高内涵药物筛选方法的研究及应用

,3(,100050):;;:R965.1:A:0513-4870(2005)06-0486-05:2004208218.:(30171148);(02203ZP08).3Tel/Fax:[phone],E2mail:[e-mail]nganditsapplicationZHANGLi,DUGuan2hua3(InstituteofMateriaMedica,ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing100050,China)Keywords:highcontentscreening;drugdiscovery;cell2basedassay(high2throughputscreening,HTS)2080,1998,[1,2](high2contentscreening,HCS)1,,20,,,,,,,,,,,[3]10(ultrahigh2throughputscreening,Ultra2HTS),,,,1997,,,[4-6],,684ActaPharmaceuticaSinica2005,40(6):486-490,,,,,,96,2,,,,,,(ArrayScanÒandKineticScanTMHCSReader)Cellomics,CCD(chargecoupleddevices),,,G[7-9]2.1,,,SRB(sulforhodamineB),MTT(32[4,52dimethylthiazol222yl]22,52diphenyltetrazoliumbromide),52(bromodeoxyuridine,BrdU)3HMTT,(MTT)(formazan),,,,,DNA,,DNA,;,,VogtA[10],Hoechst3334(EX350/EM461),MitoTracker(EX556/EM573),AlexaFluor488(EX494/EM519),,ArrayScanII(),,,,DNA,[11]ArrayScanÒ(normalhumandermalfibroblast,NHDF),Ki267,BrdU,Phospho2Rb,H3(Phospho2histoneH3)4[12]Ki267,H3MBrdU,DNA,pRb,,pRbGasprriF,FluorolinkTMCy2TM(EX489/EM506nm)FluorolinkTMCy2(EX489/EM506nm),FluorolinkTMCy3TM(EX554/EM566nm),DAPI(4,62diamidino222phenylindole,EX359/EM461nm)H3ArrayScanÒBrdUH3,784:ELISAArrayScanÒ,ArrayScanÒ,4,4,,H3MBrdUS,2.2GG(G2protein2coupledreceptors,GPCRs),G,GPCR,[13-15](greenfluorescentprotein,GFP)CellomicsArrayScanÒGFPArrayScanÒhGR2GFP,GPCRGPCRJELLY(AequoriaVictoria),GFP65-6722GFP,GFP395nm488-490nm,,,507-511nm,GFPSer65Thr(enhancedgreenfluorescentprotein,EGFP)(enhancedbluefluorescentprotein,EBFP)(enhancedyellowfluorescent,EYFP)(enhancedcyanfluorescentprotein,ECFP),,GFP,GPCRC[16],[17]GPCR,pH(Cyaninedyes)[18]CypHer25pH,(pKa611)GPCRCypHer25,2GPCRpH714,GPCR,GPCRpH,2.3G,,,TAMRA,[19](DSPases)[20],HeLaU937,,DNAHoechst33342,caspases2X2rosamine(MMP),ArrayScanÒ,,caspase23,,,[21]3,,,,ELISA[22]884ActaPharmaceuticaSinica2005,40(6):486-490VogtA[10],,4,IL21,IL28TNF2,,3,NDGADiclofenacIL21TNF2,IL28LPS(4h),,352NDGADiclofenac,,,3,,,,,,[23]5,,,,,3841536,,,,,,,,,,,,,,,200015[24],,,GFP,,GFP,,,,,,,,,References[1]DuGH,HuJJ,XiaLJ,etal.Developmentandstatusofdrugdiscovery[J].ActaPharmSin(),1998,30(11):166-169.[2]ZhangL,ShangNY,DuGH.Establishmentandapplicationofhighthroughputscreeningfordiscoveryofnewantidepressants[J].ChinPharmJ(),2003,38(4):263-266.[3]ConnickJH.Aflexibletechnologyplatformtoexplorevaluabledrugtargets[J].BiochemSocTrans,2002,30(4):786-788.[4]GiulianoKA,DeBiasioRL,DunlayRT,etal.High2contentscreening:anewapproachtoeasingkeybottlenecksinthedrugdiscoveryprocess[J].JBiomolScreen,1997,2(3):249-259.[5]GiulianoKA,TaylorDL.Fluorescent2proteinbiosensors:newtoolsfordrugdiscovery[J].TrendsBiotechnol,1998,16(3):135-140.[6]DoveA.Screeningforcontenttheevolutionofhighthroughput[J].NatBiotechnol,2003,21(8):859-984:864.[7]HaskinsJR,RowseP,RahbariR,etal.Thiazolidinedi2onetoxicitytoisolatedhepatocytesrevealedbycoherentmultiprobefluorescencemicroscopyandcorrelatedwithmultiparameterflowcytometryofperipheralleukocytes[J].ArchToxicol,2001,75(7):425-438.[8]GiulianoKA.High2contentprofilingofdrug2druginterac2tions:cellulartargetsinvolvedinthemodulationofmicro2tubuledrugactionbytheantifungalketoconazole[J].JBiomolScreen,2003,8(2):125-135.[9]ConwayBR,MinorLK,XuJZ,etal.Quantitativeanalysisofagonist2dependentparathyroidhormonereceptortraffickinginwholecellsusingafunctionalgreenfluorescentproteinconjugate[J].JCellPhysiol,2001,189(3):341-355.[10]VogtA,KalbEN,LazoJS.Ascalablehigh2contentcytotoxicityassayinsensitivetochangesinmitochondrialmetabolicactivity[J].OncolRes,2004,14(6):305-314.[11]MinguezJM,GiulianoKA,BalachandranR,etal.Synthesisandhighcontentcell2basedprofilingofsimplifiedanaloguesofthemicrotubulestabilizer(+)2discodermolide[J].MolCancerTher,2002,1(14):1305-1313.[12]GasparriF,MarianiM,SolaF,etal.QuantificationoftheproliferationindexofhumandermalfibroblastcultureswiththeArrayScanhigh2contentscreeningreader[J].JBiomolScreen,2004,9(3):232-243.[13]ConwayBR,DemarestKT.TheuseofbiosensorstostudyGPCRfunction:applicationsforhigh2contentscreening[J].ReceptorsChannels,2002,8(5-6):331-341.[14]GurwitzD,HaringR.Ligand2selectivesignalingandhigh2contentscreeningforGPCRdrugs[J].DrugDiscovToday,2003,8(24):1108-1109.[15]ConwayBR,MinorLK,XuJZ,etal.QuantificationofG2proteincoupledreceptorinternatilizationusingG2proteincoupledreceptor2greenfluorescentproteinconjugateswiththeArrayScantrademarkhigh2contentscreeningsystem[J].JBiomolScreen,1999,4(2):75-86.[16]MilliganG.High2contentassaysforligandregulationofG2protein2coupledreceptors[J].DrugDiscovToday,2003,8(13):579-585.[17]KainSR.Greenfluorescentprotein(GFP):applicationsincell2basedassaysfordrugdiscovery[J].DrugDiscovToday,1999,4(7):304-312.[18]AdieEJ,KalinkaS,SmithL,etal.ApH2sensitivefluor,CypHer5,usedtomonitoragonist2inducedGprotein2coupledreceptorinternalizationinlivecells[J].Biotechniques,2002,33(5):1152-1154,1156-1157.[19]BlommelP,HansonGT,VogelKW.Multiplexingfluorescencepolarizationassaystoincreaseinformationcontentperscreen:applicationsforscreeningsteroidhormonereceptors[J].JBiomolScreen,200