Y-27632-DataSheet-生命科学试剂-MedChemExpress

Hotline:400-820-3792Inhibitors•Agonists•ScreeningLibrarieswww.MedChemEY-27632Cat.No.:HY-10071CASNo.:146986-50-7分⼦式:C₁₄H₂₁N₃O分⼦量:247.34作⽤靶点:ROCK;Apoptosis作⽤通路:CellCycle/DNADamage;Cytoskeleton;StemCell/Wnt;TGF-beta/Smad;Apoptosis储存⽅式:-20°C,protectfromlight,storedundernitrogen*Insolvent:-80°C,6months;-20°C,1month(protectfromlight,storedundernitrogen)溶解性数据体外实验DMSO:50mg/mL(202.15mM;Needultrasonic)扫描⼆维码，H2O:5mg/mL(20.22mM;ultrasonicandwarmingand运⽤溶解⽅案计算器heatto60°C)获得适合您实验体系的溶解⽅案MassSolvent1mg5mg10mgConcentration制备储备液1mM4.0430mL20.2151mL40.4302mL5mM0.8086mL4.0430mL8.0860mL10mM0.4043mL2.0215mL4.0430mL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，并请注意储备液的保存⽅式和期限。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%DMSO40%PEG3005%Tween-8045%salineSolubility:≥2.5mg/mL(10.11mM);Clearsolution此⽅案可获得≥2.5mg/mL(10.11mM，饱和度未知)的澄溶液。以1mL⼯作液为例，取100μL25.0mg/mL的澄DMSO储备液加到400μLPEG300中，混合均匀；向上述1/4www.MedChemEwww.MedChemE2.体系中加⼊50μLTween-80，混合均匀；然后继续加⼊450μL⽣理盐⽔定容⾄1mL。请依序添加每种溶剂：10%DMSO90%(20%SBE-β-CDinsaline)Solubility:≥2.5mg/mL(10.11mM);Clearsolution此⽅案可获得≥2.5mg/mL(10.11mM，饱和度未知)的澄溶液。以1mL⼯作液为例，取100μL25.0mg/mL的澄DMSO储备液加到900μL20%的SBE-β-CD⽣理盐⽔⽔溶3.液中，混合均匀。请依序添加每种溶剂：10%DMSO90%cornoilSolubility:≥2.5mg/mL(10.11mM);Clearsolution此⽅案可获得≥2.5mg/mL(10.11mM，饱和度未知)的澄溶液，此⽅案不适⽤于实验周期在半个⽉以上的实验。以1mL⼯作液为例，取100μL25.0mg/mL的澄DMSO储备液加到900μL⽟⽶油中，混合均匀。BIOLOGICALACTIVITY⽣物活性Y-27632⼝服有效的，ATP竞争性的ROCK-I和ROCK-II抑制剂，Ki分别为220nM和300nM。Y-27632诱导凋亡(apoptosis)。Y-27632通过上⽪-间充质过渡样调节引发⼈诱导多能⼲细胞(hIPSCs)选择性地分化为间胚层谱系。IC50&TargetROCK-IROCK-IIPKNCitronkinase220nM(Ki)300nM(Ki)3.1μM(Ki)5.3μM(Ki)PKCαPKA73μM(Ki)25μM(Ki)体外研究Y-27632inhibitstheROCKfamilyofkinases100timesmorepotentlythanotherkinasesincludingproteinkinaseC,cAMP-dependentkinaseandmyosinlightchainkinase.Y-27632prolongsthelagtimeanddelaystheappearanceofBrdU-labeledcellsinaconcentration-dependentmanner,delaysofabout1and4harenoticedintheSwiss3T3cellstreatedwith10and100μMY-27632,respectively[1].Y-27632promotesneuronaldifferentiationofadiposetissue-derivedstemcells(ADSCs).Comparedto1.0and2.5μMY-27632inducedgroups,percentagesofneuroal-likecellsachievedapeakinthe5.0μMY-27632inducedgroup[2].体内研究Y-27632(5and10mg/kg)significantlyprolongstheonsettimeofmyoclonicjerkswhencomparewithsalinegroup.Y-27632(5and10mg/kg)significantlyprolongstheonsettimeofclonicconvulsionswhencomparewithsalinegroup[3].TreatmentwithDimethylnitrosamine(DMN)causesasignificantdecreaseinratbodyandliverweight(DMN-Sgroup)comparedwithcontrolanimals(S-Sgroup).OralY27632(30mg/kg)essentiallypreventsthisDMN-inducedratbodyandliverweightloss(DMN-Ygroup)[4].PROTOCOLKinaseAssay[1]RecombinantROCK-I,ROCK-II,PKN,orcitronkinaseisexpressedinHeLacellsasMyc-taggedproteinsbytransfectionusingLipofectamine,andisprecipitatedfromthecelllysatesbytheuseof9E10monoclonalanti-MycantibodycoupledtoGprotein-Sepharose.Recoveredimmunocomplexesareincubatedwithvariousconcentrationsof[32P]ATPand10mgofhistonetype2assubstratesintheabsenceorpresenceofvarious2/4www.MedChemEwww.MedChemEconcentrationsofeitherY-27632orY-30141at30°Cfor30mininatotalvolumeof30μLofthekinasebuffercontaining50mMHEPES-NaOH,pH7.4,10mMMgCl2,5mMMnCl2,0.02%Briji35,and2mMdithiothreitol.PKCaisincubatedwith5μM[32P]ATPand200μg/mLhistonetype2assubstratesintheabsenceorpresenceofvariousconcentrationsofeitherY-27632orY-30141at30°Cfor10mininakinasebuffercontaining50mMTris-HCl,pH7.5,0.5mMCaCl2,5mMmagnesiumacetate,25μg/mLphosphatidylserine,50ng/mL12-O-tetradecanoylphorbol-13-acetateand0.001%leupeptininatotalvolumeof30μL.Incubationisterminatedbytheadditionof10μLof43Laemmlisamplebuffer.Afterboilingfor5min,themixtureissubjectedtoSDS-polyacrylamidegelelectrophoresisona16%gel.ThegelisstainedwithCoomassieBrilliantBlue,andthendried.Thebandscorrespondingtohistonetype2areexcised,andtheradioactivityismeasured[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.CellAssay[1]HeLacellsareplatedatadensityof3×104cellsper3.5-cmdish.ThecellsareculturedinDMEMcontaining10%FBSinthepresenceof10mMThymidinefor16h.AfterthecellsarewashedwithDMEMcontaining10%FBS,theyareculturedforanadditional8h,andthen40ng/mLofNocodazoleisadded.After11.5hoftheNocodazoletreatment,variousconcentrationsofY-27632(0-300μM),Y-30141,orvehicleisaddedandthecellsareincubatedforanother30min[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.AnimalMice[3]Administration[3][4]Male,inbredSwissalbinomice(2-3monthsold)weighing25-30gareused.Miceareinjectedwithasub-convulsivedoseofPTZ(35mg/kg,i.p.)(onMondays,WednesdaysandFridays)ofeachweekforatotalof11injections.AftereachPTZinjection,miceareobservedfor30minandtheoccurrenceofconvulsiveactivityisrecorded.After30min,themicearetheninjectedwitheitherFasudil(25mg/kg,i.p.)orY-27632(5mg/kg,i.p.)andreturnedtotheirhomecagesuntilthenextinjection.ControlmiceforFasudilandY-27632receivessaline.Rats[4]MaleWistarKindArats(200-250g)areused.DMN(1g/mL)isdilutedtentimeswithsaline(finalconcentration1%)and10mg/kgperdayofDMNisinjectedintraperitoneally(i.p.)onthefirst3daysofeachweekfor4weeks.Y27632isgivenorallyonceperdayatadoseof30mg/kgfor4weeksstartingonthedayofthefirstinjectionofDMN.Thedoseof30mg/kgcorrectshypertensioninseveralratmodelswithouttoxicity.Twentyratsarerandomizedintofourexperimentalgroups(n=5ineachgroup)asfollows:(1)S-S(injectionofsalinei.p.andoraladministrationofsaline);(2)S-Y(injectionofsalinei.p.andoraladministrationofY27632);(3)DMN-S(DMNi.p.andoraladministrationofsaline);(4)DMN-Y(DMNi.p.andoraladministrationofY27632).Theratsareweighedeveryweek.Theyaresacrificedattheendofthefourthweekandtheliverisexcised.Inaddition,abloodsampleistakenimmediatelybeforetheratsaresacrificed.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.户使⽤本产品发表的科研⽂献•Science.2020Dec4;370(6521):eaay2002.•CellStemCell.2020Oct1;27(4):646-662.e7.•CellStemCell.2020Jul2;27(1):125-136.e7.•CellStemCell.2020Jun4;26(6):845-861.e12.3/4www.MedChemEwww.MedChemE•AdvSci(Weinh).2020Jun17;7(15):1903583.Seemorecustomervalidationsonwww.MedChemEREFERENCES[1].IshizakiT,etal.PharmacologicalpropertiesofY-27632,aspecificinhibitorofrho-associatedkinases.MolPharmacol.2000May;57(5):976-83.[2].XueZW,etal.Rho-associatedcoiledkinaseinhibitorY-27632promotesneuronal-likedifferentiationofadulthumanadiposetissue-derivedstemcells.ChinMedJ(Engl).2012S