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Neuropharmacology 60 (2011) 93e101

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

Exploration of structure-based drug design opportunities for mGluRs

Sid Topiol*, Michael Sabio, Michelle UbertiLundbeck Research USA, 215 College Road, Paramus, NJ 07652, USA

a r t i c l e i n f o

Article history:Received 21 July 2010Accepted 3 August 2010

Keywords:GPCRsmGluRsTransmembrane domain

* Corresponding author.E-mail address: Sid.Topiol@gmail.com (S. Topiol).

0028-3908/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.neuropharm.2010.08.001

a b s t r a c t

The metabotropic glutamate receptors (mGluRs) are a subset of the Class C G-Protein Coupled Receptors(GPCRs). Recently, an emerging strategy for drug-discovery efforts targeting mGluRs has been to developcompounds acting at the so-called allosteric site in the 7-transmembrane (7TM) domain, common to allGPCRs, rather than the extracellular (EC) domain containing the orthosteric glutamate-binding site. Weexamine herein some of the intrinsic relative merits of targeting these two domains. Comparisons aremade among amino-acid sequences in the two domains and among X-ray structures and homologymodels of the EC domain. We show that there is greater sequence diversity in the EC domains than in thetransmembrane (TM) domains. Thus, contrary to generally accepted descriptions of there being greaterevolutionary pressure to preserve the EC domain, it is the 7TM domain that is more highly conserved.Within the EC domain, the glutamate-binding site of the Venus flytrap region has hitherto received themost attention as a target site. Analysis of examples of the three-dimensional structures of the ECdomains at the glutamate-binding site reveals differences as well, thereby supporting the viability oftargeting the EC domain, even at the glutamate-binding site, for drug discovery. To exemplify thisstrategy, we present examples of active compounds identified via high-throughput docking in the ECregion.

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1. Introduction

The most prominent class of target proteins for drugs are the so-called G-Protein Coupled Receptors (GPCRs). While much has beenlearned in recent years about the structure and function of theseproteins at the atomic level, there remain significant fundamentalquestions to be addressed. For instance, on the one extreme, the longsought-after X-ray structures of the 7-transmembrane (7TM) regionof ligand-mediated GPCRs finally started to emerge with the publi-cation of the b2-adrenergic receptor (Cherezov et al., 2007;Rasmussen et al., 2007; Rosenbaum et al., 2007). Others soon fol-lowed (Hanson et al., 2008; Jaakola et al., 2008; Serrano-Vega et al.,2008) and marked the beginning of a significant means for enlight-enment in this area (Weis andKobilka, 2008;Mustafi and Palczewski,2009; Topiol and Sabio, 2009; Sabio and Topiol, 2010). It is alsobecoming clear that GPCRs can adopt multiple states in response todifferent ligands, which, in turn, lead to a complexity of effects onmultiple pathways (Galandrin et al., 2007, 2008; Kenakin, 2007;Kobilka and Deupi, 2007; Violin and Lefkowitz, 2007; Audet andBouvier, 2008). As a reflection, the term “7TM receptors” is nowoften used in place of “GPCRs.” The Class C GPCRs, and in particular

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the mGluR subset of these, have more recently emerged as animportant target for numerous central nervous system (CNS) indi-cations including schizophrenia, Parkinson’s disease, Fragile Xsyndrome, anxiety, depression, and others. Here too, there have beensignificant advancements coupled with needs for increased under-standing. In addition to the 7-transmembrane spanning domain,which is the hallmark of GPCRs, Class C GPCRs contain an extracel-lular (EC) domain comprised of the “Venus flytrap” (VFT) and the“Cysteine-rich” (C-rich) regions (Fig.1).Whereas endogenous ligandsof Class A GPCRs act in the 7TM region, the binding site for endoge-nous ligands of Class C GPCRs (e.g., glutamate for mGluRs) is locatedin the VFT domain. The atomic-level details of how ligand binding inthe VFT region leads to signal propagation to G proteins at theintracellular (IC) region of the 7TM domain is an area of ongoingresearch (Kniazeff et al., 2004; Parnot and Kobilka, 2004; Rondardet al., 2006). For mGluRs, analogy to traditional approaches to drugdiscovery would suggest that the protein site with which to interactfor ligand modulation would be the binding site of the orthostericligand, i.e., the VFT domain. As instances of X-ray structures of theVFTs of the three different groups of the mGluRs [Group I (mGluR1),Group II (mGluR3), andGroup III (mGluR7)] havenowbeenpublished(Kunishima et al., 2000; Tsuchiya et al., 2002; Jingami et al., 2003;Muto et al., 2007), this domain has considerable appeal froma structure-based design perspective. Moreover, the 7TM X-raystructures determined to date are all of Class A and therefore are

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Fig. 1. Class C GPCRs consist of a 7-transmembrane spanning domain and an extracellular domain comprised of the “Venus flytrap” and the “Cysteine-rich” regions, as schematicallyrepresented here. Glutamate, the endogenous ligand, binds to the active site in the Venus flytrap region, which may adopt an “open” or “closed” conformation, as defined withrespect to the position of this region’s two lobes.

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extremely challenging to use as templates for generating homologymodels of mGluRs. Nevertheless, there has been a growing focus onthe design of allosteric ligands for mGluRs. Positive (PAM) andnegative (NAM) allosteric modulators of the mGluRs are generallybelieved to act via 7TM interaction, which is “allosteric” to the“orthosteric” VFT. Various arguments have been posed for pursuingthis paradigm. The difficulty in obtaining selective orthosteric ligandsis perhaps the principle justification for one argument, i.e., that the7TM region will have greater opportunity for selectivity because ofgreaterdiversity inherent in this “allosteric”domain. Thewidely citedrationale for this is the “evolutionary pressure” (Kew, 2004;Niswender et al., 2005; Ritzén et al., 2005; Grigoriadis et al., 2009;Lindsley et al., 2009) to preserve the domain where the endoge-nous ligand binding occurs, i.e., the EC domain. Indeed, the discoveryof MPEP, a selective mGluR5 negative allosteric modulator, andcompoundsderived from it have fueled efforts in this direction (Slassiet al., 2005; Bach et al., 2007; Gasparini et al., 2008). Other reasonsgiven for targeting the 7TM region is that, particularly for positivemodulation of the mGluRs, allosteric ligands would not be prone toreceptor desensitization and internalization issues ascribed toorthosteric agonists. Moreover, PAMs should have minimal effect onthe tone of the receptor. From a structure-based perspective, theavailability of X-ray structures for the VFT region begs the question offindingways to capitalizeon theseX-ray structures. Indeed, a numberof investigations using these X-ray structures or homology modelsderived from themhave been performed to rationalize the activity ofknown ligands as well as to design new ones (Clark et al., 1997;Malherbe et al., 2001; Bertrand et al., 2002; Yao et al., 2003;Costantino, 2006; Frauli et al., 2007; Monn et al., 2007; Lundströmet al., 2009; Selvam et al., 2010). In the present work, we employsequence- and structure-based approaches to challenge the rationalefor this emerging trend to emphasize the 7TM as the primary targetfor the design of mGluR drug candidates.

2. Methods

2.1. Sequence analyses

Sequence alignments of the mGluR 7TM and EC domains were conducted withPSI-BLAST (Altschul et al., 1997) using sequence information from the Swiss-Prot(SP) database (Bairoch et al., 2004): (1a) h.mGluR1 EC, SP entry Q13255, residues19e592; (2a) h.mGluR2 EC, SP entry Q14416, residues 19e567; (3a) h.mGluR3 EC, SPentry Q14832, residues 23e576; (4a) h.mGluR4 EC, SP entry Q14833, residue33e587; (5a) h.mGluR5 EC, SP entry P41594, residues 22e579; (6a) h.mGluR6, EC, SPentry O15303, residues 25e585; (7a) h.mGluR7 EC, SP entry Q14831, residues35e590; (8a) h.mGluR8 EC, SP entry O00222, residue 34e583; (1b) h.mGluR1 TM,SP entry Q13255, residues 593e840; (2b) h.mGluR2 TM, SP entry Q14416, residues568e819; (3b) h.mGluR3 TM, SP entry Q14832, residues 577e828; (4b) h.mGluR4TM, SP entry Q14833, residues 588e847; (5b) h.mGluR5 TM, SP entry P41594,residues 580e827; (6b) h.mGluR6 TM, SP entry O15303, residues 586e845; (7b)

h.mGluR7 TM, SP entry Q14831, residues 591e850; and (8b) h.mGluR8 TM, SP entryO00222, residues 584e843.

2.2. Homology modeling and docking

By using the X-ray structure of the open form of mGluR1 (PDB entry 1ISS, chainA) as a template and the Prime module within the Maestro software suite(Schrödinger, 2008), we constructed a homologymodel of the open form of mGluR5.We performed a high-throughput docking study by using the Glide module inMaestro (Schrödinger, 2008) and 350,000 compounds (representing 800,000structures of various protonation or stereochmical configurations) from our corpo-rate database to dock into the mGluR5 homology model.

We also performed docking calculations using the mGluR3 X-ray structure (PDBentry 2E4U, chain A) to dock LY404040 (Monn et al., 2007) after preparing the X-raystructure by using the Protein Preparation module in Maestro (Schrödinger, 2008).

2.3. In vitro calcium mobilization assay

The cDNA for rat metabotropic glutamate receptor 5 was a generous gift fromProf. S. Nakanishi (Kyoto University, Kyoto, Japan). The rat metabotropic glutamatereceptor 5 (rmGluR5) was stably expressed in a HEK 293 cell line and grown inDulbecco’s Modified Eagle Medium (DMEM) (Invitrogen Carlsbad, California) withsupplements (10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin,100 mg/ml streptomycin, and 0.75 mM G1418) at 37 �C, 5% CO2. Twenty-four hoursprior to the experiment, cells were seeded into 384-well black wall microtiter platescoated with poly-D-lysine for assay the following day. Just prior to the assay, mediawas aspirated and cells were dye-loaded (25 ml/well) with 3 mM Fluo-4/0.01%pluronic acid in assay buffer (HBSS ¼ Hank’s buffered saline-check: 150 mM NaCl,5mMKCl,1mMCaCl2,1 mMMgCl2, plus 20mMHEPES, pH 7.4, 0.1% BSA and 2.5mMprobenicid) for 1 h in 5% CO2 at 37 �C. After excess dye was discarded, cells werewashed in assay buffer and layered with a final volume equal to 30 ml/well. Basalfluorescence was monitored in a fluorometric imaging plate reader (FLIPR, Molec-ular Devices Sunnyvale, California) with an excitation wavelength of 488 nm and anemission range of 500e560 nm. Laser excitation energy was adjusted so that basalfluorescence readings were approximately 10,000 relative fluorescent units. Cellswere stimulated with an EC80 concentration of glutamate in the presence ofa compound to be tested, both diluted in assay buffer, and relative fluorescent unitswere measured at defined intervals (exposure ¼ 0.6 s) over a 3 min period at roomtemperature. Basal readings derived from negative controls were subtracted from allsamples. The maximum change in fluorescence was calculated for each well.Concentrationeresponse curves derived from the maximum change in fluorescencewere analyzed by nonlinear regression (Hill equation). A negative allosteric modu-lator will be identified from these concentrationeresponse curves if a compoundproduces a concentration-dependent inhibition in the glutamate response.

3. Results

3.1. Analysis of the 7-transmembrane vs. extracellular regions

A pervasive challenge in drug design is to ensure that the activityof the drug is selective for the desired target. This challenge is mostpronounced in the context of selectivity within a family of targetsthat, by definition, are highly homologous. For example, ligandsacting at a given serine protease or protein kinase are generally prone

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to have activity across the target family, thereby requiring great carein designing compounds without this cross reactivity. For themGluRs, this is the case as well. All of these proteins are normallytriggered by the binding of glutamate, the endogenous ligand, in theactive site located in the so-called Venus flytrap region of the extra-cellular domain (Fig. 1). As a result, it is expected that beyondglutamate, other compounds binding to this site at any of themGluRswould have a significant likelihood of invoking similar bindingcharacteristics (affinityanddegree of activation/inactivation) at othermGluRs. Indeed,manycompoundsdomanifest cross reactivity. Theseobservations have led to an emergingmGluRdrug-discovery strategytargeting the 7TM region. The rationale for this strategy is that theVFT region contains the active site for the endogenous ligand gluta-mate to which all mGluRs must respond, which, in turn, creates anevolutionary pressure to preserve the similarity of this region acrossthe mGluRs. As the endogenous ligand does not bind in the 7TMregion, there should therefore be little evolutionary pressure topreserve the 7TM regions across themGluRs (Kew, 2004; Grigoriadiset al., 2009; Niswender et al., 2005; Ritzén et al., 2005; Lindsley et al.,2009). This, in turn, should translate to an enhanced opportunity tocreate selective compounds for the 7TM region. Support for thisstrategy comes from the discovery of selective, 7TM-acting mGluRligands.While the “evolutionary pressure” argument appears sound,and the existence of 7TM-selective ligands agrees with the potentialfor greater selectivity in the 7TM region, herein we challenge thisargument. We note that the discovery of 7TM-selective ligands doesnot preclude selectivity in the EC region. Moreover, much of thesearch for EC-binding drugs predates the availability of the afore-mentioned X-ray structures.

Our first layer of analysis is to examine the sequence relation-ships of the mGluRs. In Table 1, we summarize the results for pair-wise alignments of all of the mGluRs. Separate alignments wereperformed within the 7TM and EC domains. Within Group I, we seethat the sequence identity between mGluR1 and mGluR5 is 71% inthe EC domain and 79% in the 7TM domain. Thus, contrary toexpectations based on the evolutionary argument above, there isactually greater sequence identity in the 7TM region. ComparingmGluR1 of Group I with mGluR2 of Group II, one observes that thesequence identity is 44% in the EC region and 50% in the 7TMregion. While the sequence identities between mGluR1 andmGluR2 (of Groups I and II, respectively) are less than thosebetween mGluR1 and mGluR5 (both of Group I), in this case again,

Table 1Percentage Identities of Human mGluR-x (x ¼ 1,8) Extracellular (EC) and Transmembran

(A) EC %ID Group I Group II

(B) TM %ID mGluR1 mGluR5 mGluR2

Group I mGluR1 (A) 100%(B) 100%

mGluR5 (A) 71% (A) 100%(B) 79% (B) 100%

Group II mGluR2 (A) 44% (A) 42% (A) 100%(B) 50% (B) 47% (B) 100%

mGluR3 (A) 44% (A) 43% (A) 68%(B) 44% (B) 44% (B) 75%

Group III mGluR4 (A) 43% (A) 41% (A) 46%(B) 44% (B) 47% (B) 54%

mGluR6 (A) 40% (A) 38% (A) 47%(B) 43% (B) 44% (B) 50%

mGluR7 (A) 39% (A) 39% (A) 44%(B) 47% (B) 47% (B) 53%

mGluR8 (A) 42% (A) 42% (A) 44%(B) 45% (B) 47% (B) 53%

a In all sequence comparisons, TM %ID � EC %ID. Values in italics (all equal to 100%)represent comparisons within groups.

the sequence identity of the 7TM region exceeds that of the ECregion. As can be seen in Table 1, these two examples of EC-to-TMsequence comparisons illustrate two absolutely consistentpatterns. (1) In all cases, within either domain, the sequenceidentities are greater within a group than between groups and ofsimilar degree as reported by others (e.g., Kanuma et al., 2010). Thisis consistent with expectations and the groupings of the proteins.(2) More surprisingly, in each and every case, the sequence identity inthe 7TM region is greater than or equal to that in the EC region. This isan apparent contradiction to the argument that evolutionarypressure leads to greater commonality in the EC region and,thereby, to reduced inherent capacity for the design of selectiveligands. While the “causes” for these sequence relationships aredebatable, continuing with evolutionary terminology, it is inter-esting to speculate that the pressure to maintain commonalityamong themGluRs in the region closest towhere they interact withtheir downstream partner (7TM), the G protein, is greater than thepressure to preserve the glutamate-binding site, which regulatesthe GPCR/G-protein interaction from a distance. Regardless of theexplanation, looking at the domains as a whole at the sequencelevel, the popular argument of greater variability in the 7TMdomain of mGluRs seems to be unfounded.

3.2. Structural comparison of the EC ligand-binding regions aspotential selectivity sources

3.2.1. Closed, active forms of mGluR1 and mGluR3While the sequence alignments, as discussed above, give infor-

mation about the entire domains, it is interesting to explorewhetherthere are differences within these domains at potential ligand-binding sites. At present, there are no X-ray structures of the 7TMregions of any of the mGluRs. Consequently, any analysis of potential7TM binding sites would require the use of homology models builtusing the X-ray structures of the Class AGPCRs as templates. Becausethese are only distally related to the mGluRs, such an analysis wouldbe questionable. For the EC regions, where X-ray structures spanningall three groups have become available, we can much more readilyexamine these questions structurally. In Fig. 2, we show side-by-siderepresentations of two closed-form structures of the glutamate-binding site in theVFT regions of theX-ray structures ofmGluR1 (PDBentry 1EWK, chain A) and mGluR3 (PDB entry 2E4U, chain A) fromGroups I and II, respectively. The most obvious source of ligand

e (TM) Sequences.a

Group III

mGluR3 mGluR4 mGluR6 mGluR7 mGluR8

(A) 100%(B) 100%

(A) 47% (A) 100%(B) 47% (B) 100%(A) 47% (A) 69% (A) 100%(B) 47% (B) 76% (B) 100%(A) 46% (A) 69% (A) 66% (A) 100%(B) 47% (B) 78% (B) 74% (B) 100%(A) 46% (A) 75% (A) 70% (A) 73% (A) 100%(B) 49% (B) 82% (B) 77% (B) 86% (B) 100%

represent self comparisons which are 100% identical by definition. Values in bold

Fig. 2. The close similarity of active sites is shown in these side-by-side representations of two closed-form structures of the glutamate-binding site in the Venus flytrap regions ofthe X-ray structures of mGluR1 (PDB entry 1EWK, chain A) and mGluR3 (PDB entry 2E4U, chain A) of Groups I and II, respectively. However, there are also a number of amino aciddifferences in contact with the bound glutamate molecules, indicating differences in binding characteristics that could be exploited to introduce ligand selectivity.

Fig. 3. Different binding characteristics are observed in two open, inactive forms of mGluR X-ray structures from different groups, i.e., mGluR1 (PDB entry 1EWK, chain B) andmGluR7 (PDB entry 2E4Z, chain A) of Groups I and III, respectively. In the “open“ Venus flytrap conformation, the glutamate ligand sits close to, and interacts directly with the“upper” N-terminal lobe in mGluR1. The open form of mGluR7 contains a molecule of the buffer 2-(N-morpholino)ethanesulfonic acid in place of glutamate. Residues near theligands differ in these two proteins. Thus, Tyr74, Trp110, Ser164, and Ser186 of mGluR1 are replaced by, respectively, Asn74, Ser110, Gly158, and Ala180 in mGluR7.

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selectivity would be differences in the amino acids of the twoproteins at potential ligand-binding sites.While a number of bindingsites may be considered from examination of the existing X-raystructures (Topiol and Sabio, 2010), we limit the present work toconsiderations of only the glutamate-binding site (“active site”) toevaluate what its characteristics suggest about ligand binding andselectivity opportunities. As depicted in Fig. 2, while the active sitesare very similar, with many identical amino acids anchoring theglutamatemolecules in the two structures, there are also a number ofdifferences in the amino acids in contact with the bound glutamatemolecules, indicatingdifferences in binding characteristics that couldbe exploited to introduce ligand selectivity. Striking support fordifferent active-site properties comes from a comparison of glutamicacid itself in these two proteins. While the amino-acid backbone ofboth glutamate molecules overlap, the side-chain d acids sit slightlydifferently. Thus, in the mGluR1 X-ray structure, one oxygen atom ofthe glutamate backbone carboxyl moiety hydrogen bonds to theSer165 hydroxyl group oxygen atom, while the other carboxyl oxygenatom hydrogen bonds to both the backbone nitrogen atom of Ser165and to the hydroxyl group of Ser164 via a through-water (H2O17)hydrogen bond. The amino nitrogen atom of glutamate formshydrogen bonds with an oxygen atom of the acid of Asp318, thehydroxyl oxygen atom of Thr188, and the backbone nitrogen atom ofSer186. One oxygen atomof the side-chain d acid of glutamate is closeto the side-chain amino nitrogen atom of Lys409. All of these inter-actions are replicatedwith identical amino acids in themGluR3X-raystructure. However, other interactions of the glutamate ligand differbetween the mGluR1 and mGluR3 structures. A pair of hydrogenbonds betweenone side-chain d oxygen atomof the glutamate ligandand the hydroxyl group of Tyr74, and the other d oxygen atom ofglutamate to Arg78 via a through-water (H2O46) interaction isreplaced by two hydrogen bonds involving the d acid oxygens ofglutamate and Arg68 in the mGluR3 structure (the latter

Fig. 4. In the “closed“ Venus flytrap conformation of mGluR1 (PDB entry 1EWK, chain A)glutamate in the binding site are the same.

corresponding to Arg78 of mGluR1). Tyr74 of mGluR1 has beenreplaced by Arg64 in mGluR3. These differences, as well as othersdiscussed below, coincide with the different binding affinity of theglutamate ligand itself in these two complexes (Schoepp et al., 1999;Bertrand et al., 2002).

3.2.2. Open, inactive forms of mGluR1 and mGluR7A similar comparison to that of the closed, active forms of

mGluR1 and mGluR3, can be made between two open, inactiveforms of mGluR X-ray structures from different groups, i.e., mGluR1(PDB entry 1EWK, chain B) and mGluR7 (PDB entry 2E4Z, chain A)of Groups I and III, respectively (Fig. 3). The open form of the X-raystructure of mGluR1 contains a bound glutamate ligand. In thisopen form of the VFT region, the glutamate ligand sits close to, andinteracts directly with the “upper” N-terminal lobe. At the back-bone portion of glutamate, both the acid and amino groups formtwo hydrogen bonds with the protein. One of the acid oxygenatoms hydrogen bonds to the backbone NH of Ser165 while theother acid oxygen atom hydrogen bonds to the Ser165 hydroxyl sidechain. The amino group of glutamate forms hydrogen bonds withthe side-chain hydroxyl group of Thr188 and with the backbonecarbonyl oxygen atom of Ser186. The open form of mGluR7 containsa molecule of the buffer 2-(N-morpholino)ethanesulfonic acid inplace of glutamate. Compared to the amino-acid region of theglutamate structure, the same interactions described above in theopen structure of mGluR1 would be possible for mGluR7. However,even in this region, there are nearby residues that differ in thesetwo proteins. Thus, Tyr74, Trp110, Ser164, and Ser186 of mGluR1 arereplaced by, respectively, Asn74, Ser110, Gly158, and Ala180 inmGluR7. At the d carboxyl side of glutamate in the mGluR1 struc-ture, there is a hydrogen bond between a glutamate oxygen atomand Tyr74 and another (i.e., through-water H2O31) between theother acid oxygen atom and Arg78. Arg78 is preserved in mGluR7,

and mGluR5 (PDB entry 3LMK, chain A), each in Group I, the direct interactions of

Fig. 5. As shown in this comparison of the X-ray structure of the open form of mGluR1 (PDB entry 1ISS, chain A) with a homology model of the open form of mGluR5, residues thatdiffer are accessible from the glutamate subsite; i.e., Asp324 and Glu325 of mGluR1 versus Tyr311 and Asp312 of mGluR5, respectively. Thus, at or near the glutamate-binding site, thesecomparisons suggest that within this group, potential sources of selectivity are more readily identifiable in the open form than in the closed form.

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but Tyr74 becomes Asn74. Thus, these two open forms have cleardifferences in the features responsible for binding in the glutamatesite, which suggest sources for creating selective inhibitors.

3.2.3. Closed and open forms of mGluR1 and mGluR5The above comparisons of pairs of structures between two

groups of mGluRs can be extended to analyses within mGluR

Fig. 6. The antagonist nature of LY341495 is readily revealed from a structural analysisof the recent X-ray structure of the ligand bound to the Venus flytrap region of mGluR1(PDB entry 3KS9 chain A) in the “open” conformation. The upper portion of LY341495,which closely resembles glutamate, sits in the glutamate pocket, and the remaining9-methyl-9H-xanthene fragment projects downwards into the lower lobe, preventingthe Venus flytrap from closing.

groups. Fig. 4 shows the glutamate-binding sites of mGluR1 (PDBentry 1EWK, chain A) and mGluR5 (PDB entry 3LMK, chain A), i.e.,two structures within Group I. The interactions of the glutamateligand in mGluR1 have been described earlier. Unlike the compar-ison of this structure with that of mGluR3 (of Group II) above, thedirect interactions of glutamate in the binding site of mGluR5 arethe same as that of mGluR1. Thus, there are no obvious sequencedifferences which could be expected to provide selectivity betweenmGluR1 and mGluR5 in the glutamate-binding site of the closedform of the VFT, particularly for ligands similar to glutamate. Asindicated earlier, other sites on the protein may still offer more

Fig. 7. Docking calculations with an mGluR3 X-ray structure (PDB entry 2E4U, chain A)are helpful in understanding the affinity of the agonist LY404040, a potent, mGluR2/3-selective ligand for which no mGluR complexed X-ray structure exists. Also, the use ofan existing X-ray structure, i.e., mGluR1 (PDB entry 1EWK, chain A), allows one torationalize the ligand’s selectivity for mGluR2/3 (Group II) by observing the effect oftwo coordinated mutations in these two mGluRs: Tyr150 and Ser100 in mGluR3 arereplaced by Ser164 and Trp110 in mGluR1, respectively.

Fig. 8. High-throughput docking studies using an “open” conformation homology model of mGluR5 (of Group I) identified active molecules that are significantly different fromknown glutamate-like ligands in chemical structure as well as polar nature. Two examples of these hits, having IC50 values of 5 mM and 1 mM, are shown.

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obvious sources of selectivity. Interestingly, the antagonistLY367385, which is closely related to glutamate, is selective formGluR1 over mGluR5 at the extracellular region (Clark et al., 1997).LY367385 would be expected to bind at the active site, in theproximity of the same amino acids for both mGluR1 and mGluR5.One possible explanation (Costantino et al., 1999) is that residuesmore distal from the ligand induce subtle alterations in this bindingsite. A similar effect could then be possible for analogous agonists,i.e., even at the active site.

It is interesting to consider a similar comparison for open formsof the proteins. As there are presently no pairs of X-ray structures ofopen forms of mGluRs within the same group, we compare the X-ray structure of the open form of mGluR1 (PDB entry 1ISS, chain A)with a homology model we have generated for the open form ofmGluR5. The primary interactions of glutamate in mGluR1 havebeen described above. Note that there is also an X-ray structure ofmGluR1 containing the antagonist analogue of glutamate, (S)-(a)-methyl-4-carboxyphenylglycine (S-MCPG). While the hydrogen-bonding patterns are similar to those described for glutamate in theclosed and open X-ray structures of mGluR1, the aromatic ring of S-MCPG cannot be accommodated in the closed form, therebyensuring an open form of the VFT, which, in turn, is responsible forits antagonist action. Considering the glutamate-bound openstructure of mGluR1, in contrast with the comparisons of the X-raystructure of mGluR1 and the mGluR5 homology model in theclosed forms, we now see that residues that differ between themare accessible from the glutamate subsite (Fig. 5). Thus, Asp324 andGlu325 of mGluR1 become Tyr311 and Asp312 of mGluR5, respec-tively. Thus, at or near the glutamate-binding site, these compari-sons suggest that within this group, potential sources of selectivityare more readily identifiable in the open form than in the closedform. This would suggest that a intra-group-selective antagonistwould be more easily obtained than an agonist for this site. In

addition, it hints that selectivity sources could be available as oneinvokes binding away from the core of the active site.

3.2.4. Retrospective structural analysis of function, affinity, andselectivity of known ligands in the EC domain of mGluRs

The early pursuit of ligands modulating mGluR activity predatesthe availability and deployment of the recent EC X-ray structures.With these X-ray structures having become available, it is inter-esting to retrospectively examine how to rationalize the activitiesof these compounds and what insights can be gained into howthese X-ray structures might play into more efficient design in thefuture. We consider here two such mGluR compounds that areclosely related to glutamate and act on the EC region. LY341495 isa potent mGluR2 antagonist with activity at other mGluRs(Kingston et al., 1998). LY404040 is a potent, selective mGlu2/3agonist (Monn et al., 2007).

As is known for other GPCRs, the mGluR receptors are incontinuous equilibrium between active and inactive states. The VFTregion of the EC domain exists in at least two conformations. The“open” conformation has the two well separated lobes (Fig. 1,)whereas the two lobes are in close proximity in the “closed”conformation. Inducing the open state of the VFT tends to shift theprotein towards the inactive conformation, whereas inducing theclosed conformation tends to shift the equilibrium towards theactive state. While other factors are involved with the complexstructure/function mechanism, e.g., interactions as mGluR dimers,these are beyond the scope of this analysis. Figs. 2 and 3 (left side ofeach) show the closed and open conformations, respectively, ofmGluR1 with glutamate bound in the active site, illustrating thestriking conformational changes associated with these two states.From this comparison, it is clear that a ligand that can bind in theglutamate site so as to “wedge” the VFT into an open conformationwould impede the closing/activation process, i.e., act as an

S. Topiol et al. / Neuropharmacology 60 (2011) 93e101100

antagonist. The recent X-ray structure of the antagonist LY341495bound to the VFT of mGluR1 (PDB entry 3KS9 chain A) clearly offersthe explanation for this activity through the requirement of anopen conformation of the VFT. As seen in Fig. 6, the upper portion ofLY341495, which closely resembles glutamate, sits in the glutamatepocket. The remaining 9-methyl-9H-xanthene fragment ofLY341495 projects downwards into the lower lobe, preventing theVFT from closing. Thus, the antagonist nature of the ligand is readilyrevealed from such a structural analysis.

To understand the activity of the agonist LY404040, a potentmGluR2/3-selective ligand for which no mGluR complexed X-raystructure exists, we performed docking calculations using themGluR3 X-ray structure (PDB entry 2E4U, chain A).

The docked structure, shown in Fig. 7, allows an understandingof the affinity. The LY404040 structure shown is consistent with thestereochemical preference of the sulfinyl moiety (Monn et al.,2007). In addition to being able to “overlap” with glutamatethrough the atoms in commonwith glutamate, the cyclized sulfinylstructure provides a stabilizing hydrophobic interaction withTyr222, while its sulfinyl oxygen atom forms a hydrogen bond withthe backbone NH of residue Ser278. These interactions clearlyexplain the affinity of this potent compound. The use of existing X-ray structures also allows us to explore the selectivity for mGluR2/3(Group II). In Fig. 7, an X-ray structure of mGluR1 (PDB entry 1EWK,chain A) has been overlapped with the structure of LY404040docked into mGluR3. Two coordinated mutations in these twomGluRs play a critical role here. Tyr150 and Ser100 in mGluR3 havebeen replaced by Ser164 and Trp110 in mGluR1, respectively.The general nature of the active site is maintained with the struc-tural interchange of Trp110 of mGluR1 sitting in the same region asTyr150 in mGluR3. However, the differences in the fine detailsprovide a profound effect on the selectivity of LY404040. Thearomatic ring of Trp110 of mGluR1 is approximately perpendicularto that of Tyr150 of mGluR3. As a result, the aromatic ring of Trp110 inmGluR1 would point head-on into the cyclized sulfinyl componentof LY404040, precluding its binding and providing the source of itsselectivity. Taken together, these X-ray structures readily providea clear explanation of both the affinity and selectivity of this ligand,a task far more challenging to achieve in the absence of these X-raystructures.

3.2.5. Prospective exploration of structure-based design in the ECdomain

The examples given thus far employ structure analyses in theEC regions to explain the activity of known ligands. As a means ofvalidating whether structure-based studies in the EC domain canbe effective in the prospective discovery of new ligands, wedeployed our homology model of mGluR5 (of Group I) for dockingstudies. The open form of an X-ray structure of mGluR1 was usedin efforts to identify potential antagonists. In Fig. 5, we show theactive site of this model superimposed on the X-ray structure ofmGluR1. As described above, there are two residues near theglutamate-binding site which differ between mGluR1 andmGluR5 (encircled in green). Rather than center our search fornew ligands in the glutamate location, we selected a regionbetween the glutamate molecule and these two “mutations”described earlier. The goal of this specific centering of the dockingstudy was to provide access for any active compounds identified inour docking to these potential variable residues. The potential forintroducing selectivity at a later optimization stage would then befacilitated. Additionally, in the interest of finding ligands that aremost likely to have CNS-like properties and that differ fromglutamate, the docking protocol was tuned to raise the contribu-tion of hydrophobic effects to the scoring/selection of ligands. Ahigh-throughput docking study was then performed using the

Glide docking system. From our corporate database, 350,000compounds (800,000 structures) were docked into this model. Ofthe 100 top-scoring docked compounds, 45 were tested in a FLIPRassay. Of these, 15 exhibited greater than 30% inhibition at 30 mM,and 8 of these 15 initial hit compounds had activities with IC50values <10 mM. Fig. 8 shows two examples of these hits, havingIC50 values of 5 mM and 1 mM. As per the described protocol, theligands reach into the region with two variable residues and aresignificantly different from known glutamate-like ligands inchemical structure as well as polar nature. These results demon-strate that structure-based approaches in the EC region canprovide productive means for identifying new, active ligands formGluRs.

4. Perspective

The mGluRs are an increasingly more popular target for drug-discovery efforts in a number of areas. Along with the other Class CGPCRs, they have a unique tertiary architecture within the GPCRs.Specifically, the endogenous ligand, glutamate, binds in the VFTregion of the EC domain, which also contains the C-rich regionthrough which it is linked to the 7TM domain. Early efforts todiscover new ligands for these targets preceded the availability ofX-ray structures of either domain and were thereby limited tohomology models of distally related proteins. Under thesecircumstances, i.e., without accurate three-dimensional structuresto guide efforts into new chemistries, it is not surprising that initialactivity in this area evolved around close analogues of glutamate.The absence of such structural information made it particularlydifficult to develop ligands that are selective among the mGluRs,which are, by definition, highly homologous. With the discovery ofselective ligands, such as MPEP, which act at the allosteric 7TMdomain, the attractive argument that the 7TM regions should bemore varied and therefore more suitable subtargets for selectivityhas become widely accepted. The sequence analysis providedherein contradicts this premise. As there are presently X-raystructures of the VFT but not of the 7TM regions, for use in drugdesign, it becomes important to explore this potential. Ourcomparisons herein of various X-ray structures suggest that suchselectivity could be achieved, even at the glutamate-binding site ofthe VFT. Furthermore, we illustrate that such differences readilyexplain both the activity and selectivity of previously obtainedglutamate analogues (e.g., LY404040) acting at the glutamate site.As shownwith the analysis of LY404040, selectivity can be achievedby very few residue differences at the active site, provided there isthe opportunity to capitalize on them. Consideration of other sitesof the VFT, or possibly even the cysteine-rich region linking the VFTto the 7TM, may provide still additional opportunities to developselective ligands. Indeed, multiple binding sites have been impli-cated for ligand binding to other Class C GPCRs, the sweet tastereceptors (Xu et al., 2004). Moreover, based on the sequence andstructural analyses provided herein, and considering that there areno 7TM X-ray structures available to date for the mGluRs, the VFTregion appears to offer significant opportunities for structure-baseddesign. To illustrate the use of the VFT for identifying newcompounds, we have used a homology model of the VFT region ofmGluR5 for in silico screening via high-throughput docking andobtained initial hits of ligands that are active at micromolar levels.

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