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Allosteric Modulation of Family 3 GPCRs

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Page 1: Allosteric Modulation of Family 3 GPCRs

Allosteric Modulation of Family 3 GPCRsTobias Noeskea, b, Aleksandrs Gutcaitsc, Christopher G. Parsonsa and Tanja Weila*a Merz Pharmaceuticals GmbH, Eckenheimer Landstraße 100, D-60318 Frankfurt am Main, Germany, Phone: þþ49/69/1503478,Fax: þþ49/09/5962150, E-mail: [email protected]

b Johann Wolfgang Goethe-Universit6t, Institut f8r Organische Chemie und Chemische Biologie, Marie-Curie-Str. 11, D-60439Frankfurt am Main, Germany

c Latvian Institute of Organic Synthesis, 21 Aizkraukles, Riga LV 1006, Latvia

Keywords: GPCR, Family C GPCRs, Transmembrane helix, Homology model, Ligand binding,Mutation analysis

Received: May 4, 2005; Accepted: August 24, 2005

DOI: 10.1002/qsar.200510139

AbstractThis article reviews recent advances towards an understanding of the transmembranestructure, its activation and binding sites of family 3 G-Protein-Coupled Receptors (3-GPCRs). First, the prediction of their 3D structure via homology models based on thecrystal structure of bovine rhodopsin will be discussed. Then, different kinetic mechanismsof ligand-binding inside the heptahelical domain are presented before an overview ofmost common structural motifs of 3-GPCR allosteric modulators will be given. Here,special focus lies in the characterization of their binding pocket within the transmembranedomain as well as their interaction with crucial amino acids. The overview of availablemutation data inside the ligand-binding domains as well as the influence of exchangedamino acids on the activity of different receptors leads to the question whether allostericligands of family 3 GPCRs bind to the same binding pocket and if there exists a commonbinding site of family 1 GPCRs and family 3 GPCRs.

1 Introduction: Family 3 GPCRs

G-Protein-Coupled Receptors (GPCRs) are the largestfamily of cell-surface receptors involved in signal transmis-

sion. They have been successful during evolution in recog-nizing a wide range of stimuli from photons to large glyco-proteins [1]. These receptors transduce extra-cellular sig-nals in cellular responses via heterotrimeric G proteins.

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Abbreviations: G-Protein-Coupled Receptors (GPCRs); Hepta-helical Domain (HD); Calcium-Sensing Receptors (CaSRs); g-Aminobutyric Acid Type B Receptors (GABABR1/2s); Extrac-elluar Domain (ECD); Intracellular Domain (ICD); Metabo-tropic Glutamate Receptors (mGluRs); Bacteriorhodopsin(BR); Bovine Rhodopsin (bRho); Bacterial Periplasmic AminoAcid-Binding Proteins (PBPs); Nuclear Magnetic Resonance(NMR); Transmembrane Helix (TM); 5-Methylene-6a-naphtha-len-2-ylmethy l-hexahydro-cyclopenta[c]furan-1-one (BAY36-7620) [61]; 3-Ethyl-6-piperidin-1-yl-1,2,3,4-tetrahydro-thioxanth-en-9-one (Bayer Compound); 2-Hydroxyimino-1a,2-dihydro-1H-7-ox a-Cyclopropa[b]naphthalene-7a-carboxylic Acid Phenyla-mide (PHCCC); 4-Chloro-N-[2-(1-naphthalen-1-yl-ethylamino)-cyclohexyl]-benzamide (Calhex 231) [74, 75]; (1aRS,7aRS)-2-Hy-droxyimino-1a, 2-Dihydro-1H-7-oxacyclopropa[b]naphthalene-7a-carboxylic Acid Ethyl Ester, (þ /� )-1 (CPCCOEt) [92];N,N’-Bis-(3-methoxy-benzylidene)-hydrazine (DMeOB) [81]; 1-(3,4-Dihydro-2H-pyrano[2,3-b)quinolin-7-yl)-2-phenyl-1-etha-none (R214127) [66, 93]; 1-[2-Cycloheptyloxy-2-(2,6-dichloro-phenyl)-vinyl]-1H-[1,2,4]triazole (Ro 64-5229) [73]; 3-Chloro-8-[3-(5-hydroxymethyl-[1,2,3]triazol-1-yl)-phenyl]-2-pyrrolidin-1-yl-5,7-dihydro-benzo[b][1,4]diazepin-6-one (Roche Compound)[73]; 2-Quinoxaline-carboxamide-N-adamantan-1-yl (NPS 2390)

[64]; 2-Chloro-6-[3-(1,1-dimethyl-2-naphthalen-2-yl-ethylamino)-2-hydroxy-propoxy]-benzonitrile (NPS 2143) [74, 75]; 2-[4-(In-dan-2-ylamino)-5,6,7,8-tetrahydro-quinazolin-2-ylsulfanyl]-etha-nol (LY 456066) [94]; (1-Ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahy-dro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile(EM-TBPC) [28]; 2-Methyl-6-(phenylethynyl)-pyridine (MPEP)[67]; 3-(2-Methyl-thiazol-4-ylethynyl)-pyridine (MTEP) [77]; N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)-pyrid-3-yl-methylamine (LY 487379) [95]; 3,5-Dimethyl-1H-pyr-role-2,4-dicarboxylic Acid 2-Propyl Ester 4-(1,2,2-trimethyl-pro-pyl) Ester (GSK compound) [96, 97]; 2-(3-Methoxy-4-pyridin-3-yl-phenyl)-imidazo[1,2-a]pyridine (Merck compound 1) [70]; 3’-Fluoro-5’-(5-pyridin-2-yl-tetrazol-2-yl)-biphenyl-2-carbonitrile(Merck compound 2) [69]; 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (Merck compound 3) [98, 99]; Diphenylacetyl-carbamic Acid Ethyl Ester (Ro 01-6128) [62]; (9H-Xanthene-9-carbonyl)-carbamic Acid Butyl Ester (Ro 67-4853) [62]; 2-(4-Fluoro-phenyl)-1-(toluene-4-sulfonyl)-pyrrolidine (Ro 67-7476)[62]; N,N’-Bis-(3-fluoro-benzylidene)-hydrazine (DFB) [81]; [3-(2-Chloro-phenyl)-propyl]-[1-(3-methoxy-phenyl)-ethyl]-amine(NPS R-568) [100]; N,N’-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS 39783) [84, 85]

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Several different signal transduction pathways as well assecond messengers are involved in GPCRsP function,which are even different among the subtypes of a given re-ceptor. This might be also one reason for the broad thera-peutic potential of GPCRs. Numerous diseases have beenlinked to specific mutations within the genes encodingGPCRs, marking these receptors as targets for specifictherapeutic interventions [2, 3]. Today 50 % of all recentlylaunched drugs are targeted against GPCRs with annualworldwide sales exceeding $30 billion in 2001 [4].All GPCRs share a common central domain composed

of seven transmembrane helices, the Heptahelical Domain(HD, Figure 1).On the basis of sequence similarity, mammalian GPCRs

have been classified into three major categories, namelythe rhodopsin/b-adrenergic receptors (family 1) whichcontain many receptors for classical neurotransmitters, thesecretin receptor family (family 2) comprising receptorsfor distinct hormones and peptides, and the metabotropicglutamate receptors (family 3) [5]. Apart from Metabo-tropic Glutamate Receptors (mGluRs), family 3 G-pro-tein-coupled receptors (3-GPCRs) comprise the g-Amino-butyric Acid Type B Receptors, (GABABR1 and GABAB

R2) [6, 7], the parathyroid Calcium-Sensing Receptors(CaSRs) [8], and the vomeronasal receptors [9], e.g., sometaste and putative pheromone receptors [10]. Glutamatereceptors mediate excitatory transmission on the cellularsurface through initial binding of glutamate [11, 12]. ThemGluR family comprises eight subtypes and several splicevariants, which have been cloned and named mGluR1 – 8according to the succession of the molecular cloning [13].These eight receptors are further subdivided into threegroups based on sequence homology, pharmacology andtransduction mechanism: Group I (mGluR1 andmGluR5), Group II (mGluR2 and mGluR3) and GroupIII (mGluR4, mGluR6, mGluR7 and mGluR8). The CaSRis an ion-sensing GPCR that is allosterically regulated byextracellular calcium and different aromatic amino acidse.g., l-phenylalanine and l-tyrosine [14, 15]. GABAB is in-

volved in the presynaptic inhibition of transmitter releaseand mediates the slow synaptic inhibition by increasingthe potassium conductance responsible for long-lasting in-hibitory postsynaptic potentials [6, 7].3-GPCRs are characterized by an Extracelluar Domain

(ECD), a HD consisting of seven transmembrane heliceswhich are linked by six alternating extracellular and intra-cellular loops and an Intracellular Domain (ICD) whichcontains the C-terminus and the G-protein interactionsites (Figure 1). They possess a typical but unique feature:a large extracellular ligand-binding domain (ECD) thatshares some sequence similarities with bacterial Periplas-mic Amino Acid-Binding Proteins (PBPs) [16]. The ECDis characterized by a bilobate structure and adopts a closedconformation on agonist binding in the cleft that separatesboth lobes and is often called “Venus flytrap module” [17,13]. This stands in contrast to most other GPCRs, wherenatural ligand binding occurs in the HD. The orthostericbinding sites of 3-GPCRs are well understood today, whichis mainly due to the success in crystallizing the ECD ofmGluR1 with and without glutamate associated [18] aswell as due to detailed mutation studies of both mGluRs[19], GABAB [20] and CaSR [21]. In contrast, little isknown about the 3D structure and dynamics of the HD orthe binding mode of allosteric modulators. However, inthe past, there has been much effort in the identificationof new allosteric modulators, especially in the mGluRarea. This is mainly due to the fact that ligands binding inthe HD possess more drug-likeness than their analoguesinteracting with the orthosteric site. In particular, the ap-plication of HTS technologies in pharmaceutical industryfacilitated the discovery of agonists and antagonists bind-ing exclusively in the HD of the receptor.This review will attempt to show the current knowledge

of homology modelling of the HD, structural motifs of al-losteric modulators as well as their binding sites inside theHD of 3-GPCRs with special emphasis on mGluRs andCaSR. Furthermore, receptor – ligand interactions, theirbinding pockets as well as receptor activation on a struc-tural basis will be discussed briefly. The role of the HD inreceptor dimerization and G protein coupling will not bewithin the scope of the review.

2 3D Structure of the HD of Family 3 GPCRS

The exact knowledge of the 3D structure of a given targetis a key concern in drug discovery since it facilitates a bet-ter understanding of ligand binding, which could be usedfor a rational design of novel ligands as prospective drugcompounds [22]. To gain an insight into the 3D structureand binding sites of proteins the application of specialtechniques such as X-ray crystallography, electron micros-copy and Nuclear Magnetic Resonance (NMR) are state-of-the-art. However, the expression, purification and crys-tallization of membrane proteins remains a challenging

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Figure 1. Schematic representation of 3-GPCR receptors.

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process which impedes their structure elucidation [23 – 25].Therefore, only a high-resolution X-ray structure of an in-active state of bovine rhodopsin (bRho) and bacteriorho-dopsin (BR) are available so far [26, 27]. In the past, theX-ray structure of bRho in particular has been applied as atemplate for building homology models of a given GPCR.

2.1 Is the HD of bRho a Useful Template for Family 3GPCRs?

All GPCRs bear a similar basic membrane topology. Sev-en transmembrane segments (H1 –H7), predominantlyhelical, are linked together sequentially by extracellular(EC1, EC2 and EC3) and cytoplasmic loops (C1, C2 andC3). The transmembrane helices are tilted to varying de-grees with respect to the putative plane of the membranebilayer. Despite the common heptahelical architecture oftheir transmembrane domains, GPCRs are characterizedby a relatively low sequence identity (less than 20%), es-pecially when amino acid sequences of two GPCRs fromdifferent families are compared such as family 1 and 3.However, it has been demonstrated recently that the back-bone of the bRho (family 1) appears to be a reasonabletemplate for building a model of the HD of group ImGluRs and the CaSR (family 3) [13, 31, 28 – 30]. In thecase of GABAB receptors, their extraxcellular domain hasbeen investigated in detail [13, 31 – 33], as well as the rolesof receptor dimers in G-protein signaling and coupling ef-ficacy [34]. However, to the best of our knowledge, no ho-mology model of the transmembrane region of GABAB

receptors has been published to date.The validity of using the bRho template despite the low

sequence similarities is supported by the fact that the HDof mGluRs behaves like any other family 1 GPCR in termsof G-protein coupling and regulation by various types of li-gands [35]. Like bRho, the HD of mGluRs constitutivelycouples to G proteins and is negatively and positivelyregulated by ligands [35]. Furthermore, a detailed analysisof the antagonist binding sites of mGluRs has been per-formed by site-directed mutagenesis and molecular model-ling and the binding pocket was found to be equivalent tothat of retinal in bRho [36, 28]. In the past, homologymodels of mGluR and CaSR have successfully been estab-lished and applied to understand ligand binding into theHD as well as to search for new allosteric modulators [21,28 – 30, 37, 38]. Miedlich et al. constructed a homologymodel of the CaSR based on bRho (Figure 2) and con-ducted a refinement by manually docking a non-competi-tive agonist or antagonist into the putative binding site ofthe receptor [29]. Meanwhile, Petrel et al. published amodel of CaSR (based on bRho) where an allosteric an-tagonist was docked into the binding pocket [21]. Despitethe fact that both models deviated considerably in severalhelices they were shown to be valid to explain mutationdata based on the interaction of allosteric agonists and an-tagonists within the pocket of CaSR [21, 39].

It has been shown recently that application of homologymodels with the aim of performing a virtual screening fornew allosteric binders requires special knowledge of thefunctional activity of the ligand which was used during theconstruction of the homology model [40, 41]. Since the X-ray structure of bRho corresponds to the ground state inwhich retinal is covalently bound [42] and since GPCRsare known to adopt different conformational states [43],the inactive state of the receptor resembles the “antago-nist-bound” instead of the “agonist-bound” state [41]. Inthis way, antagonist-bound state models of hGPCRs haveproven to be suitable for virtual screening of GPCR antag-onists thus indicating the reliability of such homologymodels [41]. This suggests that bRho is a particularly suita-ble template for the screening of antagonists even for re-ceptors that significantly differ from bRho [41]. However,with respect to the quality of those models, difficulties inexactly predicting helix orientation remain to be solvedand the structure of the intracellular and extracellular se-quences connecting the helices are usually not reliable.

2.2 Activation of 3-GPCRs

GPCRs oscillate between various inactive and active con-formations [43]. In the case of rhodopsin, the ground-statechromophore, 11-cis retinal, holds the HD in an inactivestate [42]. Absorption of a photon by cis retinal causes itsisomerization to all-trans retinal, leading to a conforma-tional change and subsequent activation [42]. For the fami-ly-1 GPCRs, it is well documented that the TM6 helixplays an important role in the receptor activation [44]. Viaspin labeling and cystein cross-linking studies on bRho it

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Figure 2. Homology model of the CaSR.

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has been demonstrated that upon rhodopsin activation arigid-body movement of the cytoplasmic end of TM6 awayfrom TM3 takes place [45 – 47].Family 3 GPCRs bear a large ECD that is comprised of

two globular lobes linked by a hinge region, which adoptseither an open or a closed conformation. It turns out thatmovements of ECD are likely to affect the separation ofthe HD and ICD and hence activate the receptor [18]. Asan integral part of the process of receptor activation, ago-nist binding is thought to induce a conformational changein the receptor that facilitates receptor –G-protein interac-tions. However, after ligand binding to the ECD, the mo-lecular events associated with a change in the conforma-tion in the HD are still hypothetical. Ray suggested twodifferent modes of action: Upon ligand binding, the closedconformation of the ECD might fulfill a torsional changethat is propagated through the first transmembrane helixleading to a conformational change of the HD core do-main of the receptor for G-protein activation [48]. As asecond possibility, ligand binding might evoke a closure ofthe ECD, which engages new contacts with the EC loopsof the HD propagating a conformational transition to theHD and consequent G-protein activation [48]. To date,changes in conformation for signal transduction are stillunder investigation. However, with respect to critical heli-ces involved in receptor activation, it has been reported re-cently, that mutation experiments within the binding siteof allosteric antagonists of mGluR I point to a critical roleof the TM6 helix in the receptor activation process [28]which could be similar to the mode of activation reportedfor rhodopsin. In the following, current knowlegde on li-gand binding into the allosteric site of family 3 GPCRswill be discussed briefly with respect to the kinetics of thereceptors, their binding sites as well as most commonstructural motifs.

3 Characterization of the Ligand-Binding DomainsInside the TMD

3.1 Kinetic Considerations of Ligand Binding into theAllosteric Site

To date, there are several different ligands known to inter-act with GPCRs [49, 50]. They can be classified accordingto their place of action and their resulting pharmacologicalactivities. In this article, ligand binding at a site differentfrom the orthosteric site, the so-called allosteric site willbe discussed exclusively. Such “exogenous allosteric modu-lators” could act as allosteric agonists, neutral antagonistsor inverse agonists [51]. Exogenous agonists increase or-thosteric agonist affinity and efficacy, whereas exogenousantagonists decrease orthosteric affinity and efficacy. Fur-thermore, exogenous antagonists either act as neutral an-tagonists, which have the same affinity to both the inactiveand active conformational state or as inverse agonists [52]by stabilizing the inactive state and therefore inhibitingthe constitutive activity of the receptor [36]. To contributeto these multiple ways of receptor activation and ligandbinding, new kinetic models had to be postulated in orderto understand the observed pharmacological profile ofGPCRs.Figure 3 shows three dose-response curves of various

concentrations of a receptor agonist. The presence of afixed concentration of an orthosteric antagonist results ina rightward shift of the dose-response curve (decrease inaffinity) whereas an allosteric antagonist leads to a signifi-cantly diminished maximal response (decrease in efficacy).The Allosteric Ternary Complex Model (ATCM) is the

simplest model describing the reversible binding of orthos-teric and allosteric ligands with their respective bindingsites at the free receptor [53, 54]. Since ligands have been

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Figure 3. Difference in dose response for orthosteric antagonists compared to allosteric modulators.

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identified that evoke their effects either indirectly, bymodulating the affinity and consequently the efficacy ofthe orthosteric ligand, or directly by activating the recep-tor in the absence of an orthosteric ligand, the necessity ofpostulating a new model has arisen which could be under-stood as an extension of the ATCM [55]. Figure 4a showsthis extension of the ATCM, which incorporates allostericinteractions on a receptor that is able to exist in either anactive or an inactive state in a schematic way. However,even this model does not recognize all possible ligand – re-ceptor interactions.A further extension of the ATCM model was developed

as a combination of the ATCM and the model that de-scribes binding of ligands to PBPs [56, 57]. SeveralGPCRs, especially mGluRs, bear an orthosteric ligand-binding domain that is loosely coupled to the effector do-main in the HD [58]. In this case, the HD domain is ableto reach its active state even if the B domain stays open inan inactive state. In consequence, constitutive activity ofthese receptors might result from a spontaneous activity oftheir HD rather than from a spontaneous closure of the li-gand-binding domain. Some other GPCRs such as theGABAB receptors only bear a tight coupling of these twodomains, indicating that in case of GABAB receptors ago-nist affinity correlates with the conformation of the E do-main, whereas in case of mGluRs agonist affinity is con-stant irrespective of the conformation of the E domain(Figure 4b) [58]. Since mGluRs, in contrast to GABAB re-ceptors, possess a cysteine-rich region connecting the ECDwith HD, Parmentier et al. raised the hypothesis that looseand tight coupling might be due to the fact that the cys-teine-rich region allows the HD domain to reach its activestate, even if the ECD domain remains open [58].

3.2 Characterization of the Binding Sites of AllostericModulators

In recent years, a substantial number of potent allostericinhibitors and potentiators of family 3 GPCRs has beenidentified (Figure 5). Their binding sites have been deter-mined to reside exclusively within the HD of mGluR,CaSR or GABAB, far away from the othosteric site in theECDs of the receptors [36, 59 – 64]. In contrast to the or-thosteric binding site of mGluRs which is well conservedduring evolution, there was no selective pressure to main-tain the “artificial” allosteric binding sites. Therefore, mostallosteric modulators appear as structurally diverse ligandsand several of them bear a high selectivity for a given re-ceptor subtype. Via site-directed mutagenesis, specific resi-dues responsible for the subtype selectivities of several li-gands have been identified which also enables a character-ization of their binding site in the HD [36, 60, 62, 63].

3.3 Antagonist Binding in the HD

Until now, several structurally diverse and highly potentmGluR1 antagonists have been reported [62, 64]. Amongthose, CPCCOEt was one of the first subtype-selectivenon-competitive mGluR1 antagonists with low micromo-lar affinity (hmGluR1b) [60, 65]. It turned out that Thr-815 and Ala-818 of mGluR1b mediate subtype-selectiveinhibition of CPCCOEt [60]. Both residues are only foundin mGluR1 but not in the homologous position at the sub-types mGluR2 to 8, which further support the observedlack of interaction of CPCCOEt in functional assays ofother cloned mGluR subtypes. Further mGluR1 antago-nists such as R214127 [66], NPS 2390, LY 456066 or EM-TBPC have shown highest affinities down to the sub-nano-molar level. Binding of EM-TBPC to mGluR1 was report-

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Figure 4. a) This extension of the ATCM incorporating allosteric interactions on a receptor that is able to exist in either an activeor an inactive state; O describes orthosteric ligand and A denotes allosteric ligand. R corresponds to the inactive receptor whereasR* stands for the receptor in the active state. b) Ligands and modulators interacting orthosterically and allosterically with the extrac-ellular and the transmembrane region, respectively. Bc/Bo (K1): the ligand-binding domain in a closed and open state, respectively,and the corresponding equilibrium constant when the E domain is closed; E*/E (K2): heptahelical effector domain either activated orinactivated and the corresponding equilibrium constant when the B domain is open; KL: dissociation constant for an orthosteric ligandto the B domain; KM: dissociation constant for an allosteric ligand to the E domain.

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ed to involve Val-757 and Thr-815. The latter residue isalso involved in CPCCOEt binding whereas conversion ofAla-818 did not affect EM-TBPC binding (Table 1) [28,60, 62]. Based on a homology model, Malherbe et al. sug-gested that the aromatic ring of EM-TBPC interacts withthe cluster of aromatic residues formed from Trp-798, Phe-801 and Tyr-805, thereby blocking the movement of theTM6 helix, which is crucial for receptor activation [28]. In-terestingly, they found that radio-labeled EM-TBPCshowed high affinity for rat mGluR1 (rmGluR1) but onlyvery low affinity for human mGlu1 and none for mGlu5.Val-757 was identified as the critical residue for the bind-ing selectivity of EM-TBPC at the rat versus humanmGlu1 receptor since all other mGlu receptors bear leu-cine at this position. It is worthy of note that the absenceof one additional methyl group (valine versus leucine) al-ready leads to a considerable decrease in affinity of EM-TBPC for hmGluR1 and the observed selectivity of this li-gand for rmGluR1. Since NPS 2390, CPCCOEt andBAY36-7620 were shown to displace binding of radio-la-beled R314127 to mGluR1a it was suggested that most ofthe mGluR1 antagonists share the same binding pocket in-volving TM 5– 7 (Figure 8, see later) [64].MPEP was the first mGluR5 antagonist that was report-

ed to bind to mGluR5 with nanomolar affinity [67]. At thebeginning, most of the allosteric mGluR5 antagonists re-vealed an MPEP-like structure and only recently, new an-tagonists with different scaffolds have been reported [68 –71]. In this context, Renner et al. reported a successful ap-proach of scaffold hopping starting from MPEP-like deriv-atives by applying CATS3D descriptors. A flexible phar-macophore-based alignment of different mGluR5 inhibi-tors (Figure 6a) was used for a virtual screening of com-mercial databases and resulted in the discovery of new,moderately active inhibitors with diverse scaffolds not re-sembling the classical MPEP-like structure [72]. However,since these inhibitors bear activity in the low micromolarrange a reconvergence to the classical MPEP structuremight occur upon iterative refinement.In the past, mainly MPEP was investigated for the char-

acterization of mGluR5 and crucial determinants of thesubtype selectivities of MPEP have been identified inTM3 and TM7 [35, 36, 60]. In two recent studies, severaladditional residues in TM5 and TM6 of mGluR5 havebeen demonstrated to contribute to the binding of MPEP(e.g., Table 1, Figure 6b). Pagano et al. have shown thatMPEP and the mGluR1 antagonist CPCCOEt bind tooverlapping binding pockets in the TM region of mGluR1and mGluR5, respectively, but interact with different non-conserved residues [36]. Their models suggest that the pyr-idine ring of MPEP occupies the same space between TM7and TM3 as the benzene ring of CPCCOEt. However, oth-er parts of these antagonists do not overlap and imply in-teractions with different TM helices. Recently, Malherbeet al. reported a similarity between the critical residues inthe TM6 region involved in MPEP-binding site with those

of EM-TBPC pointing to a common mechanism of inhibi-tion shared by both antagonists [38].The synthesis of selective allosteric mGluR2 antagonists

has been published recently and Figure 5 depicts some ofthe most common structural motifs [73]. However, no mu-tation study has been performed and, therefore, their bind-ing pocket has not been characterized so far. Antagonistsfor all other mGluR subtypes have not been reported eventhough it is likely that there also exists a similar bindingpocket for agonist and antagonist binding. The main rea-son for the lack of appropriate ligands could be attributedto a low interest of pharmaceutical companies to performa HTS at these receptors in the past.Based on a homology model of the CaSR and a detailed

mutation analysis [21] crucial amino acids for allosteric an-tagonists Calhex 231 and NPS 2143 [74, 75] were found inTM2, TM3, TM5, TM6 and TM7 [21, 29]. In particular, ex-change of Phe-668, Arg-680, Phe-684 and Glu-837 attenu-ated responses of CaSR [21]. These results have been in-terpreted with the formation of a salt bridge of the carbox-ylic group of Glu-837 in TM7 via the protonated aminogroup in NPS 2143 as predominant interaction. Further in-teractions include Phe-668 (TM2) and Phe-684 (TM3)

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Table 1. Overview of crucial amino acids interacting with known3-GPCR ligands.

Receptorligand

TM2 TM3 TM4 TM5 TM6 TM7

Rhodopsincis Retinal

Glu-113Ala-117Thr-118Gly-121Glu-122

Met-207His-211Phe-212

Phe-261Trp-265Tyr-268Ala-269Ala-272

Ala-292

3-GPCR AntagonistsCaSRCalhex 231

Phe-684Phe-688

Trp-818 Glu-837Ile-841

CaSRNPS 2143

Phe-668 Phe-684Arg-680

Glu-837

rmGluR1CPCCOEt**

Met802Thr805

hmGluR1CPCCOEt

Thr815Ala818

rmGluR1EM-TBPC

Val-757 Trp-798Phe-801Tyr-805

Thr-815

rmGluR5MPEP

Pro-654Tyr-658

Leu-743 Thr-780Trp-784Phe-787Tyr-791

Ala-809

hmGluR5MPEP

Pro-655Ser-658

Ala-810

3-GPCR AgonistsrmGluR1Ro 67-7476

Ser-687 Val-757

hmGluR2LY487379

Ser-688Gly-689

Asn-735

hCaSRNPS R-568

Phe-668 Phe-684 Glu-837

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Figure 5. Overview of structurally different 3-GCPR antagonists.

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forming close hydrophobic contacts and p-stacking withboth allosteric antagonists and Arg-680 (TM3) interactingwith the hydroxyl group of the NPS 2143 [21, 29]. Accord-ing to the best of our knowledge no allosteric GABAB an-tagonists have been reported so far.

3.4 Inverse Agonists

All mGluR antagonists are divided into two categories:They could be either neutral antagonists or inverse ago-nists [36]. The first mGlu receptor inverse agonist identi-fied was MPEP. Later, Bay36-7620 and R214127 werefound to act as inverse agonists by inhibiting mGluR1aconstitutive activity [64, 66]. In contrast, CPCOOEt wasreported to be a neutral antagonist, and it was postulatedthat this ligand prevents the ECD from activating the HDrather than stabilizing it in an inactive form [76]. There-fore, CPCCOEt is considered as an inhibitor of the intra-molecular signaling mechanism, disconnecting the crosstalk between the two domains of the receptor. One proba-ble reason for this behavior was presented by Litschiget al., who connected the pharmacological behavior withthe binding pocket of CPCCOEt which appears exclusive-ly in TM7 [60]. Since both crucial amino acids ofCPCCOEt (Thr-815 and Ala-818) are located at the ex-tracellular surface of the TM7, Litschig proposed that byinteracting on the top of TM7, CPCCOEt prevents the ac-tivation of the HD domain by the glutamate/ECD com-plex probably due to steric hindrance [60], which could beinterpreted analogously to the possible mode of receptoractivation already suggested by Ray [48].

3.5 Agonist Binding in the HD

Figure 7 shows positive modulators of 3-GPCRs based ondifferent structural motives. It was suggested that these en-hancers bind to and stabilize the activated receptor states[28, 62, 76]. Knoflach et al. have described a novel class ofligands Ro 67-7476, Ro 01-6128 and Ro 67-4853 acting aspositive allosteric modulators of the mGlu1 receptor [62].

A detailed mutational analysis revealed that in particularVal-757 in the TM5 of the receptor is responsible for theenhancing effect of both Ro 01-6128 and Ro 67-7476 [62].Interestingly, Ro 01-6128 and the structurally different Ro67-7476 bear only high affinity for rat mGluR1 whereasRo 67-4853, structurally similar to Ro 01-6128, exhibitedactivity at all h/rmGluR1 and rmGluR5 suggesting a dif-ferent binding mode for this compound. Further criticalamino acids are located in TM3 and TM5 of mGluR1, athomologous residues where MPEP interacts with themGluR5 receptor, e.g., close to the inverse agonist bindingsite [36, 62]. It has been shown that the position of valine(Val-757) is critical not only for the enhancing effect ofpositive allosteric modulation of rat mGlu1 [62] but alsofor negative modulation (MPEP, EM-TBPC) [28, 38].Therefore, even though Ro 67-7476, EM-TBPC andMPEP belong to different chemical series (Figure 7), thisresult indicated that this amino acid occupies a strategicposition to gate the effect of positive and negative alloster-ic modulation [38, 77].Furthermore, allosteric inhibitors of mGluR1 or

mGluR5 signaling, such as PHCCC, SIB-1893 and MPEP,have shown to be weak allosteric potentiators of mGluR4signaling [78 – 80] and a series of benzaldazine analogues(including DFB in Figure 7) have exhibited everythingfrom allosteric potentiation to allosteric inhibition to neu-tral cooperativity on mGluR5 signaling [81]. Recently, al-losteric potentiators of the mGluR5 receptor have beendescribed [82]. Most interesting examples include DFB[81] and CPPHA. Although both potentiators increase theaffinity for glutamate, only DFB partially displaces allos-teric antagonists such as the radio-labeled MPEP-deriva-tive [3H]-3-methoxy-5-(pyridin-2-ylethynyl)pyridine [81,83]. Therefore, it was proposed that CPPHA binds to a dif-ferent binding site within the HD. Since DFB shares a sim-ilar binding pocket with mGluR5 antagonists, a series ofDFB analogues were designed where a transition frompositive (DFB) via silent to negative modulation was ach-ieved [81]. The crucial substituent that determines themechanism of action was found to be the difluoro group of

QSAR Comb. Sci. 25, 2006, No. 2, 134 – 146 www.qcs.wiley-vch.de H 2006 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim 141

Figure 6. a) Flexible pharmacophore-based alignment of different MPEP inhibitors according to Renner [72]. b) Schematic repre-sentation of MPEP interacting with crucial amino acids inside mGluR5-binding pocket according to Malherbe [38].

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DFB. Substitution of the difluoro group by a dichloro sub-stituent (DCB) resulted in a “silent” or neutral agonistwithout functional activity whereas substitution with a di-methoxy group (DMeOB, Figure 5) yielded the respectiveallosteric antagonist. All these ligands were found to com-pete with each other as well as with the antagonist radio-labeled MPEP derivative [3H]methoxy-PEPy pointing tothe same binding pocket [81].One mutation analysis study of CaSR has been reported

where the binding agonist NPS R-568 was investigated.According to antagonist binding, the exchange of Phe-668,Phe-684 and Glu-837 were found to have a major influenceupon receptor activation [29]. It turned out that importantinteractions included hydrophobic contacts and p-stackingof NPS R-568 with Phe-668 and Phe-684 of TM2 andTM3. The predominant interaction is in accordance to an-

tagonist binding the salt bridge to Glu-837 (TM 7). Mied-lich et al. suggested that due to structual similarity of the li-gands the binding site of the allosteric agonist NPS R-568might overlap with that of NPS 2143 [29]. Recently, Urwy-ler et al. reported the identification of CGP7930 and its al-dehyde analogue CGP13501 as positive modulators ofGABAB receptor function [84, 85]. However, their bind-ing sites still remain to be determined.

3.6 Is There a Common Binding Pocket for All Family 1and 3 GPCRs?

In order to answer this question it is essential to elucidatecommon features of all family 1 and 3 GPCRs, i.e., isoster-ic amino acids in their binding pockets as well as structuralsimilarities of their allosteric modulators. The binding sites

142 H 2006 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim www.qcs.wiley-vch.de QSAR Comb. Sci. 25, 2006, No. 2, 134 – 146

Figure 7. Overview of structurally different C-GCPR agonists.

Full Papers Tobias Noeske et al.

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of mGluR1, mGluR5 and CaSR allosteric modulatorshave been shown to include residues in TM2, TM3, TM5,TM6 and TM7 of the respective receptors (Table 1) [21,28, 38]. Hence, all these allosteric modulators of family 3GPCRs appear to bind in a crevice composed by thesefour transmembrane segments (Figure 8). Furthermore, ithas been shown that CaSR agonist and antagonists com-pete for the same binding site and it was proposed in thesame paper that also agonist binding sites of mGluRs andGABAB overlap significantly with those of the respectiveantagonist [27]. The results of these studies lead to the as-sumption that there is a general allosteric site in the HD offamily 3 GPCR mediating both allosteric potentiation andinhibition [29].A comparison of critical residues involved in the binding

of mGluR antagonists such as CPCOOEt, EM-TBPC andMPEP as well as Calhex 231 to CaSR, an accumulation ofaromatic amino acids became evident. Malherbe et al.found the residue Trp-798 in TM6 of mGluR1 to be essen-tial, since it was highly conserved in all mGluRs, CaSRand in family 1 GPCRs [21, 28, 38]. Furthermore, consider-ing family 1 GPCRs, it is well documented that the TM6helix plays an important role in the receptor activation[44]. In case of bRho it has been reported that upon rho-dopsin activation, a rigid body movement of the cytoplas-mic end of TM6 away from TM3 is required and that theTrp-265 serves to transmit the chromophore motion toTM6 helix [86]. Therefore, Trp was suggested to play a piv-otal role by acting as a switch for the transition of the re-ceptor between different allosteric states [86]. Other aro-matic amino acids such as Phe-801 and Tyr-805 in case ofEM-TBPC or Phe-821 and Phe-688 in case of Calhex 231were also assumed to play an important role since theywere found in proximity to Trp-798 in the TM6 helix.Thus, Malherbe et al. proposed that the aromatic clusterformed from Trp-798, Phe-801 and Tyr-805 of rmGluR1 in-teracts with the aromatic ring of the antagonist, e.g., EM-TBPC, blocking the Trp-798 movement in TM6 helix,which is required for receptor activation and consequentlystabilizes the inactive conformation of the receptor [28].The same mechanism was proposed for the action ofMPEP [28]. Similar to rmGluR1, rmGluR5 bears severalaromatic amino acids in the putative MPEP binding pock-et such as Phe-658 in the TM3 helix and Trp-798, Phe-787and Tyr-791 in the TM6 helix. It was suggested that MPEP,via its interactions with this network of the aromatic resi-dues could also prevent the movement of the TM6 helixrelative to the TM3 helix. The fact that both MPEP, EM-TBPC and Calhex 231 have homologous contact sites withtheir respective receptors in the TM6 helix (a region high-ly conserved among all mGlu receptors) points at a com-mon mechanism of inhibition shared by those antagonistsand some striking similarities to 1-GPCR modulation [21,28]. Furthermore, conservation in the position of thesecritical residues was also observed in the previously re-

ported ligand recognition sites for h2 adrenergic, hA3 ade-nosine, or h5-HT4 receptors [87 – 89].Therefore, the allosteric binding site crevice of mGluR

shares similar structural features as the agonist bindingsite in the monoamine family 1 GPCRs [86]. However,LY487379, an allosteric potentiator of mGluR2 function,has recently been demonstrated to bind to residues inTM5 as well as in TM4, indicating that other TMs can par-ticipate in the binding of allosteric modulators as well (Fig-ure 8) [63].Another similarity between family 3 and family 1

GPCRs is the occurrence of inverse agonism and somestriking similarities in the binding pocket of inverse ago-nists. In this context, Joubert reported that three residuesof human 5-hydroxytryptamine type 4 receptor, Asp-100,Trp-272 and Phe-275, are important molecular determi-nants for the effects of inverse agonists [91]. Two of theseresidues were found to be homologous to the critical resi-dues Trp-798 and Phe-787 that Malherbe et al. identified inthe MPEP-binding pocket of rmGlu5 and a similar obser-vation was also noted previously for the EM-TBPC-bind-ing pocket of rmGlu1 [28]. Thus, it was suggested that Trp-798 and Phe-787 are part of network involved in the in-verse agonist activity of MPEP and probably EM-TBPCwhich remains to be demonstrated [28].One striking difference of mGluR and some 1-GPCR is

the finding of Lavreysen et al. that the driving force formGluR1 binding is not electrostatic in nature which is incontrast to results obtained for monoamine GPCRs (e.g.,dopamine receptor), which are often strong bases and bindin a cationic form [64]. However, in case of CaSR antago-nists ionic interactions were reported to play an important

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Figure 8. Allosteric sites at the 7TMs of mGluR1, mGluR2 ormGluR5. Residues or regions determined to be crucial for thesubtype selectivities of allosteric inhibitors CPCCOEt, MPEP,EM-TBPC, BAY36-7620 and allosteric potentiators Ro 67-7476and LY487379 are depicted. The two residues in TM3 shared byRo 67-7476 and MPEP in their interactions with mGluR1 andmGluR5, respectively, are given as mGluR1 residue/mGluR5residue according to Jensen [90]).

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role and strong bases were shown to bind in their chargedform [21, 29].

4 Future Trends and Challenges

This article reviewed the current status of family 3 GPCRhomology modeling of the heptahelical domain, highlight-ing available mutation data within the binding domain ofallosteric ligands. During recent years, important progresshas been made in the discovery of several structurally di-verse allosteric modulators of mGluR1, mGluR5 andCaSR activating or deactivating family 3 GPCRs. The exis-tence of such ligands, particularly in the mGluR and CaSRarea, facilitated a better understanding of their mode ofaction as well as the process of receptor activation via theHD. It has been shown that there is a considerable similar-ity of key amino acids in the ligand binding domain of al-losteric modulators of 1-GPCRs and 3-GPCRs suggestinga similar pocket as well as mode of action. Since first datafrom behavioral pharmacology already propose the suita-bility of some family 3 GPCR modulators for several CNSindications, the discovery of novel modulators bearingstructurally diverse scaffolds will be a key concern. There-fore, the knowledge of the topology of family 3 bindingsites as well as the mechanisms leading to antagonism, ag-onism or inverse agonism will be highly crucial.

Acknowledgements

The authors would like to thank Prof. Gabriele Costantino(University of Perugia, Italy), Mr. Steffen Renner (Univer-sity of Frankfurt, Germany) and in particular Prof. GisbertSchneider (Schneider Consulting GbR, Germany and Uni-versity of Frankfurt, Germany) for many valuable discus-sions on this exciting receptor family.

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