Hotline:400-820-3792Inhibitors?ScreeningLibraries?Proteinswww.MedChemEPF-06751979Cat.No.:HY-112157CASNo.:1818339-66-0分?式:C??H??F?N?O?S?分?量:455.5作?靶點(diǎn):Beta-secretase作?通路:NeuronalSignaling儲(chǔ)存?式:Powder-20°C3years4°C2yearsInsolvent-80°C6months-20°C1month溶解性數(shù)據(jù)體外實(shí)驗(yàn)DMSO:150mg/mL(329.31mM;Needultrasonic)Ethanol:50mg/mL(109.77mM;Needultrasonic)MassSolvent1mg5mg10mgConcentration制備儲(chǔ)備液1mM2.1954mL10.9769mL21.9539mL5mM0.4391mL2.1954mL4.3908mL10mM0.2195mL1.0977mL2.1954mL請(qǐng)根據(jù)產(chǎn)品在不同溶劑中的溶解度選擇合適的溶劑配制儲(chǔ)備液；?旦配成溶液，請(qǐng)分裝保存，避免反復(fù)凍融造成的產(chǎn)品失效。儲(chǔ)備液的保存?式和期限：-80°C,6months;-20°C,1month。-80°C儲(chǔ)存時(shí)，請(qǐng)?jiān)?個(gè)?內(nèi)使?，-20°C儲(chǔ)存時(shí)，請(qǐng)?jiān)?個(gè)?內(nèi)使?。體內(nèi)實(shí)驗(yàn)請(qǐng)根據(jù)您的實(shí)驗(yàn)動(dòng)物和給藥?式選擇適當(dāng)?shù)娜芙?案。以下溶解?案都請(qǐng)先按照InVitro?式配制澄的儲(chǔ)備液，再依次添加助溶劑：(為保證實(shí)驗(yàn)結(jié)果的可靠性，澄的儲(chǔ)備液可以根據(jù)儲(chǔ)存條件，適當(dāng)保存；體內(nèi)實(shí)驗(yàn)的?作液，建議您現(xiàn)?現(xiàn)配，當(dāng)天使?；以下溶劑前顯?的百分?指該溶劑在您配制終溶液中的體積占?；如在配制過程中出現(xiàn)沉淀、析出現(xiàn)象，可以通過加熱和/或超聲的?式助溶)1.請(qǐng)依序添加每種溶劑：10%DMSO>>40%PEG300>>5%Tween-80>>45%saline1/3MasterofBioactiveMolecules—您?邊的抑制劑?師www.MedChemESolubility:≥2.75mg/mL(6.04mM);Clearsolution2.請(qǐng)依序添加每種溶劑：10%DMSO>>90%cornoilSolubility:≥2.75mg/mL(6.04mM);ClearsolutionBIOLOGICALACTIVITY?物活性PF-06751979?種有效的，腦滲透性的β-淀粉樣前體蛋?裂解酶1(BACE1)抑制劑，IC50為7.3nM。IC50&TargetIC50:7.3nM(BACE1),194nM(BACE2)[1]體外研究PF-06751979showsimprovedselectivityoverBACE2(IC50=194nM)inbinding(27-fold)relativetotheliteratureexamplesandacrossmultiplechemicalseriesinBACE1program.PF-06751979alsoinhibitsBACE1andBACE2inafluorescentpolarization(FP)assaywithIC50sof26.9nMand238nM,respectively.PF-06751979hasexcellentpotencyatBACE1inbindingorFPassayformatsalongwithcellularactivitylookingatproductionofsAPPβinH4cellswithanIC50of5nM[1].體內(nèi)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?邊的抑制劑?師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