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MethodsandResources
MethodsandResourcesreportnovelmethods,substantialimprovementstocurrentmethodologies,orinformationaldatasets.
AcademicEditor:AnnStock,UMDNJ/RobertWoodJohnsonMedicalSchool,UnitedStatesofAmerica
Received:February6,2018;Accepted:May2,2018;Published:May21,2018
Funding:DeutscheForschungsgemeinschaft(grantnumberSFB1078B1).Thefunderhadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.iNEXT(grantnumberWP1).Thefunderhadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.SwissNationalScienceFoundation(grantnumber200020_159707and200020_146757).Thefunderhadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.EuropeanResearchCouncil(grantnumber741863,FASTER).Thefunderhadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
Competinginterests:Ihavereadthejournal’spolicyandtheauthorsofthismanuscripthavethefollowingcompetinginterests.AlexMacpherson,FabienLecomte,RichardD.Taylor,TimNorman,MartaWestwood,BenCossins,JamesWhite,RobertGriffin,ChristineProsser,SebastianKelm,AlistairHenry,RichardTaylor,andAlastairD.Lawsonareallcurrentorformeremployeesand/orshareholdersinUCBPharma.NicolasBasseisanemployeeand/orshareholderinSanofi.HervéDebovesisanemployeeand/orshareholderinEvotec.JohnPorterisanemployeeand/orshareholderinMidatechPharma.KatharineCainisanemployeeand/orshareholderinVertex.AmyH.SullivanandDavidFoxIIIare/wereemployeesand/orshareholdersinBerylliumDiscovery.ThisworkwasfundedbyUCBPharma.
Abbreviations:β2m,β2-microglobulin;APS,AdvancedPhotonSource;AUC,analyticalultracentrifugation;BMRB,BiologicalMagneticResonanceDataBank;CD8,clusterofdifferentiation8;CLS,CanadianLightSource;COOT,CrystallographicObject-OrientedToolkit;CP,CrossPolarization;CSP,chemical-shiftperturbation;ECD,extracellulardomain;FcRn,neonatalFcreceptor;FcRnECD,extracellulardomainoftheneonatalFcreceptor;HSA,HumanSerumAlbumin;IgG,ImmunoglobulinG;MAS,magic-angle-spinning;MHC1,classImajorhistocompatibilitycomplex;ORF,OpenReadingFrame;PDB,ProteinDataBank;r.m.s.d.,rootmeansquaredeviation;SPR,SurfacePlasmonResonance;TCR,Tcellreceptor;TIPS,titerlessinfected-cellspreservationandscale-up
ThesolubleextracellulardomainofneonatalFcreceptor(FcRnECD,PDBcode1EXU)isaheterodimercomposedofβ2m(green)andα-chain(blue)withacavityattheinterfacebetweenthetwoproteins.FcRnisinvolvedintheregulationofHSA(orange)andIgG(red)levels.ThebindingofbothHSAandIgGtoFcRnispHdependent,whichprovidesamechanismforproteinhomeostasisthroughendosomaltrafficking.β2m,β2-microglobulin;FcRn,neonatalFcreceptor;FcRnECD,extracellulardomainoftheneonatalFcreceptor;HSA,HumanSerumAlbumin;IgG,ImmunoglobulinG;PDB,ProteinDataBank.
NoregionsneartheIgGbindingsitewerefoundbySiteMap.Basedonouranalysis,shouldanorthostericpocketforanIgGblockingsmallmoleculeexist,itislikelytobetransientinnatureandnotstabilizedinthecrystallattice.
Oursequenceanalysisbroadlysupportsthenotionthatregionsoftheproteinimportantforstructuralintegrityorfunctionareconserved,inparticulartheinterfacebetweentheα-chainandβ2m.Itcontainskeycontacts,suchasthosebetweenD53β2m,Q34α-chain,andS37α-chain,andconservationofthesecontactspreservesthestructuralintegrityofthenoncovalentlylinkedheterodimer.
Confirmedhitsfromthefragment-screenweresoakedintoFcRnECDcrystalsthathadbeengrownunderacidicconditions.Basedonthederivedcrystalstructures,weaimedtofurtherimprovebothaffinityandsolubilityoftheligand.
(A)Theproteincrystallizedasadimercomposedoftwoβ2m(darkgreyandgreen)andtwoα-chain(lightgreyandblue)molecules.(B)Attheinterfaceofβ2mandtheα-chain,UCB-FcRn-303(grey)occupiesabindingpocketwithGlycine,Cysteine,hydrophobic(Leucine),charged(Histidine,Aspartate),andpolaruncharged(Serine,Glutamine)residues.β2m,β2-microglobulin;FcRn,neonatalFcreceptor;FcRnECD,extracellulardomainoftheneonatalFcreceptor.
Inordertodeterminewhethertheligandmightinducesmallconformationaland/ordynamicchangesinregionsbeyondthedirectbindingsite,wehaveusedorthogonalsolution-basedtechniques,whichwillavoidconformationalrestrictionsimposedbycrystalpacking.
(A)ThesolubleFcRnECD(42kDa)wassedimentedbyultracentrifugationat100,000xgdirectlyintoa0.7mmMASNMRrotorusingahome-madefillingtool.(B)2D15N-1Hcorrelationspectrumrecordedat100kHzMASoffullyprotonated[13C,15N]-labeledFcRnECD.(C)Typicallinewidthsof1H(1)and15N(2)atfull-width-half-maximum(FWHM)ofaselectedcrosspeakfromthe15N-1Hspectrum.β2m,β2-microglobulin;FcRn,neonatalFcreceptor;FcRnECD,extracellulardomainoftheneonatalFcreceptor.
Insummary,(H)CANH,(H)CA(CO)NH,and(H)CBCANHspectrarepresentanacceptablebasisforobtainingbackboneresonanceassignmentsandallowedustoexploitCSPsasmonitorsforstructuralalterationsinFcRnECDuponUCB-FcRn-303binding.
Inthecontextofadruggabilitystudy,weidentifiedacompoundof<500Damolecularweight,UCB-FcRn-303,thatbindstotheextracellulardomainofFcRnwithlowμMaffinity.Theconservedbindingsiteislocatedattheinterfaceofβ2mandtheα-chain,featuringatunnel-likecavitywithsolventaccessfromtwodifferentsides.Thepocketwasidentifiedbycomputationalchemistrymethodsinatheoreticaldruggabilityexaminationandwascorroboratedbyfragmentscreening,X-raycrystallography,andNMRspectroscopy.Therespectivecrystalstructuresshowadimerofheterodimers,witha1:1stoichiometryfortheligand/proteincomplex.
ThepresentedMASNMRmethodologyprovidesanappealingapproachforstructuralinvestigationsoflarge,solubleproteinsexpressedinmammalianorothertypesofeukaryoticcellsinwhichdeuterationischallenging,especiallyincaseswhenglycosylationiscrucial.Moreover,itextendstherangeofNMRapplicationstopharmacologicallyrelevanttargets,whichareofteninaccessiblebysolution-stateNMRmethodsduetosize.TheMASNMRapproachworkswithhighmolecularweighttargetsandisindependentofthephysicalstateofthesample,providingspectralcomplexitycanbehandled,e.g.,byapplyingappropriatelabellingconcepts.Thisfacilitatesprotein-ligandinteractionstudiesbyNMRforanytypeofproteinorbiomolecularcomplex,makingpreviouslyintractablepharmacologicaltargetsaccessible,suchastheribosome,Gprotein-coupledreceptors,andthelike.OurinvestigationshowsthatMASNMRcomplementsX-raycrystallographyinstructure-baseddrugdiscoverycampaigns.Itisparticularlyusefultoexploreallostericchangesbeyondsmallmoleculebindingsites,whichmaybedifficulttoobservebyX-raycrystallography.
Thecodingsequencesofextracellulardomain(ECD)(aminoacids1–297)ofhumanFcRnα-chainandhumanβ2mweresynthesizedbyEntelechon(Entelechon,Regensburg,Germany).Theα-chainfragmentwasclonedintotheexpressionvectorpMH(UCB)andtheβ2msequencewasclonedintopMK(UCB)asHindIIIandEcoRIfragments.BothvectorsweredigestedwithSalIandNotIandtherelevantfragmentsexcisedandligatedtogenerateavectorcontainingboththeα-chainandβ2mgenes(pM-ECDFcRn-B2M).
ThevectorwasfurtherdigestedwithSalIandaneomycincassettewasligatedintogenerateadoublegenevectorwithantibioticselection(pMFcRnECD-B2M-Neo).
HEK293cellsweretransfectedwithpMFcRnECD-B2M-Neousing293fectin(Thermofisher)accordingtothemanufacturer’sinstructions.Transfectedcellswereincubatedinastaticincubatorat37°Cand8%CO2for24hours.Thecellswerethendilutedtotherequiredconcentrationwithmediumsupplementedwith0.5mg/LG418(Invitrogen)andsubsequentlydividedinto1-mLpoolsin24-wellplatesandincubatedinastaticincubatorat37°Cwith8%CO2.Every7days,themediumwasremovedfromeachwellandreplacedwithfreshmedium.Afterafurther14days,thepoolsthatexhibitedcellgrowthweretransferredto6-wellplates.Thesewereexpandedupto50-mLculturesinE250flasksinshakingincubators.TodeterminetotalexpressionoftheFcRnECDheterodimercomposedofβ2mandα-chain,batchovergrowsweresetupandincubatedfor10days.
Samplesofthe50-mLbatchovergrowsupernatantswereanalyzedforFcRnECDexpressionusingwesternblotting.15μLofeachsupernatantwasrunona(denaturedTris/Glygelwesternblot)alongsideknownconcentrationsofpurifiedhumanFcRnasacontrol.
Thehighestexpressingpoolwasexpanded(withoutNeomycinselection)upto10-LscaleinaWaveBioreactor20/50EHTat37°C,8%CO2for4days,atwhichpointthetemperaturewasreducedto32°Candincubatedforafurther6days.Thecellculturewasharvestedbycentrifugation(1,000xgfor1hour)andsupernatantputthrougha0.22-μmfilter.
MammaliancellculturesupernatantcontainingtheheterodimerFcRnECDwasconcentratedwitha10,000MWCOmembraneusingtangentialflowfiltration(Centramate,Pall)orAmiconstirredcell(Millipore),dependingonscale.Thesamplebufferwasexchangedinto50mMsodiumphosphate,pH5.8,30mMNaClbydilutingandconcentrating.FcRnECDwasloadedontoaKappaSelect(GEHealthcare)column,whichhadbeenpreloadedwithanIgG4monoclonalantibodytobindFcRnECD,beforewashingwith50mMsodiumphosphate,pH5.8,30mMNaCl,andelutingwith50mMsodiumphosphate,pH8.0,30mMNaCl.ElutionfractionswereanalyzedbySDS-PAGEandrelevantfractionspooled.
ThesamplewasconcentratedusinganAmiconspinconcentrator(Millipore),10,000MWCOmembrane.TheproteinwaspurifiedbyGelFiltrationwithaSuperdex200(GEHealthcare)columnusing25mMsodiumphosphate,pH7.4,100mMNaCl.PeakfractionswereanalyzedbySDS-PAGEbeforepoolingandconcentratingifrequired.
SPRwascarriedoutusingBIAcore4000instruments(GEHealthcare).ReagentsincludingCM5sensorchips,N-hydroxysuccinimide(NHS),N-ethyl-N-(3-dimethylaminopropyl)carbodiimide(EDC),andethanolamineHCl,10mMsodiumacetatebuffers(pH5.0,pH4.5)andHBS-P(10xbuffer)wereobtainedfromGEHealthcare.
FcRnECDwasdilutedinto10mMsodiumacetatebuffer,pH5.0andimmobilizedonaCM5SensorChipviaaminecouplingchemistrytoacapturelevelofapproxmimately4,000responseunits.Compoundswerescreenedina10-pointtitrationfrom200μM,at2%DMSO,pH6.0,andthesurfacewasre-immobilizedaftereach384-wellplate.Injectionswereperformedataflowrateof10μL/min.
Alldataweredouble-referencedforblankinjectionsandreferencesurface,followingstandardprocedures.BothdataprocessingandfittingwereperformedusingActivityBasetemplateprotocolsdevelopedinhouse.
Alibraryofapproximately1,100fluorine-containingfragmentswerecocktailedintogroupsof12ensuringnooverlapof19Fsignals.Cocktailswereinitiallypreparedataconcentrationof4.2mMind6-DMSOanddilutedto800μMinPBSpH7.4beforeafinaldilutionto40μMligandconcentration(1%d6-DMSO)ineitherPBScontaining10%D2O(forcontrolsamples)or20μMFcRnECDcontaining10%D2O(forproteinsamples).NMRspectrawereacquiredat25°ConaBruker600MHzAVIII-HDspectrometerequippedwithaQCI-FcryoprobeandaSampleJetautosampler.DatawerecollectedusingaCPMGpulsesequencewithatotalechotimeof160msacrossasweepwidthof126ppmwithanacquisitiontimeof1s.AllspectrawereprocessedusingTopSpin3.2.FragmentswereconsideredbinderstoFcRnECDwhenthe19FsignalintensitywassignificantlyreducedinthespectrawithFcRnECDpresentcomparedtothespectrarecordedintheabsenceofprotein.
STDNMRsampleswerepreparedwithaligandtoproteinratioof50:1(500μMligand,10μMFcRnECD)in500μLphosphatebufferedsaline,pH7.4(90%H2O,10%D2O)with5%d6-DMSOtohelpsolubilizetheligand.STDNMRspectrawererecordedusingaBrukerAvanceIIIHD600MHzspectrometerequippedwitha5mmQCI-FCryoprobe.DatawereacquiredandprocessedusingthestandardBrukersoftwareandwerecollectedat298K.Theproteinwassaturatedinthemethylregionofthespectrumat0ppm,andoff-resonancesaturationwasperformedat33ppm.Aseriesof120EBurp2pulses(50mseach)wereappliedwitha4-μsdelaybetweeneachpulse,resultingintotalsaturationtimeof6s.Proteinsignalswereremovedbyapplyingaspinlockof100ms.Interleavedon-andoff-resonancedatawererecorded,processedseparately,andthenthedifferencespectraobtainedbysubtractingtheon-fromtheoff-resonancespectra.Datawerezerofilledonceandanexponentialmultiplicationwindowfunctionapplied(LB2Hz).
InordertosearchforcrystallizationconditionsforFcRnECDexpressedininsectcells,sitting-dropvapordiffusioncrystallizationtrialsweresetupat291Kusingavarietyofcommercialspare-matrix(RigakuReagents:JCSG+,Wizard1/2,Wizard3/4;HamptonResearch:CrystalScreenHTIndex;MolecularDimensions:PACT,Morpheus,Proplex;Microlytics:MCSG1)using0.4μLofproteinsolutionat5mg/mLthatweremixedwith0.4μLofreservoirsolutionandequilibratedagainst80μLofreservoirsolution.TheinitialcrystallizationtrialsproducedsmallkitecrystalsinseveralconditionsthatcontainPEG3350,PEG6000,orPEG8000atlowpH.LowpHcrystalsofFcRnECDwereproducedinanoptimizedcrystallizationconditionscreen(Rigaku)containing12%–16%PEG6000,100mMCitricAcid/AmmoniumcitratetribasicpH3.00–3.09.CrystalsofFcRnECDappearedwithin24hoursandgrewlargerovertime,typicallyto50–150micronsinsize.Thecrystalswereharvestedusing20%glycerolasacryoprotectantandflashfrozeninliquidnitrogenpriortodatacollection.
FcRnECDwasexpressedusingastableHEK293cellline,asdescribedabove,butthegrowthmediawasreplacedwithBioexpress6000media(CambridgeIsotopeLaboratories).
A0.5-mLsampleof100μM[13C,15N]-labeledFcRnECDina25mMNa2HPO4,100mMNaCl,50μMEDTA,0.02%(w/v)NaN3bufferatpH7.4containing3%DMSOwasdirectlyultracentrifugedintoa0.7mmMASNMRrotorat100,000xgand4°Cfor40hoursusingaswingingbucketultracentrifugerotor.Dedicatedhome-madefillingtoolswereusedforthisstep.Bothbottomandtopcapsofthe0.7mmMASrotorweregluedtoavoidthelossofliquidorremovalofcapsduringMAS.Excessproteinandliquidafterultracentrifugationwasremovedbeforeclosingthe0.7mmNMRMASrotor.ForexperimentstodetectCSPs,3mMUCB-FcRn-303wasaddedtoa0.5-mLsampleof100μM[13C,15N]-labeledFcRnECDina25mMNa2HPO4,100mMNaCl,50μMEDTA,0.02%(w/v)NaN3bufferatpH7.4containing3%DMSO(ligand:proteinratioof30:1).Theligand-boundsamplewassedimentedintoasecond0.7mmMASrotorunderthesameconditionsastheligand-freesample.
Allsolventsandreagentswereusedasreceivedfromcommercialsuppliers,unlessnotedotherwise.ThecompoundswerenamedusingtheBioviaDraw2016package(IUPAC).
NMRspectrawererecordedonaBrukerAvanceIIIHD500MHzor250MHzspectrometer.
Thechemical-shifts(δ)reportedaregiveninpartspermillion(ppm)andthecouplingconstants(J)areinHertz(Hz).Thespinmultiplicitiesarereportedass=singlet,d=doublet,t=triplet,q=quartet,dd=doubletofdoublet,ddd=doubletofdoubletofdoublet,dt=doubletoftriplet,td=tripletofdoublet,andm=multiplet.
uPLC-MSwasperformedonaWatersAcquityUPLCsystemcoupledtoaWatersAcquityPDAdetector,anELSdetectorandanMSD(ScanPositive:150–850).Method(pH3):PhenomenexKinetix-XBC18(2.1x100mm,1.7μm)column.ElutionwithalineargradientofWater+0.1%FormicacidandAcetonitrile+0.1%Formicacidataflowrateof0.6mL/min.ChiralSFCanalysis:WatersThar3100SFCsystemconnectedtoWaters2998PDAdetector,ChiralcelOD-H25cm.ChiralSFCseparation:WaterTharSFCsystemwithaWatersTharFDMpump,WatersTharAliasautoinjector,WatersTharfractioncollectorandaWaters2998PDAdetector.
1-[7-(3-fluorophenyl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-yl]ethanone(CAS691368-95-3)waspurchasedasaracematefromLifeChemicalsandthemixtureseparatedbychiralchromatographyusingaChiralpakADphase(100*500),300mL/minwithanheptane/isopropanol(8/2)system.1.21Gofstartingmaterialledtorespectively577mgand588mgofseparatedisomers.ChiralanalyticalSFC:RT=8.22min,100%ee;RT=10.40min,100%ee.
Astirredsolutionof3,5-difluorobenzaldehyde(0.1mL,0.946mmol),pentane-2,4-dione(0.146mL,1.42mmol,1.5eq.),and4H-1,2,4-triazol-3-amine(119mg,1.42mmol,1.5eq.)inN,N-dimethylformamide(1.5mL)wasirradiatedinamicrowaveoven(upto200W)at150°Cfor60min.Thereactionmixturewaslefttocooldowntoambienttemperatureandwater(6mL)wasaddedleadingtotheformationofaprecipitate.Theresultingsolidwascollectedbyfiltration,rinsedwithwater(2x1mL)andcyclohexane(2x1mL),thentrituratedinhotacetonitrile(1mL)anddriedinvacuotoafford94mg(34%yield)ofthetitlecompoundasapaleyellowsolid.
The1HNMRanalysisyielded(500MHz,DMSO-d6)δ10.89(s,1H),7.70(s,1H),7.14(t,J=9.1Hz,1H),6.98(d,J=6.3Hz,2H),6.46(s,1H),2.46(s,3H),2.20(s,3H).uPLC-MS:[M+H]+m/z=291,RT=2.38min(99%).
Theracemate(50mg)wasseparatedbychiralpreparativechromatographyonaChiralpakASV(50*490)phase,80mL/minwithaheptane/ethanol(9/1)systemtoafford24mgand19mgofthepureenantiomers.ChiralanalyticalSFC:RT=10.89min,100%ee;RT=15.16min,97.8%ee.
Toastirredsolutionof3,5-difluorobenzaldehyde(150mg,1.06mmol),methyl3-oxo-3-(pyridin-3-yl)propanoate(265mg,1.48mmol,1.4eq.),and4H-1,2,4-triazol-3-amine(124mg,1.48mmol,1.4eq.)inN,N-dimethylformamide(1.5mL)wasaddedchloro(trimethyl)silane(0.268mL,2.11mmol)dropwise.Thereactionmixturewasthenirradiatedinamicrowaveoven(upto200W)at130°Cfor60minutes.Thereactionmixturewaslefttocooldowntoambienttemperatureandwater(6mL)wasaddedleadingtotheformationofaprecipitate.Theresultingsolidwascollectedbyfiltration,rinsedwithwater(2x1mL)andcyclohexane(2x1mL)thenrecrystallizedfromacetonitrile(2mL)anddriedinvacuotoafford238mg(59%yield)ofthetitlecompoundasapaleyellowsolid.The1HNMRanalysisyielded(500MHz,DMSO-d6)δ11.18(s,1H),8.67–8.61(m,2H),7.92(dt,J=7.8,1.9Hz,1H),7.75(s,1H),7.48(dd,J=7.8,4.9Hz,1H),7.20(tt,J=9.2,2.2Hz,1H),7.16–7.10(m,2H),6.50(s,1H),3.27(s,3H).uPLC-MS:[M+H]+m/z=370,RT=2.12min(97%).
AtlowpHbindingsiteswiththecapacitytoyieldhighaffinitybindingarerestrictedtothedimerinterface,withtheregiondescribedaseitheronelargeorthreedistinctpockets(pocketsA-C).AtneutralandbasicpH,transientsitesariseatthealbuminbindingsite,betweentheα1andα2helices(D),andbetweentheβ2mandtheα3domain(E).β2m,β2-microglobulin;FcRnECD,extracellulardomainoftheneonatalFcreceptor.
(JPG)
(TIF)
Thecompoundbindsattheinterfaceofβ2m(green)andtheα-chain(blue).Alsointhiscrystalstructure,asecondheterodimercanbefoundintheasymmetricunit(grey).β2m,β2-microglobulin;FcRn,neonatalFcreceptor;FcRnECD,extracellulardomainoftheneonatalFcreceptor.
ThecompoundoccupiesthesamebindingpocketasUCB-FcRn-84attheinterfaceofβ2m(green)andtheα-chain(blue).Thebindingregionisatunnel-likecavityextendingthroughtheprotein.Again,asecondheterodimerisfoundinthecrystalstructure(depictedingrey).β2m,β2-microglobulin;FcRn,neonatalFcreceptor;FcRnECD,extracellulardomainoftheneonatalFcreceptor.
β2m,β2-microglobulin;FcRnECD,extracellulardomainoftheneonatalFcreceptor;IgG,ImmunoglobulinG;PDB,ProteinDataBank.
(A)2D15N-1HcorrelationusingTROSYoffullyprotonated[13C,15N]-labeledFcRnECDmeasuredinsolution.(B)Overlayofthespectrumshownin(A)(orange)witha2D15N-1Hspectrumofsedimentedfullyprotonated[13C,15N]-labeledFcRnECDrecordedat100kHzMAS(black).FcRnECD,extracellulardomainoftheneonatalFcreceptor;MAS,magic-angle-spinning.
Sedimentationvelocityexperimentsatthreedifferentconcentrations(52μM,grey;14μM,red;4μM,blue)exhibitproteinconcentrationdependentpeaksat3.5S,5.1S,and5.3S.FcRnECD,extracellulardomainoftheneonatalFcreceptor.
FcRn,neonatalFcreceptor.
(PDF)
(XLSX)
β2m,β2-microglobulin;CSP,chemical-shiftperturbation;FcRn,neonatalFcreceptor;FcRnECD,extracellulardomainoftheneonatalFcreceptor;MHC1,classImajorhistocompatibilitycomplex.
FcRnECD,extracellulardomainoftheneonatalFcreceptor;MAS,magic-angle-spinning.
FcRnECD,extracellulardomainoftheneonatalFcreceptor.
FcRn,neonatalFcreceptor;MAS,magic-angle-spinning.
ExcellenttechnicalsupportbyNilsCremeriskindlyacknowledged.
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