GPCRpresentation091304-中国药科大学药物筛选课件

DevelopmentofNovelCell-BasedAssaysandScreeningToolsforGProtein-CoupledReceptorsMatthewH.Hsu,Ph.D.ChemiconInternational,Inc.TOOLSFORDRUGDISCOVERYTowardsProprietaryBioassaySystemsforHighThroughputScreeningTargetTargetValidationValidationHITSHITSLEADSLEADSDRUGDRUGSARSAR22oo/3/3ooScreenScreenNative/StableNative/StableCellLinesCellLinesPharmacologicallycharacterizedcellbaseassaysStableCellLinesPrimaryPrimaryScreenScreenHTSAssaysMOLECULARMOLECULARTARGETSTARGETSMolecularMolecularToolsToolsRNAandcDNALibraryWeoffertoolsfor1stand2ndscreensandHTSARƒCosttodevelopadrugtodayisover$800million,andnocompanyhasthecapacity(chemists)tolaunchneedednumberofprojectseachyeartoincreasetheirpipelineofopportunities.ƒBigpharmaiswakinguptooutsourcingtooptimizeR&Dinvestment.ƒDrugdiscoverymarketestimatedtobe$1.5billionwith20%annualgrowth.ƒGPCRsaretheleadingdrugtargetswithmorethan2000identified.HTSTargetTypesGPCR40%Kinase19%Phosphatase5%Protease10%IonChannel9%NucleicAcid8%Other9%Ref:HTSReport2000,HighTechBusinessDecisionsOver60%oftheCurrentlyMarketedDrugsAreTargetedtoGPCRsProposedGProtein-CoupledPathwaysforGPCRCalciumandcAMParethe2ndmessengersGPCRGPCRAgonistAgonistGα15/16Gα15/16PKAACACATPATPcAMPcAMPβγβγPTXPTXαi/oαi/ocAMPPLCβPLCβIP3IP3Ca2+Ca2+αqαqRac/RhoNF-κBGα12GαqGαqGαsGαiGαiGαoGαoβγβγβγβγCommonHTSFormatsforGPCRsAssayThroughputGproteincouplingReceptorbindinganyRadioligandfiltrationassay96RadioligandSPA384FP,FMAT,FRET384,1536GTPγSbinding96,384GicAMPdeterminationcAMPincrease384,1536GscAMPdecrease96Gi[Ca2+]ideterminationFluorescentindicators384GqAequorin384GqReportergeneGs,Gq,GiLuciferase384,1536β-lactamase384,1536HCS96anyUseoffluorescencedetectionandhomogeneousassayisanewtrendofHTSHigh-levelGPCRExpressiononCellSurfaceAchievedbyUsingaProprietaryExpressionSystemCXCR2-GFP/pHStransfectedinChem-1(Chemicon)CXCR2/CHOCHOCXCR2/Chem-1Chem-1FACSanalysisCXCR2-GFP/pCDNA3transfectedinCHO(Conventional)FluorescentimagingChemicon’sHigh-qualityGPCRMembranePreparations•ChemiconGPCRmembranepreparationsarecrudemembranepreparationsmadefromourproprietarystablerecombinantcelllinesofhigh-levelGPCRsurfaceexpression.•Novelmammalianexpressionsystemconsistsofaproprietaryvectorwithanon-CMVpromoterandchoiceofcelllines.•ReduceER-retention,whichresultsinincreasedcellsurfaceexpressionandsuperiorbinding.•EachbatchofmembranepreparationispharmacologicallycharacterizedbyradioligandsaturationbindingassaytodetermineBmaxandKd.•Competitionbindingtodeterminetherankorderofanarrayofagonistsandantagonistswithaknownvalues.•Optimizetheamountoftheproteinandradioligandtobeusedperassaytoachievethemaximumspecificbindingandsignaltobackgroundratio.BetterSignal/NoiseforHTS,bettercharacterizedforHTSARChemicon’sGPCRMembranesHaveHigherBmaxCXCR2/pCDNA3transfectedinCHOCXCR2/CHOMembraneSaturationBinding0.00.51.01.52.02.50100020003000400050006000700080009000TBNSBSB=TB-NSBBMAXKD27560.1484125IGroaBmax(fmol/mg)CXCR2/pHStransfectedinChem-1CXCR2/Chem-1MembraneSaturationBinding0.00.51.01.52.02.50100002000030000400005000060000TBNSBSB=TB-NSBBMAXKD511220.2787[125I]Groα(nM)Bmax(fmol/mg)MembraneradioligandsaturationbindingassayMultipleGPCRsDemonstrateHighLevelofSurfaceReceptorExpressionUsingpHSVector0.7150.4120.140.250.170.4150.2101201.5240510152025Bmax(pmoles/mgprotein)CXCR1CXCR2CXCR3CXCR4CCR5CCR6CCR7C3aRC5aRpcDNA3.1pHSChemicon’sGPCRMembranesAreBetterCharacterizedPharmacologicallyCompetitionbindingusingCXCR2/Chem-1membranes-13-12-11-10-9-8-7-6-505001000150020002500300035004000450050005500GroαIL-8EC50Groα4.349e-010IL-86.736e-011Log[M]125IGroαbound(cpm)MembraneradioligandcompetitionbindingassayCompetitionbindingusingCXCR2/CHOmembranes-13-12-11-10-9-8-7-6-5010002000300040005000GroaIL-8EC50_1EC50_2Groa2.227e-0103.433e-008IL-87.623e-0112.900e-008Log[M]125IGroαbound(cpm)GroαGroαUsingGPCRInternalizationandDesensitizationasEndPointforReceptorActivationAssayRecyclingEndosomeGFPERCppSortingEndosomeGFPGFPGFPGRKppGFPβ-ArrestinGFPβ-ArrestinGFPHCSAssaysCXCR2-GFPFusionProteinIsExpressedonCellSurfaceandInternalizedUponStimulation0min30minGroα(10nM)LowbackgroundinHigh-contentscreeningCanWeFunnelAllGPCRPathwaysToACommonAndSimpleRead-out?GPCRAgonistFLIPRreadoutFluo-3AequorinreadoutApoaequorin+CoelenterazineGqαqPLCβIP3PTXGoαi/oβγGiGα12GsGα15/16GqchimerasConventionalmethod:Co-transfectingGa15/16,GqchimerasorGiConstructionofGαqChimericProteinsReplacetheC-terminalresidueswithcorrespondingresiduesfrom:Gαi2DCGLFGqi5GαoGCGLYGqo5GαsQYELLGqs51MTLESIMACCLSEEAKEARRINDEIERQLRRDKRDARRELKLLLLGTGES51GKSTFIKQMRIIHGSGYSDEDKRGFTKLVYQNIFTAMQAMIRAMDTLKIP101YKYEHNKAHAQLVREVDVEKVSAFENPYVDAIKSLWNDPGIQECYDRRRE151YQLSDSTKYYLNDLDRVADPAYLPTQQDVLRVRVPTTGIIEYPFDLQSVI201FRMVDVGGQRSERRKWIHCFENVTSIMFLVALSEYDQVLVESDNENRMEE251SKALFRTIITYPWFQNSSVILFLNKKDLLEEKIMYSHLVDYFPEYDGPQR301DAQAAREFILKMFVDLNPDSDKINYSHFTCATDTENIRFVFAAVKDTILQ351LNLKEYNLVAAsequenceofGqLimitationstoCo-transfectingGα16orChimericGqtoPromoteCa2+ResponseforNon-GqCoupledReceptors-13-12-11-10-9-8-7-60204060801000.5ugGα161ugGα162ugGα163ugGα164ugGα165ugGα16Vectorcontrol[C5a]LogM%ofmaximalFLIPRrespons