PF-06751979-DataSheet-生命科学试剂-MedChemExpress

Hotline:400-820-3792Inhibitors•ScreeningLibraries•Proteinswww.MedChemEPF-06751979Cat.No.:HY-112157CASNo.:1818339-66-0分⼦式:C₁₈H₁₉F₂N₅O₃S₂分⼦量:455.5作⽤靶点:Beta-secretase作⽤通路:NeuronalSignaling储存⽅式:Powder-20°C3years4°C2yearsInsolvent-80°C6months-20°C1month溶解性数据体外实验DMSO:150mg/mL(329.31mM;Needultrasonic)Ethanol:50mg/mL(109.77mM;Needultrasonic)MassSolvent1mg5mg10mgConcentration制备储备液1mM2.1954mL10.9769mL21.9539mL5mM0.4391mL2.1954mL4.3908mL10mM0.2195mL1.0977mL2.1954mL请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；⼀旦配成溶液，请分装保存，避免反复冻融造成的产品失效。储备液的保存⽅式和期限：-80°C,6months;-20°C,1month。-80°C储存时，请在6个⽉内使⽤，-20°C储存时，请在1个⽉内使⽤。体内实验请根据您的实验动物和给药⽅式选择适当的溶解⽅案。以下溶解⽅案都请先按照InVitro⽅式配制澄的储备液，再依次添加助溶剂：(为保证实验结果的可靠性，澄的储备液可以根据储存条件，适当保存；体内实验的⼯作液，建议您现⽤现配，当天使⽤；以下溶剂前显⽰的百分⽐指该溶剂在您配制终溶液中的体积占⽐；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的⽅式助溶)1.请依序添加每种溶剂：10%DMSO>>40%PEG300>>5%Tween-80>>45%saline1/3MasterofBioactiveMolecules—您⾝边的抑制剂⼤师www.MedChemESolubility:≥2.75mg/mL(6.04mM);Clearsolution2.请依序添加每种溶剂：10%DMSO>>90%cornoilSolubility:≥2.75mg/mL(6.04mM);ClearsolutionBIOLOGICALACTIVITY⽣物活性PF-06751979⼀种有效的，脑渗透性的β-淀粉样前体蛋⽩裂解酶1(BACE1)抑制剂，IC50为7.3nM。IC50&TargetIC50:7.3nM(BACE1),194nM(BACE2)[1]体外研究PF-06751979showsimprovedselectivityoverBACE2(IC50=194nM)inbinding(27-fold)relativetotheliteratureexamplesandacrossmultiplechemicalseriesinBACE1program.PF-06751979alsoinhibitsBACE1andBACE2inafluorescentpolarization(FP)assaywithIC50sof26.9nMand238nM,respectively.PF-06751979hasexcellentpotencyatBACE1inbindingorFPassayformatsalongwithcellularactivitylookingatproductionofsAPPβinH4cellswithanIC50of5nM[1].体内研究PF-06751979displaysexcellentbrainpenetration,potentinvivoefficacy,andbroadselectivityoverrelatedaspartylproteasesincludingBACE2.AcuteadministrationofPF-06751979yieldsarobustdose-responsiveandtime-dependentreductionofcerebralspinalfluid(CSF)Aβx-40withpeakinhibitionat3hof>77%.TodetermineifthereductioninbrainandCSFAβismaintainedduringsustainedexposuretoPF-06751979,a5daysubchronicstudyisexecuted,dosingoncedailybysubcutaneous(SC)administration(10or50mg/kg/day).BrainandCSFsamplesarecollectedonday5,followingthelastdose.PF-06751979producesadose-responsiveandtime-dependentinhibitionofAβ42inmousebrain.Atthe50mg/kg/daydose,maximalbrainloweringis63%at7to9h.AdministrationofPF-06751979(10or50mg/kg/dayfor5days)producesadose-responsiveandtime-dependentinhibitionofAβx-40inmouseCSFresultingin77%inhibitionofCSFat3hpost-final50mg/kgdose[1].PROTOCOLKinaseAssay[1]BothBACE1andBACE2enzymaticactivityismeasuredwiththeaidofanoptimizedsyntheticpeptidesubstratebiotin-GLTNIKTEEISEISYEVEFR-C[Oregongreen]KK-OH.Uponcleavageofthepeptidesubstrate,adecreaseinfluorescencepolarizationismeasured.Compoundsaredilutedbyhalflogin100%DMSO11timeswithatopconcentrationof10mMina384-wellpolypropyleneplate.The100%DMSOdoseresponsecurveisthenaddedtoa384-wellblackassayplateas0.150μLperwell.Thefinalworkingtopconcentrationis0.1mM,andtheDMSOconcentrationis1%.Avolumeof7.5μLofBACEsubstrateisthenaddedinassaybuffer(100mMsodiumacetate,pHto4.5withglacialaceticacid,0.001%Tween20).Thebackgroundwellsincolumn1ofthe384-wellassayplatereceive7.5μLofassaybuffer.Thereactionisstartedwiththeadditionof7.5μLofBACE1orBACE2enzymeinassaybuffertoallwellsexceptthebackgroundwellsincolumn1.Thefinalconcentrationofpeptidesubstrateis150nM,andthefinalconcentrationofBACE1andBACE2enzymeis0.15and2.5nM,respectively.Theassayplateissealedandincubatedat37°Cfor3or1h(BACE1orBACE2,respectively).Afterincubation,15μLofstopsolution(1.5μMstreptavidininDulbecco’sPBS)isaddedtoallwells,andtheplateisread.Percenteffectvaluesforeach2/3MasterofBioactiveMolecules—您⾝边的抑制剂⼤师www.MedChemEconcentrationofcompoundarecalculatedbasedonfluorescencepolarization(FP)readingsinthe100%effectcontrolwellscontainingnoenzymeandthe0%effectcontrolwellscontainingnocompound.Curve-fittinganalysisutilizingconcentrationsandpercenteffectvaluesforagivencompoundisplotted,andtheIC50isdeterminedusingasigmoidalfour-parameterfitalgorithm[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.CellAssay[1]TheneurogliomacelllineH4cellsaregrowninDulbecco’smodifiedEagle’smedium(DMEM)with10%fetalbovineserum(FBS)and200mMglutamax.CellsareplatedovernightintissueculturetreatedFalcon384-wellplatesatacelldensityof4500cells/wellin50μLofmedia.Thenextday,mediaisremoved,andcellsarewashedoncewithPBS,afterwhich25μLofmediaisplacedinallwells,followedbytheadditionofthedilutedPF-06751979doseresponsecurve.Thehighestconcentrationtestedis30μMwith1%DMSO.Cellsservingasthebackgroundcontrolsreceive30μMofaproprietarycompound.Compoundsareallowedtoincubatewithcellsovernightina37°Cincubator.Concurrently,384-wellblackNuncMaxisorpplatesarealsoincubatedovernightat4°Cwith10μLof4μg/mLAβantibodyincoatingbuffer(0.1Msodiumbicarbonate,pH8.8to9.0)[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.AnimalMice[1]Administration[1]Male129/svewild-typemice(20-25g)areinanonfastedstatepriortosubcutaneousdosingwithvehicleorPF-06751979usingadosingvolumeof10mL/kg.Themicearedosedsubcutaneouslyonceadayfor5dayswithPF-06751979(10or50mg/kg/day)orvehicle.Themice(n=5pergroup)arethensacrificedat1,3,5,7,14,20,and30hpostdose.Followingcardiacpunctureintoethylenediaminetetraaceticacid(EDTA)-containingtubes,wholebloodsamples(0.5-1.0mL)arecollected,andplasmaisseparatedbycentrifugation(1500×gfor10minat4°C).Thegeneratedplasmaisdistributedintoseparatetubesonweticeforexposuremeasurements(50μL)andAβanalysis(remainder).CSFsamples(8-12μL)areobtainedbycisternamagnapunctureusingasterile25gaugeneedleandcollectedwithaP-20Eppendorffpipet.CSFsamplesaredistributedintoseparatetubesondryiceforexposuremeasurements(3μL)andAβanalysis(remainder).Wholebrainisremovedanddividedforexposuremeasurements(cerebellum)andAβanalysis(leftandrighthemispheres),weighed,andfrozenondryice.Priortotheassay,allsamplesarestoredat-80°C