E-64-DataSheet-生命科學(xué)試劑-MedChemExpress

Hotline:400-820-3792Inhibitors?Agonists?ScreeningLibrarieswww.MedChemEE-64Cat.No.:HY-15282CASNo.:66701-25-5分?式:C??H??N?O?分?量:357.41作?靶點(diǎn):Cathepsin;Autophagy;Bacterial作?通路:MetabolicEnzyme/Protease;Autophagy;Anti-infection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:50mg/mL(139.90mM;Needultrasonic)掃描?維碼，H2O:36mg/mL(100.72mM;Needultrasonicand運(yùn)?溶解?案計(jì)算器warming)獲得適合您實(shí)驗(yàn)體系的溶解?案MassSolvent1mg5mg10mgConcentration制備儲(chǔ)備液1mM2.7979mL13.9895mL27.9791mL5mM0.5596mL2.7979mL5.5958mL10mM0.2798mL1.3990mL2.7979mL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:≥2.08mg/mL(5.82mM);Clearsolution此?案可獲得≥2.08mg/mL(5.82mM，飽和度未知)的澄溶液。以1mL?作液為例，取100μL20.8mg/mL的澄DMSO儲(chǔ)備液加到400μLPEG300中，混合均勻；向上述體系中加?50μLTween-80，混合均勻；然后繼續(xù)加?450μL?理鹽?定容?1mL。1/4www.MedChemEwww.MedChemE2.請(qǐng)依序添加每種溶劑：10%DMSO90%(20%SBE-β-CDinsaline)Solubility:≥2.08mg/mL(5.82mM);Clearsolution此?案可獲得≥2.08mg/mL(5.82mM，飽和度未知)的澄溶液。以1mL?作液為例，取100μL20.8mg/mL的澄DMSO儲(chǔ)備液加到900μL20%的SBE-β-CD?理鹽??溶3.液中，混合均勻。請(qǐng)依序添加每種溶劑：10%DMSO90%cornoilSolubility:≥2.08mg/mL(5.82mM);Clearsolution此?案可獲得≥2.08mg/mL(5.82mM，飽和度未知)的澄溶液，此?案不適?于實(shí)驗(yàn)周期在半個(gè)?以上的實(shí)驗(yàn)。以1mL?作液為例，取100μL20.8mg/mL的澄DMSO儲(chǔ)備液加到900μL??油中，混合均勻。BIOLOGICALACTIVITY?物活性E-64(ProteinaseinhibitorE64)?種有效的不可逆的半胱氨酸蛋?酶(cysteineproteases)抑制劑，抑制papain的IC50為9nM。IC50&TargetIC50:9nM(Papain)[1]體外研究E-64(ProteinaseinhibitorE64)isacathepsinB-specificinhibitor,anditsbindingmodeswithpapain,actinidin,cathepsinL,andcathepsinKhavebeenreviewedattheatomiclevel.E-64hasbeenwidelyusedasapotentandirreversible(covalent-type)inhibitorformanycysteineproteasessuchaspapain,ficin,actinidin,cathepsinBandL[1].TheS.cerviadultparasitesareincubatedintheKreb'sRingerbicarbonate(KRB)maintenancemediumfor8hat37°C,5%CO2with5,10,20and40μMconcentrationofE-64.E-64showsaconcentrationandtimedependentdecreaseinmotilityandviabilityoftheparasites(EC50=16μM)[2].體內(nèi)研究AbroadspectrumofexpressionofCD4andCD19isfoundexistsinboththeisletsandpancreaticlymphnodes(PLNs)andthatanti-serpinB13mAbexposurecausesasignificantshiftthatfavoredcellsexpressinglow-to-intermediateamountsofthesemarkers.However,thisshiftisabolishedinanimalsthatreceiveanti-serpinB13mAbinthepresenceoftheproteaseinhibitorE-64(ProteinaseinhibitorE64),whichmaintainsitsblockingactivityundertheexperimentalconditionsused[3].Dahlsalt-sensitive(SS)ratsarefedan8%highsaltNaCldietandintravenouslyinfusedwiththeirreversiblecysteinecathepsininhibitorE-64(1mg/day)orthevehicle(control).BoththecontrolandE-64infusedgroupsdevelopesignificanthypertensionandkidneydamage,andnodifferenceofthemeanarterialpressureandthehypertension-associatedalbuminuriaisobservedbetweenthegroups[4].PROTOCOLKinaseAssay[2]TheCathepsinBactivityisdeterminedusingZ-Arg-Arg-4mβNAassubstratewithslightmodifications.Thecrudeextractispre-incubatedat37°Cfor5minin50mMsodiumacetatebuffer,pH5.0containing1mMEDTAand5mMDTT.Thesubstrate(finalconcentration,100μM)isaddedtomakethefinalassayvolumeof1mL.Thereactionmixtureisincubatedat37°Cfor30min.ThereactionisterminatedbyaddingequalvolumeofstoppingreagentcontainingFastGarnetGBCsalt(1mg/mL),10mMpHMBand50mMEDTA,pH6.0.Theextractionofproduct,β-napthylamine(β-NA),iscarriedoutwithn-butanol.Aftercompletelayer2/4www.MedChemEwww.MedChemEseparation,theabsorbanceismeasuredinn-butanollayerandactivityiscalculatedusingmolarextinctioncoefficientofβ-napthylaminesolutionas31.5M/cmpersecat520nm.Oneunitofenzymeactivityisdefinedastheamountofenzymeliberating1μmolofβNAperminuteat37°C.Thehalfmaximalinhibitoryconcentration(IC50)iscalculatedbyplottingthegraphbetweenthedifferentconcentrationofE-64andthe%inhibitionincathepsinBactivity.Here,IC50indicatestheconcentrationoftheE-64requiredtoinhibittheparasiticcathepsinBactivitybyhalf[2].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.AnimalMice[3]Administration[3][4]TheNOD/LtJandBDC2.5Tcellreceptor(TCR)transgenicNODmiceareusedtostudytheeffectsoftreatmentwithanti-serpinB13monoclonalantibody(mAb).Four-week-oldfemaleNOD/LtJmiceareinjectedintravenouslyfourtimesoveraperiodof10dayswithanti-serpinB13mAb(100μg/injection).Inaddition,duringthesameperiod,someanimalsarealsoinjectedintraperitoneallywiththeproteaseinhibitorE64at10mg/kg/dayforseveraldays.Controlmicearetreatedwithdiluent(asterilizedPBSsolutioncontaining10%DMSO)andcontrolIgG.ThesolutionscontainingE64orDMSOarepreparedimmediatelybeforeuse.Twenty-fourhoursafterthelastinjection,themicearekilled,andcellsfromtheirlymphoidorgansandpancreaticisletsaresubjectedtoFACSanalysis.Rats[4]Seven-weekoldmaleDahlSaltSensitiverats(SS/JrHsdMcwi)areused.Briefly,8-weekoldanesthetizedSS

ratshavetheirleftfemoralarteryandveincatheterized.Bothcathetersarefixedandexteriorizedfromthe

backoftheneckandthearteriallineisconnectedtoaheparinizedsalineinfusionpumpthatisinlinewitha

bloodpressuretransducer,andthevenouslineisconnectedtoasalineinfusionpump.Animalsareallowed

360°movementusingatether-swivelsystem.Thispreparationallowedchronicvenousinfusionandarterial

bloodpressuremeasurementinconscious,freelymovingrats.Astablebaselinebloodpressureisobtained

for4dayspriortoswitchingbothgroupstoan8.0%NaCldietandthesimultaneousadditionofE-64(1

mg/day;280mMstockinDMSO)orthevehicle(DMSOinsaline)controltothevenouscatheter.DailyMAP

iscalculatedbyaveragingMAPtakeneveryminoverthebeginning3hperiodoftheratsleepcycle.

MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.戶使?本產(chǎn)品發(fā)表的科研?獻(xiàn)?CellProlif.2019May;52(3):e12609.?LWT-FOODSCITECHNOL.2018Jan,87;186-193?IntJOncol.2019Jul;55(1):331-339.?DevCompImmunol.2018Jan;78:114-123.?FEBSLett.2021Jan;595(2):169-182.Seemorecustomervalidationsonwww.MedChemEREFERENCES[1].MatsumotoK,etal.StructuralbasisofinhibitionofcysteineproteasesbyE-64anditsderivatives.Biopolyme