GPCRs: From Deorphanization to Lead Structure Identification PDF
Preview GPCRs: From Deorphanization to Lead Structure Identification
Ernst Schering Foundation Symposium Proceedings 2006-2 GPCRs: From Deorphanization to Lead Structure Identification Ernst Schering Foundation Symposium Proceedings 2006-2 GPCRs: From Deorphanization to Lead Structure Identification H. Bourne, R. Horuk, J. Kuhnke, H. Michel Editors With 59 Figures 123 SeriesEditors:G.StockandM.Lessl LibraryofCongressControlNumber:2007921597 ISSN 0947-6075 ISBN 978-3-540-48981-8 SpringerBerlinHeidelbergNewYork This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,recitation,broadcasting,reproductiononmicrofilmsorinanyotherway,and storageindatabanks.Duplicationofthispublicationorpartsthereofispermittedonly undertheprovisionsoftheGermanCopyrightLawofSeptember9,1965,initscurrent version,andpermissionforusemustalwaysbeobtainedfromSpringer-Verlag.Violations areliableforprosecutionundertheGermanCopyrightLaw. SpringerisapartofSpringerScience+BusinessMedia springer.com ©Springer-VerlagBerlinHeidelberg2007 Theuseofgeneraldescriptivenames,registerednames,trademarks,etc.inthispublication doesnotemply,evenintheabsenceofaspecificstatemant,thatsuchnamesareexempt fromtherelevantprotectivelawsandregulationsandthereforfreeforgeneraluse.Product liability:Thepublishercannotguaranteetheaccuracyanyinformationaboutdosageand applicationcontainedinthisbook.Ineveryinduvidualcasetheusermustchecksuch informationbyconsultingtherelevantliterature. Editor:Dr.UteHeilmann,Heidelberg DeskEditor:WilmaMcHugh,Heidelberg ProductionEditor:AnneStrohbach,Leipzig Coverdesign:WMXDesign,Heidelberg Typesettingandproduction:LE-TEXJelonek,Schmidt&VöcklerGbR,Leipzig 21/3180/YL–543210 Printedonacid-freepaper Preface G-protein-coupled receptors (GPCRs) play a major role in regulating theoverallhomeostasisofcomplexorganismssuchasmammalsbutare alsofoundinprimitivespeciessuchasDictyostelium(slimemold)and yeast. The GPCR superfamily is quite diverse and sequencing has re- vealedmorethan850genescomprisingapproximately3%ofthehuman genome.ThediversityoftheGPCRsisequallymatchedbythevarietyof ligandsthatactivatethem,whichincludeodorants,tasteligands,light, metals, biogenic amines, fatty acids, amino acids, peptides, proteins, nucleotides,lipids,Krebscycleintermediates,andsteroids.Becauseof theircentralroleinregulatingnormalphysiologicalresponses,GPCRs haveattractedconsiderableattentionfromthepharmaceutical industry as targets for disease. This large superfamily of proteins remains one of the most druggable targets, accounting for more than 40% of all marketedtherapeutics. Basedontheirever-growingimportance,asoutlinedabove,arecent Ernst Schering Research Foundation Workshop held in Berlin, Ger- many,focusedonGPCRs.Entitled“GPCRs:FromDeorphanizationto Lead Structure Identification,” the workshop brought together leading experts from a variety of areas to discuss recent advances in the field. Professor Henry Bourne of UCSF gave an enthralling keynote lecture entitled “G-Proteins and GPCRs: From the Beginning” This lecture chronicled the progress made from Earl Sutherland’s original Nobel VI Preface Prize-winningachievementindiscoveringcyclicAMPasasecondmes- sengermoleculetothediscoveryofG-proteinsandGapers.AsProfessor Bourneremindedus,thesebreakthroughswereallthemoreremarkable becausetheywereachievedintheabsenceofmanyofthebreakthrough technologieswealltakeforgrantednowadayssuchasmolecularcloning andPCR. Theformalpresentationsontheseconddaybeganwithalecturefrom ProfessorHartmanMicheloftheMaxPlanckInstituteinFrankfurt,Ger- many,whodiscussedapproachestoobtainproteincrystalsofGPCRs.As ProfessorMichelremindedus,despitemassiveeffortsonlyoneGPCR, bovine rhodopsin, has so far succumbed to this approach. Professor Michel’spresentationcenteredonthemethodsthatheandothergroups areusingwithexpressionsystemstotrytoovercomesomeofthehur- dlesinobtainingsufficientactiveproteintoenablecrystallization.Atthis point,theworkshopshiftedgearsandswitchedfromdiscussingmodels determined from a protein crystal of a GPCR to models derived from analysis of GPCRs by in silico methods. First up was Professor Gert VriendoftheCMBIatNijmegenwithathought-provokingpresentation centered on molecular modeling of GPCRs.Comparing predictions of GPCR structure, Professor Vriend noted that numerous problems and errors remain inherent in most of these models, even those based on thecrystalstructureofbovinerhodopsin.Hepointedoutthatalthough homologymodelingofGPCRshasreachedavogueindrugdiscovery,it needstobeapproachedwithsomecautionandisnotquiteaspredictable as its proponents would have us believe. Following this controversial viewpoint,ProfessorAlexTropshaofUNCChapelHillcontinuedina similar vein,albeitmoreoptimistically, withapresentation thatexam- inedtheroleofQSARinaidingandabettingdrugdiscoveryforGPCRs. Here the ideais that known small molecule ligands foragiven GPCR can be used to build models with which to interrogate the chemical universetodiscovernewstructuresforthatreceptor.ProfessorTropsha pointed out that ligand-based modeling has been successfully applied to a number of receptors, including those from the dopaminergic and serotinergicreceptorfamilies,whichheusedasexamples. The well-established idea that particular classes of ligand substruc- tures seem to occur quite frequently in pharmacologically success- ful small molecules was the theme developed by Dr. Robert Bywater Preface VII of Magdalen College, Oxford. The notion of privileged structures in GPCRshasbeenexploitedindrugdesignand,aspointedoutbyDr.By- water,hasbeenusefultogeneratebothreceptoragonistsandantagonists. Heconcludedhisseminarbysuggestingthattheconceptofprivileged structuresmightbeusefulindesigningdrugsfororphanGPCRswhose naturalligandshavenotyetbeenidentified.Twofurtherspeakershigh- lightedthetreasuretroveoftargetsthathaveyettobediscoveredfrom thelargepooloforphanGPCRs.Dr.AlanWiseofGSK,Harlow,UK, gave several examples of receptor deorphanization approaches used at GSK. In particular he pointed out the use of a knowledge-based ap- proach for the successful pairing of nicotinic acid with the receptor HM74.Nicotinicacidhasbeenusedclinicallyforover40yearstotreat dyslipidemiaactingonadiposecells.Expressionanalysisrevealedthat tenorphanGPCRswereexpressedinadiposetissue.Thesewererecom- binantlyexpressedandbindingexperimentsrevealedthatHM74wasthe nicotinicacidreceptor.ProfessorMarcParmentierfromtheUniversity ofBrussels,Belgium,continuedalongthesamelines,highlightingthe successful strategies for deorphanizing GPCRs currently employed in hisgroup.Reversepharmacologicalapproachesthatinvolveextraction ofputativeligandsforGPCRsfromtissueextractsthentestingthemfor activityonorphanGPCRswereafeatureofhispresentation.Anexam- ple of this was the identification of nocipeptin as the ligand forORL1 and apelin as the ligand for the APJ receptor. Dr. Parmentier finished hisseminarbypointingoutthatsomeorphanGPCRsmightonlyexist as receptor heterodimers, which would be incredibly difficult to deor- phanize. The concept of GPCR dimers was amply illustrated from his ownworkwithchemokinereceptors,forexampleCCR2/CCR5,which heshowedcanexistasheterodimers. Clearly,theexistence ofchemokinereceptorsasdimerswillhavea profound effect on drug discovery since many existing paradigms and conceptswillhavetobealteredifwearetobesuccessfulinfindingdrugs thattargetthesecomplexes. ProfessorGraemeMilliganoftheUniver- sityofGlasgow,Scotland,UK,tookusdownthisnewavenuewithhis presentationlookingattheroleofGPCRdimerizationinreceptorsignal- ing. ProfessorMilligan reminded us that theonly established example of GPCR dimers comes from atomic force microscopy of murine rod outer-segment discs that reveal that rhodopsin is organized in a series VIII Preface of parallel arrays of dimers. This organization of GPCRs as homo- or heterodimersmaybemorecommonthanweimagineandProfessorMil- liganillustratedthiswithexamplessuchasthealphaadrenergicreceptor andtheinterestingheterodimerformedbetweenthecannabinoid1and the orexin receptors, which explain the pharmacological action of the appetitesuppressantRimonabant. Dr. Rob Leurs of the Vrije Universiteit Amsterdam, The Nether- lands, reminded us that viruses have exploited human GPCRs very effectivelytoovercomehostdefensemechanismssothattheycanprop- agate.AchillingexampleistheuseofthechemokinereceptorsCCR5 andCXCR4asvehiclesofentryforHIV-1,whichgivesrisetothedeadly diseaseAIDS.Dr.LeursshowedthatvirusescouldalsoexpressGPCRs, potentiallypiratedfromtheirhosts.Anexampleofthiswasthehuman cytomegalovirus virus US28, which has been associated with chronic diseasesandmalignancies.Examplesoftargetingtheseandotherviruses withsmallmoleculeantagonistsmightopenupnewavenuesoftreatment forsomehumandiseases. Professor Eric Prossnitz of the University of New Mexico, Albu- querque,returnedusrightbacktoHenryBourne’sopeningaddresswith his discussion of receptor signaling. His theme was that GPCRs exist inalargevarietyofconformations.Thesecanbeligand-inducedorcan be induced by post-translational modifications. As Professor Prossnitz remindedus,theseconformationsareuniqueandmightrepresentnovel targetsfordrugdiscoveryandtherapeuticintervention. Twoexcellentpresentationsbyrepresentativesfromthepharmaceu- tical industry highlighted approaches in drug discovery for GPCRs. The first by Dr. Ralf Heilker from Boehringer Ingelheim, Biberach an der Riss, Germany, explained the advantages of high-content screen- ingtomonitorG-protein-coupledreceptorinternalizationasameansof drugdiscovery.High-contentscreeningisacombinationoffluorescence microscopicimagingandautomatedimageanalysis,andhasfoundin- creasinguseinmonitoringtheeffectsofcompoundsincellularsystems, forexamplereceptordesensitization,inwhichreceptorsinternalizeafter ligandstimulation.Theuseofsuchassaystopharmacologicallyprofile compounds wasverynicely demonstrated byDr.Heilker.Dr.Andreas Sewing of Pfizer outlined drug discovery approaches employed in his company to generate therapeutics targeting GPCRs. His seminar cen- Contents IX teredonhigh-throughputscreeningapproachesforrapidlydiscovering leadcompounds.Thechoiceoftheassayemployedisobviouslyimpor- tantforsuccess,asalreadydiscussedbythepreviousspeaker.Therewas further discussion on reagent generation and supply and lead-finding strategies applied to biological screening. Finally, the hit-to-lead and lead-optimizationprocesseswerediscussed. All in all, the meeting greatly exceeded all of our expectations and liveduptotheidealsoftheErnstScheringResearchFoundationWork- shop to sponsor meetings that bring together a critical mass of top scientistsworkinginimportantareasinanintimatesettingthatfosters thefreeexchangeofknowledgeandideas. H.Bourne R.Horuk J.Kuhnke H.Michel Contents G-ProteinsandGPCRs:FromtheBeginning H.R.Bourne . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ModelingGPCRs A.C.M.Paiva,L.Oliveira,F.Horn,R.P.Bywater,G.Vriend . . . 23 QSARModelingofGPCRLigands: MethodologiesandExamplesofApplications A.Tropsha,S.X.Wang . . . . . . . . . . . . . . . . . . . . . . . 49 PrivilegedStructuresinGPCRs R.P.Bywater . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 DesigningCompoundLibrariesTargetingGPCRs E.Jacoby . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 OrphanSevenTransmembraneReceptorScreening M.J.Wigglesworth,L.A.Wolfe,A.Wise . . . . . . . . . . . . . . 105 TheRoleofGPCRDimerisation/Oligomerisation inReceptorSignalling G.Milligan,M.Canals,J.D.Pediani,J.Ellis, J.F.Lopez-Gimenez . . . . . . . . . . . . . . . . . . . . . . . . 145
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