义翘讲堂《激酶活性检测与药物筛选：SignalChem科学家的深度解析》

KinaseAssayTechnologies&OverviewofSignalChemAssayPlatformsEricYaoSr.Scientist&Teamlead,AssayDev.SinoBiological/SignalChemBiotechSeptember2024Kinasebiochemistryandfunctions1Kinaseasanimportantdrugtargetclass2Kinaseassaytechnologies3SignalChemassayplatformsandkinaseassaycasestudies4CONTENTSinoBiological,Inc.Kinasebiochemistryandfunctions01SinoBiological,Inc.SinoBiological,Inc.Kinasescatalyzesubstratephosphorylation4ProteinsLipidsCarbohydratesSinoBiological,Inc.Kinasesignalingcontrolscellularactivities5MutationOverexpressionOver-productionofligandImage:/wiki/KinaseKinaseasanimportantdrugtargetclass02SinoBiological,Inc.NumberofagentspercategoryandhighestreachedphaseSinoBiological,Inc.CardiovasculardisordersDigestivesystemdisordersEyedisordersImmunesystemdisordersInfectiousdiseasesMetabolicdisordersNervoussystemdisordersOtherdisordersRenaldisordersRespiratorytractdiseasesCancersAttwoodMMetal.20217SinoBiological,Inc.8AttwoodMMetal.2021SinoBiological,Inc.9TheneedforactivekinaseHighactivityLowactivityKinaseassaytechnologies03SinoBiological,Inc.SinoBiological,Inc.11KinaseassayreadoutsSinoBiological,Inc.12BiochemicalkinaseassayplatformsPhospho-peptidedetectionFilterBindingLanthaScreen™IMAP®-FPPhosphoSens®MobilityShiftScintillationProximityLance®UltraAlphaScreen®ADPdetectionADP-Glo™Transcreener®Adapta™ATPdetectionKinase-Glo™EasyLite™SinoBiological,Inc.13Detectionofphospho-peptideSinoBiological,Inc.14FilterbindingassayProsdirectdetectionofsubstrate,extensivelyvalidated,sensitiveConsrequireswashing,low-throughput,radioactivewaste,requirespositively-chargedsubstrateImage:UpstateGroup,Inc.2004SinoBiological,Inc.15LanthaScreenassayPros“mix-and-read”,HTS-compatible,highlysensitiveConsrequiresspecificantibody,highcostofreagentsImage:SinoBiological,Inc.16IMAP-FPassayPros“mix-and-read”,simpleworkflow,multiplexable,HTS-compatibleConssusceptibletocompoundinterference,substratemustbesmallpeptideImage:SinoBiological,Inc.17PhosphoSensassayPros“mix-and-read”,continuousmeasurement,HTS-compatibleConssusceptibletocompoundinterference,substratemustbesmallpeptideCornell-KennonSetal.2024SinoBiological,Inc.18MobilityshiftassayProslowsampleconsumption,easeofautomation,HTS-compatibleConssusceptibletocompoundinterference,substratemustbeshortpeptidewithsinglephosphorylationsiteImage:/SinoBiological,Inc.19DetectionofADPSinoBiological,Inc.20ADP-GloassayProsHighlysensitive,universal,highATPtolerance,HTS-compatible,label-freeConsCouplingenzymesmayproducefalsepositives,interferencefromATPaseImage:/SinoBiological,Inc.21Sensitivity&linearityofADP-GloassayZegzoutiHetal.2009SinoBiological,Inc.22Transcreener/AdaptaassayProsVersatile,HTS-compatible,variousdetectionmodesConsAntibody-dependent,specificityissuesAdaptaImage:SinoBiological,Inc.23DetectionofATPSinoBiological,Inc.24Kinase-Glo&EasyLiteassayProsSimpleandshortworkflow,universal,HTS-compatibleConsNegativecorrelation-smallassaywindow,narrowlinearrange(EasyLite,10nM-10uMATP)Kinase-GloLightImage:/SummaryofcommonkinaseassaysAnalyteAssaynameAssaytechnologyAssayprincipleProsConsPhospho-peptideFilterBindingAssayRadioactivity33P-labeledphospho-peptideiscapturedonnitrocellulosepaperDirectdetectionofphospho-peptide,sensitiveRadioactivewastedisposal;lowthroughputScintillationProximityAssay33P-labeledpeptide-biotiniscapturedonadetectionbeadwhichscintillatesfromproximitytotheradioisotopeHomogeneous,sensitiveRadioactivewastedisposal;costofSPAbeadsLanthaScreenTR-FRETEuropiumorTerbium-chelateddonorlinkedwithanti-phosphoantibody.Fluorophore-labelledpeptideasacceptorSensitive,highthroughputAntibody-dependent,highcostofreagentsLanceUltraAlphaScreenLuminescencePhospho-peptidebringsdonorandacceptorbeadstoproximitythroughcoatedconjugates.Uponexcitation,donorbeadexcitesoxygenwhichreactswithacceptorbeadgeneratingsignalHighlysensitive,canuseproteinorpeptidesubstrateCostofreagents;antibody-dependent,orsusceptibletophosphateinterferenceIMAP-FPFluorescencepolarization(FP)Fluorophore-labeledphospho-peptidebindstometalcoatedbeadsandgenerateshighFPsignalSimpleworkflow,multiplexableCompoundinterference;substratemustbeshortpeptidePhosphoSensFluorescence(FI)Phospho-peptidebringsMg2+totheproximityoflabeledfluorophore(SOX)whichgeneratesenhancedfluorescencewhenboundtotheMg2+Continuousassay,highATPtolerance,highthroughputCompoundinterference;substratemustbeshortpeptideMobilityShiftAssayTime-gatedfluorescencedetectionofsubstrateandproductduetotheirdifferentelectrophoreticmobilitieslowsampleconsumption,easeofautomationCompoundinterference;substratemustbeshortpeptidewithsinglephospho-siteADPADP-GloAssayLuminescenceQuantificationofkinasedependentADPproductionbyconvertingittoATPandATPismeasuredinaluciferasereactionHighlysensitive,universal,label-free,highATPtoleranceCouplingenzymesmayproducefalsepositives;ATPaseinterferenceTranscreenerFP/Fl/TR-FRETADPfromthekinasereactionreplaceswithfluorophore-labeledADPboundtoanti-ADPantibodyVersatile,multipledetectionmodesAntibody-dependent,highcostofreagents;non-specificrecognitionAdaptaTR-FRETEuropium-chelateddonorlinkedwithanti-ADPantibody.Fluorophore-labelledADPasacceptorSensitive,highthroughputATPKinase-GloAssayLuminescenceATP-dependentluminescentsignalfromluciferaseconversionofluciferin.KinasedependentdepletionofATPisquantifiedSimpleworkflow,inexpensive,universalSignalinversecorrelation-smallassaywindowEasyLite04SinoBiological,Inc.SignalChemassayplatformsandkinaseassaycasestudiesSinoBiological,Inc.27SignalChemactiveenzymeproductsCategoryProductGroup#ofProductsActiveenzymesKinases1163Phosphatases40EpigeneticEnzymes132UbiquitinEnzymes98ArginineDeiminases7GProteins52Proteases3Phosphodiesterases23OtherEnzymes–Oxidoreductases,Transferases,Hydrolases19TotalProducts1537AssaytechnologyAssaynameAnalyteProteinfamilyLuminescenceADP-GloADPKinaseAMP-GloAMPUbiquitinatingenzyme(E1,E2,E3)MTase-GloS-AdenosylHomocysteine(SAH)MethyltransferaseGTPase-GloGTPGTPaseHDAC-GloLuciferinHistonedeacetylasePDE-GlocAMP,cGMPPhosphodiesteraseROS-GloH2O2HistonedemethylaseSuccinate-GloSuccinateDUBLitLuciferinDeubiquitinaseUV-VisabsorbanceBIOMOLGreenPhosphatePhosphatasepNAcolorimetricassayp-NitroanilideProteaseFluorescence/TR-FRETCleavageassay,LanthaScreenFluorescent-orphospho-peptideProtease,kinaseImmunoassayELISAAcetylatedorcitrullinatedpeptideBromodomain,acetyltransferase,argininedeiminasePROTACassayUbiquitinatedproteinUbiquitinE3ligaseSinoBiological,Inc.29CompoundscreeningIdentifythroughcompoundlibraryscreenings,candidatesthataffectthetargetinthedesiredway,so-called“hits”,or“leads”Developingcompoundscreeningassaysrequires:IdentificationofrelevantenzymeandsubstrateformsMeasurementsofkineticparametersCharacterizationofco-factorsandbuffersChoiceofdetectiontechnologyModeofaction(MOA)studyADP-Glokinaseassay-casestudiesSinoBiological,Inc.30EnzymekineticsStreamlinedinhibitorprofilingModeofactionSinoBiological,Inc.31Determinationofkineticparameters

ZegzoutiHetal.2013SinoBiological,Inc.3270nM2694nM396nM5nM7nM4.8nMPKAATPcompetitiveinhibitorH-89PKAATPnon-competitiveinhibitorPKIZegzoutiHetal.2010ATPcompetitivevsnon-competitiveinhibitionSinoBiological,Inc.33HennekJetal.2016KinaseSelectivityProfilingSystems(KSPS)SinoBiological,Inc.34KSPS:standardizedkinaseactivitiesSinoBiological,Inc.35KSPS:single-dose&dose-responseprofilesSinoBiological,Inc.3