Hotline:400-820-3792Inhibitors?ScreeningLibraries?Proteinswww.MedChemEAST487Cat.No.:HY-15002CASNo.:630124-46-8Synonyms:NVP-AST487分?式:C??H??F?N?O?分?量:529.56作?靶點(diǎn):RET;FLT3;VEGFR;c-Kit;Bcr-Abl作?通路:ProteinTyrosineKinase/RTK儲存?式:Powder-20°C3years4°C2yearsInsolvent-80°C6months-20°C1month溶解性數(shù)據(jù)體外實(shí)驗(yàn)DMSO:≥100mg/mL(188.84mM)*"≥"meanssoluble,butsaturationunknown.MassSolvent1mg5mg10mgConcentration制備儲備液1mM1.8884mL9.4418mL18.8836mL5mM0.3777mL1.8884mL3.7767mL10mM0.1888mL0.9442mL1.8884mL請根據(jù)產(chǎn)品在不同溶劑中的溶解度選擇合適的溶劑配制儲備液；?旦配成溶液，請分裝保存，避免反復(fù)凍融造成的產(chǎn)品失效。儲備液的保存?式和期限：-80°C,6months;-20°C,1month。-80°C儲存時(shí)，請?jiān)?個(gè)?內(nèi)使?，-20°C儲存時(shí)，請?jiān)?個(gè)?內(nèi)使?。體內(nèi)實(shí)驗(yàn)請根據(jù)您的實(shí)驗(yàn)動物和給藥?式選擇適當(dāng)?shù)娜芙?案。以下溶解?案都請先按照InVitro?式配制澄的儲備液，再依次添加助溶劑：(為保證實(shí)驗(yàn)結(jié)果的可靠性，澄的儲備液可以根據(jù)儲存條件，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)1/3MasterofBioactiveMolecules—您?邊的抑制劑?師www.MedChemE1.請依序添加每種溶劑：10%DMSO>>40%PEG300>>5%Tween-80>>45%salineSolubility:≥2.5mg/mL(4.72mM);Clearsolution2.請依序添加每種溶劑：10%DMSO>>90%(20%SBE-β-CDinsaline)Solubility:≥2.5mg/mL(4.72mM);Clearsolution3.請依序添加每種溶劑：10%DMSO>>90%cornoilSolubility:≥2.5mg/mL(4.72mM);ClearsolutionBIOLOGICALACTIVITY?物活性AST487?種RET激酶抑制劑，IC50為880nM，抑制RET?磷酸化，及下游效應(yīng)器激活，也抑制Flt-3，IC50為520nM。IC50&TargetIC50:880nM(RET),170nM(KDR),790nM(Flt-4),500nM(c-Kit),520nM(Flt-3),20nM(Abl)[1]體外研究AnumberofotherkinasesarealsosimilarlyinhibitedbyAST487(NVP-AST487)intheinvitrokinaseassays,includingKDR(IC50=170nM),Flt-4(IC50=790nM),Flt-3(IC50=520nM),c-Kit(IC50=500nM),andc-Abl(IC50=20nM).AST487potentlyinhibitsthegrowthofhumanthyroidcancercelllineswithactivatingmutationsofRETbutnotoflineswithoutRETmutations.BothGDNF/GFRα1andpersephin-inducedcalcitoninmRNAaremarkedlyinhibitedbycoincubationwith100nMofAST487inMTC-Mcells[1].AST487isanovel,mutantFLT3inhibitor.AST487istestedinbiochemicalassaysforinhibitionofFlt-3kinaseactivity.TheKiisdeterminedtobe0.12μM.BesidesFlt-3,NVP-AST487inhibitsRET,KDR,c-Kit,andc-AblkinasewithIC50valuesbelow1μM.TreatmentofFLT3-ITD-Ba/F3cellsandD835Y-Ba/F3cellswithAST487potentlyinhibitscellularproliferation(IC50[2].體內(nèi)研究Afterasingleoraladministrationof15mg/kgofAST487toOF1mice,ameanpeakplasmalevel(Cmax)of0.505±0.078μMSEisachievedafter0.5h.SimilarlevelsofAST487arefoundintheplasmaofmiceupto6hafteroraladministration,withaClastof21±4nMat24h.Theoralbioavailabilityiscalculatedtobe9.7%withat1/2terminaleliminationof1.5h[1].PROTOCOLKinaseAssay[1]GlutathioneS-transferase(GST)-fusedkinasedomainsareexpressedinbaculovirusandpurifiedoverglutathione-sepharose.Kinaseactivityistestedbymeasuringthephosphorylationofasyntheticsubstrate[poly(Glu,Tyr)],bypurifiedGST-fusionkinasedomainsoftherespectiveproteinkinaseinthepresenceofradiolabeledATP;theATPconcentrationsusedareoptimizedwithintheKmrangefortheindividualkinases.Briefly,eachkinaseisincubatedunderoptimizedbufferconditionsin20mMofTris-HCl(pH7.5),1to3mMofMnCl2,3to10mMofMgCl2,10μMofNa3VO4,1mMofDTT,0.2μCi[33P]ATP,1to8μMofATP,3to8μg/mLofpoly(Glu/Tyr,4:1),and1%DMSOinatotalvolumeof30μLinthepresenceorabsenceofNVP-AST487for10minatambienttemperature.Reactionsareterminatedbyadding10μLof250mMEDTA,andthereactionmixtureistransferredontoanImmobilonpolyvinylidenedifluoridemembrane.Filtersarewashed(0.5%H3PO4),soakedinethanol,driedandcountedinaliquidscintillationcounter.IC50sforAST487arecalculatedbylinearregressionanalysisofthepercentageinhibition[1].2/3MasterofBioactiveMolecules—您?邊的抑制劑?師www.MedChemEMCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.CellAssay[2]ThetrypanblueexclusionassayisusedtodetermineproliferationofcellsculturedinthepresenceandabsenceofNVP-AST487.Cellviabilityisreportedaspercentageofcontrol(untreated)cells,anddataarepresentedastheaverageof2independentexperiments,exceptwhereindicated.Errorbarsrepresentthestandarderrorofthemeanforeachdatapoint.Apoptosisofdrug-treatedcellsismeasuredusingtheAnnexin-V-FluosStainingKit.Cell-cycleanalysisisperformed[2].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.AnimalMice[1]Administration[1]Femaleathymicnudemicearekeptunderoptimizedhygienicconditions(maximumof10miceperMakrolontypeIIIcage)withfreeaccesstofoodandwater.Tumorsareestablishedbys.c.injectionof1×106and5×106ofNIH3T3-RETC634WandTTcells,respectively,in100μLofHBSSpermouse.Treatabletumors,i.e.,meantumorvolumeof100mm3,developedwithin10daysofNIH3T3-RETC634Wcellinjection,andwithin20daysofTTcellinjection.NVP-AST487isgivenp.o.,oncedailybygavage.ThecompoundisformulatedbydissolvingtheappropriateamountofpowderinN-methylpyrrolidone/PEG300(1:10v/v).Themicearerandomizedintofourtreatmentgroupsofeightmiceeach.ThefirstthreegroupsreceiveddailyoraladministrationsofNVP-AST487at50,30,and10mg/kg,respectively,for3weeks.Thefourthgroupreceivedtreatmentwithvehicle.Tumorgrowthandbodyweightsaremonitoredtwiceweekly.Tumorvolumesaredeterminedaccordingtotheformula:length×diameter2×π/6.Tumorsarecollectedandfrozeninliquidnitrogenattheendoftheefficacystudy,6hafterthelastadministration.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.戶使?本產(chǎn)品發(fā)表的科研?獻(xiàn)?CellRes.2017Dec;27(12):1441-1465.?ACSChemBiol.2017May19;12(5):1245-1256.?ProteinExpresPurif.2020Apr;168:105552.?Patent.US20210379046A1.Seemorecustomervalidat