogenous GPCR modulators The modulation of GPCRs by endogenous substrates could be perceived as one of nature’s subtle ways of

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fine-tuning neurotransmitter/hormone signalling. A classic case of endogenous GPCR modulation is the electrostatic interaction between a sodium ion and a highly conserved aspartate residue located in cytoplasmic part of TM2 of a number of family A GPCRs (Fig. 4) (Horstman et al., 1990; Neve et al., 1991, 2001; Martin et al., 1999; Schetz and Sibley, 2001; Gao et al., 2003). Neutralisation of the negatively charged aspartate by Na+ binding has been proposed to alter the conformation of the GPCR, whereby agonist binding affinities are decreased and the G-protein coupling of the receptor is inhibited. In recent mutagenesis studies of the dopamine D2 and the adenosine A3 receptors additional residues in TM1, TM3 and TM7 have been proposed to be involved in Na+ binding to the receptors as well (Fig. 4) (Neve et al., 2001; Gao et al., 2003). Zn2+ has also been shown to modulate ligand binding and functional properties of a wide range of family A GPCRs (Schetz and Sibley, 1997; Rosenkilde et al., 1998; Schetz et al., 1999; Holst et al., 2002; Swaminath et al., 2002b). In the CNS zinc is stored in synaptic vessels and co-released with neurotransmitters leading to synaptic concentrations of up to 300 ␮M (Frederickson, 1989), so its effects on the GPCRs are physiologically relevant. In contrast to the general inhibition of Na+ on a wide range of GPCRs, Zn2+ exerts a wide range of different functional effects through different sites on the ␤2 -adrenergic, dopamine, melanocortin and tackykinin NK3 receptors (Fig. 4) (Rosenkilde et al., 1998; Schetz et al., 1999; Schetz and Sibley, 2001; Holst et al., 2002; Swaminath et al., 2002a). The calcium-sensing receptor (CaR) is potentiated by low millimolar concentrations of l-amino acids, which bind to serine residues located adjacent to the Ca2+ binding site

in the ATD of the receptor (Conigrave et al., 2000; Zhang et al., 2002). Analogously, Ca2+ increases agonist potency at another family C GPCR member, the GABAB heterodimer, through association with a serine residue located close to the othosteric site of GABAB1 (Prézeau et al., 1999). Ca2+ has also been proposed as an allosteric potentiator of some mGluR subtypes but it is not certain that the effects are receptor-mediated (Kubo et al., 1998; Nash et al., 2001). Serotonergic 5-HT2 and 5-HT7 receptor signalling have been reported to be potentiated by oleic acid and oleamide (Thomas et al., 1997; Alberts et al., 2001), and the effects of the cerebral tetrapeptide Leu-Ser-Ala-Leu (5-HT-moduline) on 5-HT1B/1D receptors constitutes a fascinating case of endogenous GPCR modulation. The peptide binds with subnanomolar affinity to presynaptic 5-HT1B/1D receptors and inhibits receptor function noncompetitively (Fillion et al., 1996; Massot et al., 1996). Since the receptors control the release of 5-HT from the serotonergic neuron terminals, inhibition of this receptor constitutes an attractive mechanism to increase serotonergic signalling. Thus, synthetic compounds facilitating the interaction between 5-HT-moduline and 5-HT1B/1D receptors or mimicking the effects of the tetrapeptide could have some therapeutic perspectives. Finally, GPCR signalling is also modulated by the receptors’ interactions with intracellular adaptor and scaffolding proteins (Sheng and Sala, 2001; Pierce et al., 2002). The interactions between GPCRs and the GPCR kinase (GRK)/␤-arrestin system are responsible for the homologous (or agonist-specific) desensitisation of a considerable number of GPCRs (Pierce et al., 2002). ␤-arrestin-1 and ␤-arrestin-2 are expressed ubiquitously in multiple tissues, where they bind to intracellular regions of the

Fig. 4. Molecular basis for allosteric modulation of family A and C GPCRs. (A) Allosteric sites at family A GPCRs. The residues indicated have been determined to be crucial for the actions of different endogenous and synthetic allosteric modulators at their respective GPCRs. Only metal ion binding sites existing in wild-type receptors are shown. (B) Allosteric sites at the 7TMs of mGluR1, mGluR2 or mGluR5. Residues or regions determined to be crucial for the subtype selectivities of allosteric inhibitors CPCCOEt, MPEP, EM-TBPC, BAY36-7620 and allosteric potentiators Ro 67-7476 and LY487379 are depicted. The two residues in TM3 shared by Ro 67-7476 and MPEP in their interactions with mGluR1 and mGluR5, respectively, are given as mGluR1 residue/mGluR5 residue.

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agonist-occupied, phosphorylated GPCR. The formation of the GPCR/␤-arrestin complex facilitates the internalisation of the receptor via clathrin-coated vesicles due to its interactions with chlathrin itself and the ␤2-subunit of the clathrin adaptor protein AP-2 and through ubiquitylation, the post-translational addition of ubiquitin molecules to lysine residues in both ␤-arrestin and the GPCR (Pierce et al., 2002). In addition, ␤-arrestins function as scaffolding proteins bridging the GPCR with kinases involved in the intracellular ERK/MAPK and JNK3 phosphorylation cascades (Pierce et al., 2002). Other examples of scaffolding/adaptor proteins for GPCRs are the Homer proteins, which have a profound impact on the signalling, trafficking and clustering of mGluR subtypes 1a and 5, and the cytoplasmic proteins PICK1 and calmodulin, which appear to be key participants in the complex synaptic regulation of G␤␥-signalling by mGluR7 (Xiao et al., 2000; El Far and Betz, 2002). 3.2. Synthetic allosteric modulators of family A GPCRs Allosteric modulators have been identified for many family A GPCRs including the ␣1A and ␣2A adrenoceptors (Leppik and Birdsall, 2000; Leppik et al., 2000; Sharpe et al., 2003), adenosine receptors (Kollias-Baker et al., 1997; Gao and Ijzerman, 2000; Gao et al., 2001), chemokine receptors (Sabroe et al., 2000), dopamine receptors (Hoare and Strange, 1996), serotonin receptors (Im et al., 2003), and muscarinic receptors. The first generation of muscarinic allosteric ligands, gallamine and alkane-bisammonium compounds such as W84, were initially demonstrated to inhibit native cardiac muscarinic receptor function (Lüllmann et al., 1969; Clark and Mitchelson, 1976), and subsequently [3 H]NMS binding studies revealed that the actions of gallamine were of an allosteric nature (Stockton et al., 1983). Subsequently, a wide range of structurally diverse compounds have been shown to act allosterically (Tucek et al., 1990; Ellis and Seidenberg, 1992; Birdsall et al., 1997, 1999; Jakubik et al., 1997; Lazareno et al., 1998, 2000, 2002) (Fig. 5). In general, M2 is the most sensitive muscarinic receptor subtype to allosteric modulation, but no truly subtype-specific allosteric modulator has been identified. This can be ascribed to the high degree of amino acid sequence identity between the 5 muscarinic subtypes. Most allosteric modulators, including the negatively co-operative modulator gallamine and the positively co-operative modulator alcuronium, appear to bind through a “common allosteric site” positioned above the orthosteric site in the extracellular regions of the muscarinic receptor (Fig. 4). The second and third extracellular loops appear to be key participants in this site, but residues in the first extracellular loop and TM domains also appear to contribute (Ellis and Seidenberg, 1992, 2000; Ellis et al., 1993; Waelbroeck, 1994; Matsui et al., 1995; Tränkle and Mohr, 1997; Gnagey et al., 1999; Lazareno et al., 2000; Krejci and Tucek, 2001; Buller et al., 2002). Recently, two specific amino acid residues in M2 (Tyr177 in the second extracellular loop and Thr423 in TM7)

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were shown to be of key importance for the actions of the alkane-bisammonium and caracurine V types of allosteric ligands although other regions of the receptor appear be involved as well (Buller et al., 2002; Voigtländer et al., 2003). Other ligands including KT5720 and various WIN62,577 analogs appear not to act through the common allosteric site demonstrating that there are multiple allosteric sites on the muscarinic receptors (Lazareno et al., 2000, 2002). Selected examples of synthetic allosteric modulators identified for muscarinic receptors and other family A GPCRs are given in Fig. 5. Fig. 4A shows identified molecular interactions between selected endogenous and synthetic allosteric ligands and their receptor. 3.3. Synthetic allosteric modulators of family C GPCRs Recently, a substantial number of potent allosteric inhibitors and potentiators of family C GPCRs have been identified (Fig. 5). The allosteric modulators of mGluRs published to date are all highly selective for specific subtypes. Interestingly, the binding sites of allosteric inhibitors CPCCOEt, MPEP, BAY36-7620, EM-TBPC and R314127 and allosteric potentiators Ro 01-6128, Ro 67-7476, LY487379, NPS R-467 and NPS R-568 have been determined to reside exclusively within the 7TMs of mGluR1, mGluR2, mGluR5 or CaR, far away from the othosteric site in the ATDs of the receptors (Fig. 1) (Hammerland et al., 1998; Litschig et al., 1999; Pagano et al., 2000; Carroll et al., 2001; Knoflach et al., 2001; Schaffhauser et al., 2003; Lavreysen et al., 2003). Specific residues responsible for the subtype selectivities of CPCCOEt, Ro 67-7476 and EM-TBPC at mGluR1, LY487379 at mGluR2, MPEP at mGluR5, and Calhex 231 at CaR have been identified (Fig. 4B) (Litschig et al., 1999; Pagano et al., 2000; Knoflach et al., 2001; Schaffhauser et al., 2003; Malherbe et al., 2003a,b; Petrel et al., 2003). Initially, two amino acid residues in TM7 of mGluR1 and three residues in TM3 and TM7 of mGluR5 were identified as crucial determinants of the subtype selectivities of CPCCOEt and MPEP, respectively (Fig. 4B) (Litschig et al., 1999; Pagano et al., 2000). Subsequently, the allosteric potentiator Ro 67-7476 was shown to interact with residues in TM3 and TM5 of mGluR1, some of which corresponded to residues involved in MPEP binding to mGluR5 (Knoflach et al., 2001; Pagano et al., 2000). Binding of EM-TBPC to mGluR1 was shown to involve Val757 and Thr815 , residues also involved in Ro 67-7476 and CPCCOEt binding to the receptor, respectively (Fig. 4B) (Litschig et al., 1999; Knoflach et al., 2001; Malherbe et al., 2003a). Furthermore, NPS 2390, CPCCOEt and BAY36-7620 were shown to displace [3 H]R314127 binding to mGluR1a (Lavreysen et al., 2003). In two recent studies, numerous additional residues in TM5 and TM6 of mGluR5 have been demonstrated to contribute to the binding of MPEP, and the binding site of Calhex 231, an allosteric inhibitor of CaR signalling, has been shown to include residues in TM3, TM5, TM6 and TM7 of the receptor (these residues are not shown in Fig. 4B)

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Fig. 5. Allosteric ligands of family A and C GPCRs. Chemical structures of selected allosteric modulators of family A and C GPCRs. The GPCRs targeted by the ligands are given next to the name of the ligand. The family C GPCR modulators have been divided into inhibitors and potentiators based on their properties in functional assays.

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(Malherbe et al., 2003b; Petrel et al., 2003). Hence, all of the allosteric modulators appear to bind in a crevice composed by these four transmembrane segments in the family C GPCR (Fig. 4B). The notion of a general allosteric site in the 7TM of the family C GPCR mediating both allosteric potentiation and inhibition, much like the common allosteric site in the muscarinic receptors, is also supported by the subtle structural differences between allosteric inhibitors and potentiators of the receptors (Fig. 5). Allosteric inhibitors of mGluR1 or mGluR5 signalling, such as PHCCC, SIB-1893 and MPEP, have been shown to be weak allosteric potentiators of mGluR4 signalling (Maj et al., 2003; Marino et al., 2003; Mathiesen et al., 2003), and a series of benzaldazine analogues (including DFB in Fig. 5) have exhibited everything from allosteric potentiation to allosteric inhibition to neutral cooperativity on mGluR5 signalling (O’Brien et al., 2003). It is interesting that the majority of allosteric ligands at family C GPCR bind to the same TMs that form the agonist binding site in the monoamine family A GPCRs (Gether, 2000). However, LY487379, an allosteric potentiator of mGluR2 function, has recently been demonstrated to bind to residues in TM5 as well as in TM4, indicating that other TMs can participate in the binding of allosteric modulators as well (Fig. 4B) (Schaffhauser et al., 2003). Allosteric potentiators for another family C GPCR, the GABAB receptor, have also been identified but their binding sites remain to be determined (Urwyler et al., 2001, 2003). The high number of allosteric ligands identified for muscarinic receptors and mGluRs compared to other GPCRs is unlikely to be caused by a higher sensitivity of these receptor types to allosteric modulation, but probably reflects a more intensive search for allosteric modulators for these receptors. 3.4. Allosteric and ectopic agonists of GPCRs The trivalent metal ion Gd3+ has been demonstrated to be an allosteric agonist at CaR, where it acts through a binding site in the 7TM of the receptor (Hammerland et al., 1999). Either Gd3+ is capable of mimicking the activation signal caused by agonist binding to the orthosteric site in the ATD of CaR, or it triggers G-protein coupling through an alternate activation switch located in the 7TM. AC-42 (Fig. 5) is a partial agonist of the muscarinic receptor subtype M1 , and it is highly subtype-selective compared to other muscarinic agonists and modulators (Spalding et al., 2002). The amino-terminal and upper part of TM1 and the third extracellular loop and TM7 in M1 have been shown to be crucial determinants for the agonist activity of AC-42 (Fig. 4). It is unproven whether the AC-42 binding site overlaps with the orthosteric site, and to distinguish it from ligands binding to the common allosteric site, AC-42 was termed an ectopic ligand (meaning “away from a place”) rather than an allosteric ligand (Spalding et al., 2002). Recently, the 17-mer peptides RSVM and ASLW have been reported to display partial agonism and superag-

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onism at the chemokine receptor CXCR4, respectively (Sachpatzidis et al., 2003). In contrast to the response elicited by the endogenous agonist, SDF-1␣, the RSVMand ASLW-mediated CXCR4 signalling could not be antagonised by small molecule antagonists and antibodies, an observation that suggests that RSVM and ASLW do not bind the same site as the antagonist (Sachpatzidis et al., 2003).

4. GPCRs, GPCR dimers and GPCR-protein complexes as targets for allosteric modulators It is clear that GPCRs can be activated by mechanisms that do not involve agonist binding to the orthosteric ligand binding site. Schwartz and coworkers have engineered artificial agonistic Zn2+ binding sites in analogous positions in the ␤2 -adrenergic receptor and the distantly related tachykinin NK1 receptor (Elling et al., 1999; Holst et al., 2000). This is remarkable considering the different binding modes of the orthosteric ligands to the two receptors (Fig. 1). Furthermore, there are numerous examples of GPCRs constitutively activated by mutations of residues not involved in agonist binding or G-protein coupling (Spalding et al., 1997, 1998; Parnot et al., 2002). These phenomena suggest that it is feasible to invent a broad spectrum of agonists, antagonists and inverse agonists that bind allosterically. The crystal structures of the rhodopsin receptor and the ATD of mGluR1 have been used extensively as templates for molecular models of family A and C GPCRs and will be useful in future rational ligand design efforts (Kunishima et al., 2000; Palczewski et al., 2000; Tsuchiya et al., 2002), but it is unlikely that these models will enable rational design of allosteric ligands because the binding sites of allosteric compounds are still largely unknown. GPCRs should not be considered only as single units interacting solely with G-proteins. Homo- and heterooligomerisation of GPCRs can have a profound impact on transport, expression and functional properties of the receptors, and the association of GPCRs with intracellular scaffolding and adaptor proteins are of great importance for receptor compartmentalisation, function and regulation (Sheng and Sala, 2001; Angers et al., 2002; Pierce et al., 2002). Thus, an additional class of GPCR modulators could include ligands facilitating or obstructing these association processes. Very little is known about the structure of the GPCR dimer interfaces in family A and B GPCRs. However, the interface between the two ATDs in family C GPCR homodimer appears to be a highly sensitive region for both positive and negative allosteric modulation (Jensen et al., 2002; Tsuchiya et al., 2002). The two 7TMs in the family C GPCR homodimer have also been speculated to form an integrated dimeric complex upon receptor activation (Kunishima et al., 2000). Hence, the allosteric potentiators and inhibitors acting at this level of the receptors could exert their effects through facilitation or disruption of this process, respectively (Fig. 4).

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The interface between the GPCR and G-protein is also a target for allosteric ligands. GPCR coupling with G-proteins can be antagonised both by insect venoms and synthetic peptides derived from the G-protein sequence (Freissmuth et al., 1999). Furthermore, small molecules like suramin analogs target G-protein ␣-subunits and have been shown to uncouple G␣i /G␣o - and G␣s -proteins from their respective receptors (Hohenegger et al., 1998; Freissmuth et al., 1999). Inhibitors or enhancers of the interactions between GPCRs and scaffolding/adaptor proteins could also potentially serve as allosteric modulators of particular signalling pathways (Sheng and Sala, 2001; Zhong and Neubig, 2001). Ligands targeted at intracellular GPCR regions or their intracellular complex partners would have to be fairly lipophilic in order to penetrate the plasma membrane.

5. Therapeutic advantages of allosteric ligands 5.1. Nature of response Allosteric modulators with little inherent intrinsic activity that act by enhancing or attenuating the response elicited by the endogenous transmitter hold several advantages to the actions of conventional agonists and antagonists. Firstly, the effects of the allosteric modulator are often saturable and are therefore less likely to elicit adverse effects from overdose. Secondly, the effects of the modulator are closely linked with the physiological pulse of synaptic signalling, and thus it will only amplify or reduce the neural signal when the neurotransmitter is released into the synapse. Hence, the modulator will affect signalling in the same tone as the endogenous transmitter, which means that the GPCR will be less likely to desensitise upon sustained exposure to an allosteric potentiator compared to an agonist. 5.2. Selectivity The orthosteric sites of GPCR subtypes activated by the same endogenous transmitter exhibit a high degree of sequence conservation, making them poor sites for subtypeselective ligands. Allosteric and ectopic ligands can potentially be more selective than orthosteric ligands because they can act through nonconserved sites. The 7TM-binding allosteric ligands of specific mGluR subtypes and the M1 selectivity of AC-42 are excellent examples of this (Fig. 5). Additionally, Lazareno et al. (1998) showed that some allosteric ligands are potentiators on some subtypes, but neutral or inhibitors on others—a phenomenon they termed “absolute selectivity”. Examples of allosteric modulators that bind to receptors with different endogenous agonists also exist. Amiloride analogs are allosteric inhibitors of antagonist binding and function of ␣-adrenergic, dopamine and adenosine receptors, and SCH-202676 inhibits antagonist binding to a wide

range of family A GPCRs (Fig. 5) (Hoare and Strange, 1996; Gao and Ijzerman, 2000; Leppik and Birdsall, 2000; Leppik et al., 2000; Fawzi et al., 2001). Hence, it is possible that similar structural elements located distant from the orthosteric sites of the GPCRs could constitute a common motif for allosteric ligands at different GPCRs. Genes encoding for GPCRs are very often subjected to splice variation. Typically, the splice variations do not include regions involved in agonist recognition, and the orthosteric site is therefore often identical in the splice variants. This limits the potential for inventing splice variant-selective ligands that act through the orthosteric site. In contrast, it may be possible to find allosteric ligands that bind close to the splice site. This strategy could be very useful in targeting the GABAB heterodimer complex, where the splicing site of the GABAB1 subunit responsible for GABA binding is located far away from the orthosteric site. Subtype-selective allosteric ligands would be highly attractive in studies of the physiological roles of the two predominant GABAB receptors GABAB(1a,2) and GABAB(1b,2) . 5.3. Signalling It has been shown that different active GPCR conformations can be characterised by distinct signalling pathways (Perez et al., 1996; Palanche et al., 2001; Kenakin, 2003), and this could be beneficial for therapeutic compounds. An allosteric agonist could hypothetically give rise to a different signal than the orthosteric agonist. Furthermore, the regulation of a GPCR activated by an allosteric agonist could be substantially different from that activated by an orthosteric agonist, since the different active receptor conformations could be differentially targeted by kinases and ␤-arrestins. For example, in contrast to the orthosteric agonist the allosteric agonist ASLW does not cause internalisation of the CXCR4 receptor, which contributes to the superagonism of the compound (Sachpatzidis et al., 2003). Additionally, very distinct phosphorylation and internalisation patterns have been observed for two different constitutive active mutants of the ␣1b -adrenergic receptor (Mhaouty-Kodja et al., 1999). 5.4. Replacement of protein and peptide ligands Allosteric and ectopic agonists also offer an opportunity to activate receptors otherwise considered difficult targets for small molecule ligands, such as the peptide and hormone GPCRs in family A and B. The orthosteric sites in these receptors are composed of residues from many receptor regions located far apart from each other, making it virtually impossible for a small molecule to mimic the binding of the endogenous agonist (Fig. 1). Small molecule ligands targeted at these receptors are also attractive from a drug delivery perspective because the acidic milieu of the stomach rule out per oral administration of peptides and proteins.

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6. Conclusion Increasing numbers of allosteric GPCR ligands are being published. Subtype-selective allosteric ligands have already proven valuable pharmacological tools in in vivo investigations of the physiological roles of GPCRs, and delineation of the interactions of these ligands with their respective receptors have contributed significantly to the understanding of the signal transduction through the receptors. Considering the continued extensive use of high throughput technologies in the drug discovery programmes of drug companies, there is little doubt that several allosteric ligands will be introduced as drugs on the market in the years to come. It will be interesting to see whether the proposed therapeutic advantages of allosteric ligands compared to orthosteric ligands will give rise to clinical benefits.

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