SPR Assay Services for Drug Discovery - Reaction Biology

SPR Binding Assay Principle ... SPR analysis is an optical method measuring changes in the mass of biomolecules immobilized on a metal film. Upon binding an ... Skiptomaincontent Breadcrumb Home Services BiophysicalAssays SurfacePlasmonResonance(SPR) SPRAssayServicesforDrugDiscovery SurfacePlasmonResonance(SPR)isahighlysensitivetechniqueforaccurateanalysisoftheinteractionsoftwobiomoleculeswithrespecttobindingkineticsandaffinityaswellasbindingspecificity. ToperformSPRassays,ReactionBiologyisequippedwithtwostate-of-the-artBiacore8Kandtwo8K+instrumentswithhigh-throughputcompoundscreeningcapabilityandextraordinarydetectionsensitivity.Weperformhigh-throughputfragmentscreening,kineticsandaffinitydetermination,bindingspecificityprofilingandantibodycharacterization,etc. ReactionBiologyprovidesproject-tailoredsolutionstoassurethehighestchanceofsuccessinbiomolecularinteractionresearchusingSPRservicesbyusingabroadbiophysicsknowledgebaseandexcellentinstrumentcoverage.      TheSPRbindingassayissuitabletoadvanceanyanalyteincludingfragments,antibodies,peptides,nucleicacidsagainstanytargetclassincludingenzymesandnon-activeproteins SARS-CoV-2SproteinandACE2receptorbindingassayisavailablehere Deliverables:associationrateconstantkon,dissociationrateconstantkoff,bindingaffinityKD OurexpertshaveextensiveexpertiseinanalyzingandresolvingSPRchallengeswith‘difficulttotest'proteins.TheassayiscarriedoutatMalvern,Pennsylvania,intheUnitedStatesandperformedonafirst-come,first-servebasis. Contactanexpert Subscribetoournewsletter Meetusatyournextconference Share SPRBindingAssayPrinciple SPRanalysisisanopticalmethodmeasuringchangesinthemassofbiomoleculesimmobilizedonametalfilm.Uponbindingananalyte,therefractiveindexofthemetalfilmchanges,resultinginachangedreflectionangleoflight(thesurfaceplasmonresonancephenomenon).SPRbindingassaysarewidelyusedindrugdiscovery,fromfragmentscreeningthroughanalyterankingandin-depthkineticcharacterization. ListofSPRAssays Target HGNCSymbol Synonyms Data ACE2 ACE2 AngiotensinIconvertingenzyme2 Pleaseinquire Bcl-2-likeprotein1isoformL BCL2L1 BCL-XL Datasheet BRD4-1 BRD4 Bromodomaincontaining4,domain1 Datasheet BRD4-2 BRD4 Bromodomaincontaining4,domain2 Datasheet cGAS CGAS CyclicGMP-AMPsynthase,fragment Datasheet cGAS(AA157-522) CGAS CyclicGMP-AMPsynthase,fragment Datasheet G9a EHMT2 Histone-lysineN-methyltransferaseEHMT2,Euchromatichistone-lysineN-methyltransferase2,HLA-B-associatedtranscript8,HistoneH3-K9methyltransferase3(H3-K9-HMTase3),LysineN-methyltransferase1C Datasheet K-Ras KRAS C-K-RAS,CFC2,K-RAS2A,K-RAS2B,K-RAS4A,K-RAS4B,KI-RAS,KRAS1,KRAS2,NS,NS3,RASK2 Datasheet NSD2-SET NSD2 SETdomainofNSD2 Datasheet PRMT5/MEP50 PRMT5/WDR77complex Proteinargininemethyltransferase5,WDrepeatdomain77 Datasheet SARS-CoV-2Sprotein-ACE2interaction n.a. Spikeglycoprotein,receptorbindingdomain;AngiotensinIconvertingenzyme2 Sproteinwebpage SHP2 PTPN11 Tyrosine-proteinphosphatasenon-receptortype11,Protein-tyrosinephosphatase1D(PTP-1D),Protein-tyrosinephosphatase2C(PTP-2C),SH-PTP2,SH-PTP3 Datasheet SOS1 SOS1 SOSRas/Racguaninenucleotideexchangefactor1,sonofsevenlesshomolog1 Datasheet WildtypeEGFR(AA671-998) EGFR Epidermalgrowthfactorreceptor,Proto-oncogenec-ErbB-1,Receptortyrosine-proteinkinaseerbB-1 Datasheet AdditionalInformationforSPRBindingAssays Kineticsanalysis Kineticprofileofananalyte-targetbindingreaction. SPRdetectschangesintherefractiveindexatthesurfaceofasensorchipasaresultofmolecularmasschangesofatargetuponbindingoftheanalyte.Thetargetisimmobilizedtothesurfaceofthesensorchip.Duringtheassociationphase,theanalyteflowsoverthesurface,andbindingtothetargetismonitored.Theflowthenswitchestotherunningbuffer,andthedissociationoftheanalytefromthetargetismonitored. Example:Co-factoranaylsis Exampleofaco-factoranalysisbySPR. Inthisexamplestudy,weinvestigatedasubstratecompetitiveinhibitorthatbindstoitstargetenzyme,PRMT5/MEP50,onlyinthepresenceofSAMorSAManalogssuchasMTAandSAH.Nodose-dependentresponseswereobservedforanalytebindingtoapoprotein(leftfigure).ThebindingtotheMTA-boundtargetisrelativelyweak(KD~20µM)withfastkinetics(on/off).Thebindingaffinityincreasedby~10-foldfortheSAH-boundtarget(KD~2µM).Whiletheon-ratesaresimilarfortheMTA-andSAH-boundconditions,theoff-ratesareapproximately100Xslower.Thehighestaffinity(KD~3nM)andslowestoff-rate(100xlessthanSAH-bound)wasobservedforanalytebindingtotheSAM-boundtarget.Single-cyclekinetics,thatdonotrequireareturntobaselineinbetweendoses,wasusedduetotheslowoff-rateobservedforthiscondition.Asloweroff-rateindicateslongeroccupancyoftheanalyteonthetarget. Theanalytewastestedwith7concentrationsdepictedindifferentcolors.  Example:Bindingaffinity Exampleofbindingaffinitydeterminationofaninhibitortotwobromodomains. TheBD1orBD2domainofBRD4wereimmobilizedtothesensorchipsurface.Theanalytewasappliedtothechipsurfaceinincreasingconcentrationsapproachingsaturation. Rightgraph:Therelativeresponseatequilibriumforeachdosewasplottedagainsttheanalyteconcentrationtodeterminetheequilibriumdissociationconstant,KD.Theanalyteis10-timesmoreselectiveforBD2thanforBD1. ScreeningDetails Instruments:ReactionBiologyisequippedwithtwostate-of-the-artBiacore8Kunitsandone8K+unitallowinghigh-throughputscreeningwith8channels(4600compoundsperday)withhighsensitivity. Turnaroundtime:Westrivefora2weekdevelopmenttimeforassayswithanewtargetforscreeningand2weeksforthescreeningwithanestablishedassay.Projectsareperformedonafirst-come-first-servebasis. Screeninglocation:TheassayisperformedinMalvern,PA,USA. Samplerequirements:Thetargetcanbeprovidedbytheclient,beoneofouroff-the-shelfproteins,orbecustom-madetotherequirementsoftheclientandassay.Apurityof90%ormoreisrecommended. PleaseseeourFAQforshippinginstructions. AssayDevelopment ExampleSPRworkflowofacustomizedanalyte-targetbindingstudy: Sensorchippreparation:Attachingthetargettothesensorchip Assayvalidation:Testingthebehaviorofthetargetonthesensorchip Assayreproducibility:Testingiftheoptimizedconditionsarereproducible Proteinactivityandstabilityonthesensorchip Analytetesting Thebindingofthetestanalyte(s)ismeasured Forkinetics/affinitydeterminationeithera10concentrationmulti-cycleor5concentrationsingle-cyclemeasurementwillbeused(dependentupontheoff-ratesandregenerationconditions) Forscreeningstudies,asingleconcentrationmeasurementcanbeusedforanalyteranking Application SPR–aversatiletooltoaddressmanychallenges Targetisnotanenzymeorunknownsubstrate SPRmeasuresthedirectbindingbetweenanalyteandtargetwhichiswhythetargetdoesnotneedtobeanenzymeandnosubstrateisneeded. Co-factor/competitionstudies Theimpactofvariousco-factorsonanalyte-targetinteractioncanbetested. Fragment-basedscreening Lowmolecularmassfragmentcompounds(100–300 Da)tendtodemonstratelowbindingaffinity;thus,thecompoundsrequirescreeningatahighconcentration.ThisisbettertoleratedintheSPRplatformthaninmanybiochemicalassays. Antibodyscreening/ characterization SPRcanbeusedforantibodyaffinitydetermination,determinationofkineticparameters,epitopemapping,bindingspecificity,andcross-reactivity. SARstudies Thekineticsofdrugbindingandunbinding,especiallytheresidencetime,playacrucialroleinadrug’sinvivoefficacy. SPRcanrankthekineticselectivityofdruganalogsfortheselectionofthebestdrugcandidates.  High-informationcontent Combiningkineticinformationwithaffinityandpotencydataearlyinthedrugdiscoveryprocessensuresthatpromisingcompoundsarenotbeingdiscarded. Eliminationofpromiscuousbinders Promiscuousbinders,whichappearasfalsepositivesinbiochemicalinhibitorassays,canbeidentifiedbySPRwhenusedasthesecondaryscreeningtechnology. Plasmaproteinbinding SPR-basedassaysaresensitivehigh-throughoutoptionstoaccuratelymeasureplasmaproteinbindingofanalytes.   RelatedResources Poster SurfacePlasmonResonanceBindingStudiesofEpigeneticTargets Blog BiophysicalcharacterizationofcompoundsdisruptingtheSARS-CoV-2Spike–ACE2interactionusingSPR ViewpageBlog