cellular-thermal-shift-assay

DOI:10.1126/science.1233606,84(2013);341Scienceetal.DanielMartinezMolinaCellularThermalShiftAssayMonitoringDrugTargetEngagementinCellsandTissuesUsingtheThiscopyisforyourpersonal,non-commercialuseonly.clickinghere.colleagues,clients,orcustomersby,youcanorderhigh-qualitycopiesforyourIfyouwishtodistributethisarticletoothers here.followingtheguidelinescanbeobtainedbyPermissiontorepublishorrepurposearticlesorportionsofarticles ):July23,2013(thisinformationiscurrentasofThefollowingresourcesrelatedtothisarticleareavailableonlineat:includinghigh-resolutionfigures,canbefoundintheonlineUpdatedinformationandservices,:SupportingOnlineMaterial:canberelatedtothisarticleAlistofselectedadditionalarticlesontheScienceWebsites:cites42articlesThisarticle:ThisarticleappearsinthefollowingregisteredtrademarkofAAAS.isaScience2013bytheAmericanAssociationfortheAdvancementofScience;allrightsreserved.ThetitleCopyrightAmericanAssociationfortheAdvancementofScience,1200NewYorkAvenueNW,Washington,DC20005.(printISSN0036-8075;onlineISSN1095-9203)ispublishedweekly,exceptthelastweekinDecember,bytheScienceonJuly23,2013(Berlin,Germany)viatheJointBerlinMX-LaboratorysponsoredbytheHelmholtzZentrumBerlinfürMaterialienundEnergie,theFreieUniversitätBerlin,theHumboldt-UniversitätzuBerlin,theMax-Delbrück-Centrum,andtheLeibniz-InstitutfürMolekularePharmakologie.ThisworkwassupportedbytheDeutscheForschungsgemeinschaft(grantsSFB860toR.L.andSFB740/2toM.C.W.).S.M.J.wassupportedbyascholarshipfromtheInternationalMaxPlanckResearchSchoolProgramforMolecularBiology.CoordinatesandstructurefactorshavebeendepositedintheResearchCollaboratoryforStructuralBioinformaticsProteinDataBank()withaccessioncode4KIT.SupplementaryMaterials(22–31)7March2013;accepted9May2013Publishedonline23May2013;10.1126/science.1237515MonitoringDrugTargetEngagementinCellsandTissuesUsingtheCellularThermalShiftAssayDanielMartinezMolina,1*RozbehJafari,1*MarinaIgnatushchenko,1*TakahiroSeki,2E.AndreasLarsson,3ChenDan,3LekshmySreekumar,3YihaiCao,2,4PärNordlund1,3†Theefficacyoftherapeuticsisdependentonadrugbindingtoitscognatetarget.Optimizationoftargetengagementbydrugsincellsisoftenchallenging,becausedrugbindingcannotbemonitoredinsidecells.Wehavedevelopedamethodforevaluatingdrugbindingtotargetproteinsincellsandtissuesamples.Thiscellularthermalshiftassay(CETSA)isbasedonthebiophysicalprincipleofligand-inducedthermalstabilizationoftargetproteins.Usingthisassay,wevalidateddrugbindingforasetofimportantclinicaltargetsandmonitoredprocessesofdrugtransportandactivation,off-targeteffectsanddrugresistanceincancercelllines,aswellasdrugdistributionintissues.CETSAislikelytobecomeavaluabletoolforthevalidationandoptimizationofdrugtargetengagement.Drugdevelopmentfacesmultiplechal-lengesthatleadtohighcostsandlongdevelopmentcyclesfornewtherapeutics(1–3);meanwhile,insightsintomolecular,cellu-larandphysiologicalprocesseshaveidentifiedalargenumberofproteinsaspotentialdrugtargets(4).Therefore,methodsthatpromoteaccelerateddrugdevelopmentareurgentlyneeded.Thetherapeuticeffectofmostclinicallyavail-abledrugsisachievedthroughdirectbindingofthedrugtooneorafewtargetproteins.Thisbindingtypicallyoccursatafunctionalsiteoftheproteinandhaseitheranactivatingorin-hibitoryeffect.Theresultingmodulationofpro-teinactivityinthecontextofcellsandtissuesleadstothedesiredmolecular,cellular,andphysiologicalresponses.Theefficacyofadrugiscriticallydependentontheextentofitstar-getengagement,andadverseeffectsareoftenduetoexcessivebindingofthedrugintoxicity-pronecellsoritsoff-targetbindingtootherproteins.Targetengagementbyadrugincellsortis-suesisdeterminedbyitslocalconcentrationandbindingaffinity.Theeffectivedrugcon-centrationatthetargetdependsonpropertiescollectivelyreferredtoasADME(absorption,distribution,metabolismandexcretion),whichdictatethepharmacokineticsandpharmaco-1DepartmentofMedicalBiochemistryandBiophysics,KarolinskaInstitute,Scheelesväg2,SE-17177Stockholm,Sweden.2De-partmentofMicrobiology,TumorandCellBiology,KarolinskaInstitute,Nobelsväg16,SE-17177Stockholm,Sweden.3SchoolofBiologicalSciences,NanyangTechnologicalUniversity,61NanyangDrive,Singapore639798,Singapore.4DepartmentofMedicineandHealthSciences,LinköpingUniversity,58183Linköping,Sweden.*Theseauthorscontributedequallytothiswork.†Correspondingauthor.E-mail:[e-mail]ETSAinlysat