GW-5074-DataSheet-生命科學(xué)試劑-MedChemExpress

Hotline:400-820-3792Inhibitors?Agonists?ScreeningLibrarieswww.MedChemEGW5074Cat.No.:HY-10542CASNo.:220904-83-6分?式:C??H?Br?INO?分?量:520.94作?靶點(diǎn):Raf;Apoptosis作?通路:MAPK/ERKPathway;Apoptosis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:≥100mg/mL(191.96mM)掃描?維碼，*"≥"meanssoluble,butsaturationunknown.運(yùn)?溶解?案計(jì)算器獲得適合您實(shí)驗(yàn)體系的溶解?案MassSolvent1mg5mg10mgConcentration制備儲(chǔ)備液1mM1.9196mL9.5980mL19.1961mL5mM0.3839mL1.9196mL3.8392mL10mM0.1920mL0.9598mL1.9196mL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，并請(qǐng)注意儲(chǔ)備液的保存?式和期限。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(guò)程中出現(xiàn)沉淀、析出現(xiàn)象，可以通過(guò)加熱和/或超聲的?式助溶1.請(qǐng)依序添加每種溶劑：10%DMSO40%PEG3005%Tween-8045%salineSolubility:≥2.5mg/mL(4.80mM);Clearsolution此?案可獲得≥2.5mg/mL(4.80mM，飽和度未知)的澄溶液。以1mL?作液為例，取100μL25.0mg/mL的澄DMSO儲(chǔ)備液加到400μLPEG300中，混合均勻；向上述1/4www.MedChemEwww.MedChemE體系中加?50μLTween-80，混合均勻；然后繼續(xù)加?450μL?理鹽?定容?1mL。BIOLOGICALACTIVITY?物活性GW5074?種有效的，選擇性的c-Raf抑制劑，IC50值為9nM；對(duì)JNK1/2/3，MEK1，MKK6/7，CDK1/2，c-Src，p38MAP，VEGFR2或c-Fms等沒(méi)有作?。IC50&Targetc-Raf9nM(IC50)體外研究GW5074isapotentandspecificinhibitorofc-RafwithIC50of9nMandhasnoeffectofMKK6,MKK7,p38MAPkinaseandcdksinvitro.However,treatmentofneuronalcultureswithGW5074permitsaccumulationofactivatingmodificationsonc-RafandalsoB-Raf.TheinhibitionofLK-inducedapoptosisbyGW5074incerebellargranuleneuronsisnotMEK-ERK-dependent.GW5074delaysdown-regulationofAktactivitybutinhibitsapoptosisbyanAkt-independentmechanism.GW5074affectsRas,nuclearfactor-kappaBandc-jun.GW5074inhibitscelldeathcausedbyneurotoxinsingranulecellsandotherneuronaltypes[1].體內(nèi)研究GW5074(5mg/Kg)completelypreventsextensivebilateralstriatallesionsinducedby3-NPinmice[1].GW5074suppressessidestreamsmoke-inducedairwayhyperresponsivenessinmice[2].PROTOCOLKinaseAssay[1]Briefly,foreachassay5-10mUofpurifiedkinaseisused.ForGSK3β,cdk1,cdk2,cdk3,cdk5,thekinaseisincubatedwith1μMGW5074inabuffercontaining8mMMOPS,pH7.2,0.2mMEDTA,10mMmagnesiumacetateand[c-33P-ATP]for40minatroomtemperature.Kinaseactivityisquantifiedbymeasuring33PincorporationbyspottinganaliquotonP30filters,washingin50mMphosphoricacidandscintillationcounting.Thebuffercompositionforc-Raf,JNK1,JNK2,JNK3,MEK1,MKK6,MKK7is50mMTrispH7.5,0.1mMEGTA,10mMmagnesiumacetateand[c-33P-ATP].Thepeptidesubstratesusedareasfollows:Forc-Raf,0.66mg/mLMBP;forcdks,0.1mg/mLhistoneH1;forJNKs,3μMATF2;forMEK1,1μMMAPK2;forMKK6,1μMofSAPK2aandforMKK7,2μMJNK1α.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.CellAssay[1]Befiy,thetetrazoliumsaltMTTisaddedtotheculturesatafinalconcentrationof1mg/mL,andincubationofthecultureiscontinuedintheCO2incubatorforaHirther30minat37°C.Theassayisstoppedbyaddinglysisbuffer[20%sodiumdodecyisulfate(SDS)in50%N,N-dimethylformamide,pH4.7],Theabsorbanceismeasuredspectrophotometricallyat570nmafteranovemightincubationatroomtemperature.Theabsorbanceofawellwithoutcellsisusedasbackgroundandsubtracted.Dataarepresentedasmean±standarddeviation.StatisticalanalysisisperfomiedusingANOVAandStudent-Neuman-Keuls'test.BesidesMTTassays,viabilityisalsoquantitiedusingthetluorescein-diacetatemethodandbyDAPIstaining(whichrevealsapoptoticnucleiascondensedorfragmented).TheresultsusingtheseassaysaresimilartothoseobtainedwiththeMTTassay.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.2/4www.MedChemEwww.MedChemEAnimalTheTru-Scan?activitymonitoringsystemisusedtoassesslocomotoractivityonmiceonthedayfollowingAdministration[1]the5daysofinjectionwithsaline,3-NPoracombinationof3-NPandGW5074(7animalsineachgroup).TheanimalisplacedinaPerspexarena(25.9×25.9cm)witbinfraredbeamsspacedat0.6-inchintervalsintheX-Yplane.ThearenaisalsoequippedwithasecondinfraredbeamsystemattheZplanepositioned2.54cmabovetheX-Yplane.Inthissystemthemovementoftheanimalisaccuratelyassessedbyinterruptionsin17×17-gridsystemcreatedbytheinfraredbeamsinboththeX-YandZplanes.Theanimalisallowedtoremaininthearenafor15min,withdatacollectionperformedduringthisperiodusingtheTruScanLineinterfaceboxandTmScan99software,operatingthroughaPentiumPC.Thefollowingbehavioralparametersareselected:(i)Totalmovementsepisodes:eachmovementintheflorplaneisaseriesofcoordinatechangeswithnorestforatleastIsampleinterval;(ii)totalmovementdistance:thesumofallvectoredA-Ycoordinatechangesinthefloorplane;(iii)meanvelocity:themeanvelocityofallX-Ycoordinatechangedefinedmovements;and(iv)verticalplaneentries:thetotalnumberoftimesanypartoftheanimalenteredtheverticalplane(Zplane).MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.戶(hù)使?本產(chǎn)品發(fā)表的科研?獻(xiàn)?BiochemJ.2019Mar12;476(5):875-887.?SciRep.2020Jul7;10(1):11158.?AnnTranslMed.2019Dec;7(23):758.?HarvardMedicalSchoolLINCSLIBRARYSeemorecustomervalidationsonwww.MedChemEREFERENCES[1].ChinPC,etal.Thec-RafinhibitorGW5074providesn