義翹講堂《重組激酶全方位解析：高效生產(chǎn)、嚴(yán)格質(zhì)控與前沿應(yīng)用》

Recombinantkinaseproduction,qualitycontrolandapplicationsYuxiangHu,MDChiefScientificOfficerSinoBiological/SignalChemBiotechOctober2024Backgroundinformationofkinases1Manufacturingprocessofrecombinantkinases2Qualitycontrol3Recombinantkinaseapplications4CONTENTSinoBiological,Inc.Backgroundinformationofkinases01SinoBiological,Inc.TheWorldLeaderinKinases20+YearsofExpertiseinEnzymeandAssayDevelopmentEnzymes,Proteins,Compounds,Substrates,AssayReagents,andCustomServicesFoundedin2004LocatedinRichmond,BC,CanadaAcquiredbySinoBiologicalin2024SignalChemBiotech(PartofSinoBiological)SinoBiological,Inc.SignalChemactiveenzymeproductsCategoryProductGroup#ofProductsActiveEnzymesKinases1163Phosphatases40EpigeneticEnzymes132UbiquitinEnzymes98ArginineDeiminases7GProteins52Proteases3Phosphodiesterases23OtherEnzymes–Oxidoreductases,Transferases,Hydrolases19TotalProducts1537SinoBiological,Inc.Kinasekinome90%kinomecoverage700wild-typevariants400disease-relevantmutantkinasesSinoBiological,Inc.KinaseSubstrateProductPATPADPProteins

Lipids

Carbohydrates

SinoBiological,Inc.Serine/ThreonineproteinkinasesKinasesthatphosphorylateserineand/orthreonineresiduesonsubstratesKinasesthatphosphorylatetyrosineresiduesonsubstratesKinasesthatphosphorylatebothserine/threonineandtyrosineresiduesTyrosinekinasesDualspecificityproteinkinasesPKA,AKT,CDKs,MAPKs…EGFR,FGFR,SRC,ABL1…MEK1,DYRK3…ATPMainfunctionsofkinasesFunctionsDescriptionExamplesofKinasesSignalTransductionMediatescellularresponsestoexternalsignalsTyrosineKinases(e.g.,EGFR)CellCycleRegulationControlsprogressionthroughthecellcycleCyclin-dependentKinases(CDKs)MetabolismRegulationInfluencesmetabolicpathwaysandenergyproductionAMPKGeneExpressionModulationAlterstranscriptionfactorstoinfluencegeneexpressionVariousSerine/ThreonineKinasesCellGrowthandDifferentiationAffectsprocessesrelatedtocellgrowthanddevelopmentMAPKs,TyrosineKinasesChengHCetal.(2011).EnzymeRes,2011:794089ArditoFetal.(2017).IntJMolMed,40:271-280SinoBiological,Inc.CardiovasculardisordersDigestivesystemdisordersEyedisordersImmunesystemdisordersInfectiousdiseasesMetabolicdisordersNervoussystemdisordersOtherdisordersRenaldisordersRespiratorytractdiseasesCancersNumberofagentspercategoryandhighestreachedphaseAttwoodMMetal.(2021).NatRevDrugDiscov,20:289AbnormalkinasefunctionsareassociatedwithvariousdiseasesSinoBiological,Inc.Manufacturingprocessofrecombinantkinases02SinoBiological,Inc.ATPManufacturingprocessofrecombinant

kinasesProteinPlasmid/VirusExpressionVectorsExpressionEvaluation&OptimizationBacterial/Yeast/Insect/MammalianCellCultureProteinPurificationAffinityChromatography,IonExchange,GelFiltrationQC/QASDS,ActivityAssaysGeneSynthesis&SubcloningPrimerDesignCloningSequencingPackage&DeliveryStorageShippingSinoBiological,Inc.ATPWepreferinsectcellsforkinaseexpressionSf9,Sf21,High5Insect1.PTMs2.SolubleProteins3.HighExpressionCHO,HEKMammalian1.PTMs2.SolubleProteins3.Lowerexpression4.ExpensiveS.CerevisieaeP.PastorisYeast1.PTMs2.SolubleProteins3.HighExpression4.EasyScaleupE.coliBacteria1.“WorkHorse”2.WellEstablished3.HighExpression4.SimpleGenetics5.EasyScaleup7.LowCostSinoBiological,Inc.ATPHarvestExtractionCentrifuga-tionChromatographicMethodsDetection&AnalysisThewholeprocessiskeptoniceor4°Ctoretainactivity!ProteinpurificationSinoBiological,Inc.Qualitycontrol03SinoBiological,Inc.SDSgelrunningSizeConcentrationPurityActivitytestADP-GloOtherassaysERK2BSANewbatchExistingbatch(Internal)QualitycontrolsSinoBiological,Inc.(Datasheet)ReactionBiologywebsiteFilterbindingradiometrickinaseassayDirectdetectionofradioactivelabeledsubstrate,highspecificity,highsensitivity.Requireswashingstep,radioactivewaste,alsoneedsapositivelychargedsubstrate.SinoBiological,Inc.AhomogeneousluminescentassaytodetectADP(rangeof~1mM–0.01μM)(nowashingstep)Highsensitivity:Detect0.2pmol

ADPwithmorethan2-folddi?erenceoverthebackgroundAnyADP-generatingenzyme(e.g.,kinaseorATPase)usingupto1mMATPAlsodetectkinasewhichhasautophosphalizationeffectADP-GlokinaseassayPromegawebsiteSinoBiological,Inc.ADP-GlokinaseassayprotocolATPValidationofactivityandlot-to-lotconsistencySinoBiological,Inc.Recombinantkinaseapplications04SinoBiological,Inc.ATP>1800PUBLICATIONSINHIGHIMPACTJOURNALSSinoBiological,Inc.Pray,L(2008).NatureEducation,1(1):37ArditoFetal(2017).IntJMolMed40:271ThefirstsmallmoleculekinaseinhibitorapprovedbyFDA(2001)Gleevec(imatinib)isthefirstkinaseinhibitorapprovedbytheFDA.TargetingBCR-ABLfusionkinasefromtheinitialstudy.ItisalsoreportedtotargetPI3K,BRAF,EGFRandmTOR.Usedfortreatingchronicmyelogenousleukemia(CML),andlaterforseveraltypesofothercancerssuchasALLandgastrointestinaltumors(GIST),melanoma,lungcancerandrenaltumors.“Thebeginningofaneweraofprecisionmedicine”SinoBiological,Inc.FDAapproveddrugsandmaintargetedkinases/protein-kinase-inhibitors/BrianAetal.(2023).BiochemicalJournal480:1331SinceImatinib’sapproval,over80kinaseinhibitorshavebeenapprovedasofSeptember2024.Inthelast6years,atleast5kinaseinhibitorswereapprovedbytheFDAeachyear.Thedataprovidesacleardemonstrationofthedruggabilityofthekinaseproteinclass.SinoBiological,Inc.Developmentofin-houseAXLinhibitor:SLC-391

SinoBiological,Inc.Identifiedbyhigh-throughputscreeningbasedontheplatformusingin-housekinasesSpecificallyandpotentlyinhibitsAXLSignalinginvitroandinvivoDesirablepharmacokineticpropertiesAnti-cancerpropertiesinmiceCurrentlyatclinicaltrialphaseIIBiswasetal.(2024).Chem.Sci.15:13130Themajorchallengeusingsmallmoleculekinaseinhibitorsistheemergenceofdrugresistance.AcquiredkinasemutationSinoBiological,Inc.Acquiredmutationswithinthekinasedomainareacommondrugresistancemechanism.VariousEGFRmutantsandacquiredEGFRmutationsSinoBiological,Inc.Sharmaetal.(2007).NatRevCancer,7(3):169-181EGFRkinasemutationsanddrugsensitivityKooijmanetal.(2022).Front.Oncol.,12:953013Thefirst(gefitinib)andsecond(dacomitinib)generationEGFRinhibitorsaresensitivetoEGFR(WT)andtheexon19deletion(ex19del).TheT790Mmutationisthemostcommonlyobservedmechanismofresistance.ThethirdgenerationEGFRinhibitor(Osimertinib)isspecificallydesignedtotargettheT790Mmutation.SinoBiological,Inc.FGFRkinasemutationsanddrugsensitivityKooijmanetal.(2022).Front.Oncol.,12:953013ThesensitivityofFGFRinhibitorsvariesbetweenWTandmutantforms.AcquiredresistancemutationsareacommonresistancemechanismtoFGFRinhibitors.SinoBiological,Inc.StructuresofrecombinantkinasesindrugdesignBaiX.C.,etal.(2015).NatureMethods,12(1):29-35BrianAetal.(2023).BiochemicalJournal480:1331SinoBiological,Inc.Requirehighlypurerecombinantkinasesforstructurestudies.BothX-raycrystallographyandcryo-electronmicroscopy(Cryo-EM)aresuitabletechniques.Over300uniquekinasestructureshavebeendeterminedandareavailableintheProteinDataBank(PDB).Structureanalysisisessentialfordesigningnewdrugs,andoptimizingtheirpropertiesforimprovedefficacyandselectivity.Examplesofkinasestructure-baseddrugdevelopmentCompoundNameTargetKinase(s)TherapeuticIndicationsNotableStructuralInsightsReferencesImatinib(Gleevec)BCR-ABLChronicmyeloidleukemiaDesignedusingthestructureoftheABLkinasedomainDruker,B.J.etal.(2001).NEnglJMed,344(14):1031-1037.Gefitinib(Iressa)EGFRNon-smallcelllungcancerBasedonthestructureoftheEGFRkinasedomainHynes,N.E.,&Lane,H.A.(2005).NatRevCancer,5(4):341-354.AZD9291(Osimertinib)EGFRNon-smallcelllungcancerTargetingresistancemutationsusingstructuralinsightsYu,H.A.etal.(2013).CancerDiscov,3(12):1404-1411.Dasatinib(Sprycel)BCR-ABL,SrcfamilykinasesChronicmyeloidleukemia,othercancersDesignedusinginsightsfromBCR-ABLandothertyrosinekinasesShah,N.P.etal.(2004).CancerCell,6(3):313-325.Lapatinib(Tykerb)EGFR,HER2BreastcancerDevelopedfromstructuraldataofEGFRandHER2kinasesGeyer,C.E.etal.(2006).JClinOncol,24(18):2786-2792.SinoBiological,Inc.ApplicationareasDescriptionReferencesResearchtoolsStudysignalingpathways,cellularprocesses,andproteininteractionsindetail.Zhangetal.(2016).NatRevDrugDiscov,15(6):379-396DrugdevelopmentHelpinscreeningpotentialkinaseinhibitorsfortherapeuticuse.Cohen(2002).NatRevDrugDiscov,1(2):123-124.StructurestudiesHelptoelucidatemechanismsandguidedrugdesignefforts.RCSBPDB.(n.d.).RetrievedfromRCSBPDB.Billeteretal.(2005).CurrOpinStructBiol,15(1),:24-30.AntibodyproductionUsedasantigenstoproduceantibodiesfordiagnosticsandtreatmentofdiseases.Lee,T.etal.(2019)NatureReviewsImm