IBC’s Sixth Annual International G-Protein-Coupled Receptor (GPCR) conference,Taking Receptors from Orphans to Drugs(11–13 June 2001, Litchfield, AZ, USA),saw the highest attendance yet in thisseries, with >160 delegates. This level ofinterest reflects how the near-comple-tion of the Human Genome Project hasdefined what is required to understandthe function of every GPCR, enablingmany companies to contemplate orphanstrategies. Identifying the genes is justthe beginning; the big challenge is to attribute value to the receptors.

A major focus of the meeting was theuse of high-throughput technologies tostudy receptors on a genome-wide scalefor ligand identification and target vali-dation. Recent advances in structuralstudies could provide new opportunitiesfor rational ligand design. Another emerg-ing trend is the discovery of receptor-interacting proteins, which complicatefuture considerations of GPCRs as drugtargets.

Orphan strategies and targetvalidationA surprise from the human genome isthat there are far fewer GPCRs than waspreviously predicted. Excluding sensoryreceptors, the total number of Family A,B and C GPCRs is ∼ 400, of which only∼ 150 are ‘orphans’ for which the ligandis yet to be identified. The rationale forstudying orphans is that, historically,GPCRs have proven to be excellent drugtargets. Indeed, the list of recently li-ganded orphans, reviewed by ShelaghWilson [GlaxoSmithKline (GSK), Harlow,UK], appears to justify the investment.

Almost every case reveals an interestingbiology, and clear new drug targets areemerging. For example, the newly iden-tified receptors BLT2 and CysLT2, for theleukotrienes, LTD4 and LTB4, respec-tively, are potential targets for inflam-mation and asthma; the new purinergicP2T (P2Y12) receptor has an establishedrole in platelet aggregation and throm-bosis, and the two recently discovered

neuropeptide FF receptors are targets forthe treatment of pain. One of the mostinteresting recent pairings is the KiSS-1metastasis-suppressor protein as a putative endogenous agonist of theAxor12/GPR54 orphan receptor. Links tocancer have been made for both ligandand receptor, and their coincident tissuedistribution in placenta suggests a poss-ible role in invasive growth.

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1359-6446/01/$ – see front matter ©2001 Elsevier Science Ltd. All rights reserved. PII: S135-6446(01)01936-5884

Understanding GPCRs – from orphanreceptors to novel drugsSimon J. Dowell, Group Leader, Gene Expression and Protein Biochemistry, GlaxoSmithKline, Stevenage, Hertfordshire, UK SG1 2NY, tel: +44 1438 764987, fax: +44 1438 764488, e-mail: sd14041@glaxowellcome.co.uk

Schematic representation of a G-protein-coupled receptor bound to an unknown ligand.Ligands shown include Apelin, melanin concentrating hormone (MCH), urotensin-II (UT-II)and prolactin-releasing peptide (PRP). Reproduced, with permission, from reference 2.Illustrators: Brett Clayton and Dennis Lee.

Identification of a natural ligand is oneof the fastest means to determine thebiological role of a receptor, and compa-nies such as Synaptic PharmaceuticalCorporation (Paramus, NJ, USA) andGSK have focused much of their effortson this. Bioinformatics is the usual start-ing point, and many receptors havebeen ‘deorphanized’ solely through theirhomology to known receptors. Thus,MCH2 is identified as a new receptor formelanin-concentrating hormone, basedon its close identity with SLC-1/MCH1.Edg-1, -3, -5 and -8 are all receptors forsphingosine-1-phosphate, and Edg-2, -4and -7 are all receptors for lysophospha-tidic acid. This year, six independentgroups published the new histamine H4receptor (identified through its similarityto H3), illustrating how competitive theorphan race has become. Receptor H4 isprobably the eosinophil receptor de-scribed by Raible et al. in 1994 (Ref. 1),and is likely to trigger many new drugdiscovery projects.

With a good assay, fishing for ligandsfrom small collections can be fruitful.Theresa Branchek (Synaptic) describedthe company’s SNAP Discovery™ ap-proach, in which orphans are subjectedto a universal functional assay (UFA™)using the fluorescence imaging platereader (FLIPR). Using this method, ligands have been found for 18 of 60 receptors, a remarkable success whichBranchek puts down to the thoughtfulcomposition of Synaptic’s ligand bank.However, Vincent Dupriez (Euroscreen,Brussels, Belgium) made a case for usingnatural extracts as potential sources ofligands. Using Aequoscreen, a functionalassay based on aequorin (a calcium-sensing protein from jellyfish) Euroscreenidentified a fragment of hepatocellularcarcinoma (HCC)-1 protein as a newhigh-affinity functional agonist of CCchemokine receptor-5 (CCR5). Dupriezemphasized that other approaches wouldnot have achieved this identification.

In the absence of a ligand, value canbe conferred on a receptor in various

ways. Taqman (Applied BioSystems,Foster City, CA, USA), a high-throughputreverse transcription-polymerase chainreaction (RT-PCR) technique, enablesquantitative expression profiling of GPCRsin different tissues. Incyte Genomics(Palo Alto, CA, USA) has created GPCRmicroarrays using combinations of syn-thetic polynucleotides, representing thelargest number of GPCRs on glass, to date.These techniques allow comparativeprofiling of RNAs in, for instance, normalversus diseased tissue. Meanwhile, theresource held by Lifespan BioSciences(Seattle, WA, USA) centres on an impres-sive human-tissue bank of two millionspecimens covering every stage of all themajor diseases. They are creating a pro-prietary database in which every GPCR isprofiled in every cell type in normal anddiseased tissue using immunohistochem-istry. One of the most impressive compa-nies is Deltagen (Menlo Park, CA, USA),whose high-throughput capacity to cre-ate knockout mice is currently running atone new knockout per day. All animalsare subjected to a wide range of physio-logical tests and pathology and knockedout genes are replaced with lacZ to pro-vide expression information, all of which(including pictures) is available througha versatile web interface.

Assay technologies for GPCRscreeningOnce receptors have been identified andprioritized, a central feature of most or-phan programmes lies in the screeningstrategy. Newcomers to the field will bechallenged by a burgeoning array ofassay technologies, each claiming to beideal for orphan receptors. The big deci-sions are whether to invest in an expen-sive piece of kit, or to put faith in anemerging technology that might not befully validated.

According to Stephen Rees (GSK,Stevenage, UK), the ideal is a low cost,miniaturizable, generic, solvent-tolerant,non-radioactive single-step functionalassay with low false-positive and negative

hit rates. Unfortunately, such an assaydoesn’t exist yet! The FLIPR fromMolecular Devices (Sunnyvale, CA, USA),as used to great effect by Synaptic andGSK, has the drawbacks of its expenseand size. However, FLEXstation™,Molecular Devices’ new bench-top alter-native to FLIPR, is available at a fractionof the cost and produces excellent datathat is ideal for assay development, leadoptimization and academic research.The Aequoscreen (Euroscreen) gives aninstant ‘flash’ response, and this also re-quires specialist detection equipment,such as the Hamamatsu FDSS6000(Hamamatsu City, Japan), which allowsplate additions directly on the detector.The versatile applications of this detectorcould make it a worthwhile purchase.

Many new techniques involve the useof fluorescent or luminescent fusion pro-teins. The BRET2™ (Biosignal Packard,Montréal, Québec, Canada) uses bio-fluorescence resonance-energy transferfrom a luciferase to a green fluorescentprotein (GFP), or similar fluorescent protein, to assay proximity between twoproteins. Interactions between receptor–luciferase fusions and β-arrestin-GFP pro-vide a moderately generic assay. Manyvariations on this theme are being pre-sented, complicated by a profusion of al-ternative coloured fluorescent proteinsand luciferases, many of which are notfully evaluated. For instance, data fromGraeme Milligan (University of Glasgow,Glasgow, UK) indicated that some of thedifferent coloured fluorescent proteinsmarketed by Clontech (Palo Alto, CA,USA) have a tendency to aggregate,making interpretation of receptor behav-iour difficult.

Similarly, Biosignal’s proprietaryDeepBlueC coelenterazine (a new lu-ciferase substrate) gives improved peakseparation, but poor quantum efficiency.Assays based on fluorescent fusion- proteins are attractive in that they canprovide additional information about atarget, such as movement within a cell.They also require investment in new

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equipment that might be expensive. Theimpressive LeadSeekerTM Cell AnalysisSystem (Nycomed-Amersham, LittleChalfont, UK) is top of the price list fornew equipment, and features a high-resolution confocal-based system thatcan handle ∼ 30,000 compounds a day.On-line image analysis enables rapidconversion of images into data. Cheapermachines are available, such as theArrayscan™ (Cellomics, Pittsburgh, PA,USA), with each one suited to differentrelated applications. Time will tell whichapproaches provide the best advantagesin this crowded and competitive area.

New trends in GPCR researchBesides the human genome, the mostsignificant breakthrough in GPCR re-search in the past year was the 2.8 Å resolution crystal structure of bovinerhodopsin, presented by Ron Stenkamp(University of Washington, Seattle, WA,USA). The high B factor of the loop be-tween the transmembrane helices V andVI suggests a site of dynamic movement.Philip Yeagle (University of Connecticut,

Storrs, CT, USA) presented a novel alterna-tive approach by solving the structuresof synthetic peptides corresponding to each individual transmembrane andloop region of bacteriorhodopsin. Thefragments were assembled into a three-dimensional model that superimposedrather well onto the bacteriorhodopsinX-ray crystal structure, and took only afew months to obtain.

The identification of novel receptor-interacting proteins is also challengingpreconceptions regarding receptor be-haviour. The interaction of transcriptionfactors with GABA-B, identified by JuliaWhite (GSK, Stevenage, UK), using yeasttwo-hybrid screens, suggests novel sig-nalling mechanisms. Calcyon, a singletransmembrane protein identified byClare Bergson (Medical College ofGeorgia, Augusta, GA, USA), physicallyassociates with dopamine D1 to stimu-late intracellular calcium release. However,the blossoming literature on receptordimerization was reviewed by GraemeMilligan with a note of caution. There is little doubt that many receptors can

form homo- and/or heterodimers, how-ever Milligan’s group believes that someof the most popular techniques to showthis, such as co-immunoprecipitation,can lead to artefactual results. In short,‘if you look hard enough, everythingcomes down with everything!’ As timegoes on, clear evidence of functional significance will be required for a dimer-ization event to be credible.

The meeting was one of the moststimulating IBC GPCR conferencesthanks to the good attendance and thesense of an exploding global interest inorphan receptors. The race is clearly onto identify ligands and disease associ-ations. The winners stand to gain in-tellectual property and the chance to develop the best and most novel drugs.

Reference1 Raible, D.G. et al. (1994) Pharmacologic

characterization of a novel histaminereceptor on human eosinophils. Am. J. Respir.Crit. Care Med. 149, 1506–1511

2 Lee, D.K. et al. (2001) Orphan G-protein-coupled receptors in the CNS. Curr. Opin.Pharmacol. 1, 31–39

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