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Kinase Screening and Profilin Techniques

To assist the efforts in kinase drug discovery, Creative Bioarray offers high quality, time efficient and cost effective kinase solution for assay development, high throughput screening (HTS) and selectivity profiling.<br><br>https://dda.creative-bioarray.com/kinase-assay.html

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Kinase Screening and Profilin Techniques

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  1. Creative Bioarray DDA Platform - Drug Discovery Accelerator Kinase Screening and Profiling Boostyourkinaseresearchwiththemostcomprehensivesetofassaytechniques INTRODUCTION Kinasesarearguablyoneofthemostimportantdrugtargetsduetotherolestheirdysregulation, mutations,andoverexpressionplayinallmajorpathologies,includingmetabolic,cardiovascular,degen- erative,inflammatory,andinfectiousdiseases.Findingthebestmethodforkinasescreeningandprofiling canbeachallengingtaskandwilldependonyourresearchobjective,thetypeofkinase,availabilityofan antibody,thesizeofthekinasesubstrateandtheanalyticalinstrumentsavailable.Creative Bioarrayprovides acomprehensivesetofassaytechnologiestosolveallofyourresearchobjectives - fromtargetdiscoveryto compoundscreening,profilingandhitconfirmation.Ourbreadthofexpertiseandexperienceinkinase research enablesourscientiststosupportyoueverystepofthewaythroughthedrugdiscoveryprocess. KINASE ASSAYS Therearealargenumberofapproachestoevaluatekinasefunctionandactivity.Inrecentyears,severaldifferent typesofkinaseassaytechnologyhavebeendeveloped,eachofwhichhavetheirrelativemeritsanddrawbacks. RADIOMETRIC ASSAY Radiometrickinaseassayusing32P-labelledATPor33P-labelledATPisoneoftheoldesttechniquesusedtostudy proteinphosphorylation.TheyrelyonthetransferofradioactivephosphategroupsfromATPto the substrateby akinase,allowingdetectionofactivity.Theradiolabeledphosphateincorporated intothe substrateisdirectlyproportionaltothekinaseactivity. Radiometricassayformatsarethegoldstandardforkinasescreening,whichcanproduce reproducible,high-qualitydataanddirectlymeasureenzymeactivity.Buttheyarelabor intensiveandrequiresafedisposalofradioactivematerials,sotheyareusuallynot suitableforhigh-throughputscreening.

  2. Fluorescence Resonance Energy Transfer (FRET) Assay Fluorescenceresonanceenergytransfer(FRET)involvesthetransferofnon-radiativeenergyfromadonorfluorophoretoa close-proximityacceptorfluorophore.Whenthedonorandacceptorarespatiallyclosetoeachother,theacceptormolecule willquenchanyfluorescenceemissionfromthedonormolecule.ThefirststepoftheFRETkinaseassayistoincubatethe FRETlabeledsubstratewithATPandkinase,resultinginthetransferofγ-phosphatetothesubstratepeptide.Inthesecond reactionstep,asite-specificproteaseisaddedtothemixture.Iftheproteasecleavesanon-phosphorylatedpeptide,the fluorescentdonorandacceptorwillbesufficientlyseparatedtodisrupttheFRET,thusgeneratingafluorescentsignalthat canbemeasuredasaratioofdonoremissiontoacceptoremission. TheadvantagesofFRETareitshomogenousformatandsimpleapplicationtoHTS.However,thepeptidesubstratedesign maybedifficulttooptimizeasthedonorandacceptorfluoro-phoresneedtobewithinawell-defineddistancetotransfer energy. TIME RESOLVED FLUORESCENCE (TRF) Time-resolvedfluorescence(TRF)usesfluorophoreswithalongdecaytime,andmonitorsthefluorescenceasafunctionof timeafterexcitationbyaflashoflight.Lanthanideions,suchaseuropium,samarium,andterbium,areoftenusedinthis technologyduetotheirlongeremissionlifetimes(hundredsofmicrosecondscomparedtoseveralnanosecondsfor conventionalorganicfluorophores). Theadvantageofthistechnologyisthatcontaminatingfluorescentmoleculeswhichcontributetobackgroundnoisehavea shorterdecaytimethanlanthanideions,sothesignalismuchcleaner.ThereareseveralcommerciallyavailableTRFassays, mainlybasedeitheronELISAassayorcombinedwithFRETassay. FLUORESCENCE POLARIZATION (FP) ASSAY Fluorescencepolarization(FP),atechniquethatmonitorsmolecularmovementandrotation,isawidelyuseddetection methodforkinaseinhibitorsinHTS.Theprincipleofthisassayisthatsmallermoleculesrotatefasterthanlargermolecules. Afluorescentlylabeledpeptidesubstrateinthesolutionrotatesquicklyandsowillhavealowfluorescencepolarization. WhenthephosphorylationreactionisinitiatedbyadditionofakinaseandATP,anyphosphorylatedpeptidesubstrateswill berecognizedbythephospho-antibody.Thebindingoftheantibodywillslowdowntherotationofthemolecule(high fluorescencepolarization),andthischangeinspeedcanbemeasured. Sincethisisaone-stepassaywithoutadditionalwashingsteps,itisverysuitableforscreeningalargenumberofkinase inhibitorcandidates.However,oneofthemajordrawbacksforthisandotherfluorescence-basedassays,isthatunused fluorescentcompoundsandlabeledsubstratesmayproducehighbackgroundsignalsandfalsepositives. LUMINESCENCE DETECTION Luminescence-basedassayusesfireflyproteinluciferasetomeasuretheamountofATP.InthepresenceofATP,luciferase convertsthesubstrateluciferinintooxyluciferin,whichreleasesyellow-greenphotonoflightwithaspectralmaximumof 560nm.AlthoughthisformatshowslowsensitivityatlowATPconcentrations,thiseffectcanbecounteractedbyusinga two-stepdetectionmethodincluding1)stoppingthekinasereactionanddepletingtheremainingATP,and2)adding reagentstoconvertADPtoATP,whichismeasuredusingthecoupledluciferasereaction.Luminescencedetectionmethods canaccommodatefluorescentcompounds,buttheinhibitoryeffectofthecompoundsonluciferasemustbeconsidered.

  3. MOBILITY SHIFT ASSAY Themobilityshiftassaytakesadvantageofthefactthataphosphorylatedpeptidesubstrateismorenegativelycharged thanthesamesubstrateintheunphosphorylatedstate.Therefore,whenamixtureofthesepeptidesissubjectedtogel electrophoresis,thephosphorylatedandunphosphorylatedsubstrateswillhavedifferentmobility. Sincethedifferenceinchargebetweenthepeptideandthesubstrateiskey,theresultofmobilityshiftassayisgreatly affectedbythesizeandsequenceofthesubstrate.Therefore,thisassayisnotsuitablefortheanalysisoflargerpeptidesor wholeprotein,andworksbestwhenthepeptidesubstratecontainsonlyspecificphosphorylationsitesequences. CELL-BASED ELISA ASSAY Cell-basedfunctionalkinasescreeningprovidesareliable,quantitativemeanfordeterminingthefunctionalinhibitoryeffects ofdrugcandidatesonkinasetargetsofinterestinaphysiologicallyrelevantcellularenvironment.Thisallowsforamore predictivecharacterizationoftheorganism’sphysiologicalresponsetothedrug,andgreatlyreducesthechancesoffalse positive/negativedata. Enzyme-linkedimmunosorbentassay(ELISA)canbeusedtoquantitatekinaselevelsandassesstheiractivity.Inthisformat, thesubstrateiscapturedbythemembraneanddetectedbyaspecificanti-phosphorylatedsubstrateantibody.The detectioncanalsobeverysensitivewhenusingantibodieslabeledwithhighlyfluorescentdyes. ELISAispopularbecauseitisgenerallyfast,inexpensive,easytoaccommodatemultiplesamples,anddoesnotrequirehighly specializedequipment.However,therequirementofseparationwithmultiplewashingstepslimitstheuseofELISAinHTS.In addition,thedevelopmentofhigh-qualityphospho-specificantibodiespresentsanotherchallenge. CONCLUSION Alargenumberofcommerciallyavailablekinaseassayshavebeendevelopedinrecentyearsbasedonthetechnologies describedabove.Eachtechnologyhasitsstrengthsandweaknesses,andthemostappropriateassaywilldependontheaim oftheproject.Forhigh-throughputscreeningdrugdiscoveryprojects,methodsthatcanbeeasilyautomatedand miniaturized,suchasthefluorescence-basedassays,arethemostsuitable.Forprojectsthatrequireamorein-depthprofile ofthekinasefunction,amorerobustassaysuchasradiometricassaywillprovidemorereliableresults. REFERENCES: 1.Zegzouti,H.Goueli,S.A.KinaseScreeningandProfiling.MethodsinMolecularBiology,2016. 2.Ma,H.etal.;Thechallengeofselectingproteinkinaseassaysforleaddiscoveryoptimization.ExpertOpiniononDrug Discovery,2008,3(6):607-621. 3.Li,Y.etal.;Fluorescencedetectiontechniquesforproteinkinaseassay.AnalyticalandBioanalyticalChemistry,2008, 390(8):2049-2057. 4.Ishida,A.etal.;RecentAdvancesinTechnologiesforAnalyzingProteinKinases.JournalofPharmacologicalSciences, 2007,103(1):5-11. 5.WuJ.J.ComparisonofSPA,FRET,andFPforkinaseassays.MethodsMolBiol.2002,190:65-85.

  4. Creative Bioarray DDA Platform - Drug Discovery Accelerator USA EUROPE COMPANY: Creative Bioarray PHONE: 1-631-626-9181 FAX: 1-631-614-7828 ADDRESS: 45-1 Ramsey Road, Shirley, NY 11967, USA EMAIL: info@creative-bioarray.com COMPANY: Creative Bioarray PHONE: 44-208-144-6005 EMAIL: info@creative-bioarray.com

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