K-604-dihydrochloride-DataSheet-生命科学试剂-MedChemExpress

Hotline:400-820-3792Inhibitors•ScreeningLibraries•Proteinswww.MedChemEK-604dihydrochlorideCat.No.:HY-100400ACASNo.:217094-32-1分⼦式:C₂₃H₃₂Cl₂N₆OS₃分⼦量:575.64作⽤靶点:Acyltransferase作⽤通路:MetabolicEnzyme/Protease储存⽅式:4°C,sealedstorage,awayfrommoisture*Insolvent:-80°C,6months;-20°C,1month(sealed

storage,awayfrommoisture)溶解性数据体外实验H2O:100mg/mL(173.72mM;Needultrasonic)DMSO:62.5mg/mL(108.57mM;Needultrasonic)MassSolvent1mg5mg10mgConcentration制备储备液1mM1.7372mL8.6860mL17.3720mL5mM0.3474mL1.7372mL3.4744mL10mM0.1737mL0.8686mL1.7372mL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；⼀旦配成溶液，请分装保存，避免反复冻融造成的产品失效。储备液的保存⽅式和期限：-80°C,6months;-20°C,1month(sealedstorage,awayfrommoisture)。-80°C储存时，请在6个⽉内使⽤，-20°C储存时，请在1个⽉内使⽤。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%DMSO>>40%PEG300>>5%Tween-80>>45%salineSolubility:≥2.25mg/mL(3.91mM);Clearsolution2.请依序添加每种溶剂：10%DMSO>>90%(20%SBE-β-CDinsaline)1/3MasterofBioactiveMolecules—您⾝边的抑制剂⼤师www.MedChemESolubility:≥2.25mg/mL(3.91mM);Clearsolution3.请依序添加每种溶剂：10%DMSO>>90%cornoilSolubility:≥2.25mg/mL(3.91mM);Clearsolution4.请依序添加每种溶剂：PBSSolubility:100mg/mL(173.72mM);Clearsolution;Needultrasonicandwarmingandheatto60°CBIOLOGICALACTIVITY⽣物活性K-604dihydrochloride⼀种有效的选择性酰辅酶A：胆醇酰转移酶1(ACAT-1)抑制剂，IC50为0.45±0.06μM。IC50&TargetACAT-1ACAT-20.45μM(IC50)102.85μM(IC50)体外研究ThepotencyandselectivityofK-604forhumanACAT-1andACAT-2isexamined.TheIC50valueofK-604forhumanACAT-1is0.45μMandthatforhumanACAT-2is102.85μM,indicatingthatK-604is229-foldmoreselectiveforACAT-1thanACAT-2.Kineticanalysisindicatesthattheinhibitioniscompetitivewithrespecttooleoyl-coenzymeAwithaKivalueof0.378μM.K-604efficientlyinhibitscholesterolesterificationinhumanmacrophageswithIC50valueof68nM[1].Incellfreebiochemicalassays,K604at0.5µMinhibitsACAT1enzymaticactivityby70%withoutsignificantlyinhibitingtheACAT2enzymeactivity.ToinvestigatewhetherblockingACAT1increasesautophagyinneuronalcells,N2acellsaretreatedwithK604.Theresultshowsthatat0.1to1µM,K604inhibitsACATactivityby60-80%.NextK604isaddedtoN2acellsatconcentrationsfrom0.1to1µMfor24h,andLC3levelsareexaminedbywesternblot.TheresultshowsthatK604increasestheLC3-II/LC3-Iratio,areliablemarkerforautophagosomeformation,inadose-dependentmanner.ThenumberofthefluorescentLC3punctaissignificantlyincreasedinN2acellsafterK604treatment.Bywesternblot,K604significantlydecreasesthelevelsofp62inN2acells[2].体内研究UsingF1Bhamsters,ananimalmodelsusceptibletodiet-inducedhyperlipidemiaandatherosclerosis,theeffectsofK-604onaorticlesionareasandplasmacholesterollevelsareassessed.AdministrationofK-604doesnotaffectbodyweightorfoodconsumption.Theplasmacholesterollevelsinfat-fedhamstersare~12-foldhigherthanthoseinchow-fedhamsters,whicharesignificantlydecreasedbyK-604onlyatthehighestdosetested(30mg/kg)butnotatlowerdoses(1-10mg/kg).ThefattystreaklesionsstainwithoilredOaremarkedlyinducedbythehigh-fatdiet,whichissignificantlyreducedbyadministrationofK-604.Further,thehistologicalanalysesoftheatheroscleroticlesionsareperformed.Thefattystreaklesionsinthecontrolgrouparecharacterizedbyaccumulationoffoamymacrophagesinthesubendothelialspace.Incontrast,theareasoccupiedbyfoamymacrophagesaremarkedlyreducedbyadministrationofK-604[1].PROTOCOLKinaseAssay[1]ACATactivityisdeterminedbymeasuringtheproductionofcholesteryl[14C]oleate.MicrosomalfractionsderivedfromCHO-ACAT-1orCHO-ACAT-2cellsaredilutedwithCHAPSin0.5MKCl,0.5mMEDTA,and25mMTris-HCl(pH7.8)(bufferA)toafinalproteinconcentrationof1.0mg/mL.Themicrosomalfractions2/3MasterofBioactiveMolecules—您⾝边的抑制剂⼤师www.MedChemEcontaining9.2μgofproteinforACAT-1or4.9μgforACAT-2in10μLofbufferAaremixedwithvariousconcentrationsofK-604(0.4,0.6and0.8μM)orCI-1011in5μLofDMSO,andreconstitutedwith152.5μLofmixedmicellescontaining1.6mMCholesterol,11.2mMPhosphatidylcholine,and9.3mMTaurocholatein25,0.5mMEDTA,andTris-HCl(pH7.8).Thereactionisstartedbyadding10μLof0.45μM[14C]oleoyl-CoA,10nMfattyacid-freebovineserumalbumin(fattyacid-freeBSA)and25mMTris-HCl(pH7.8).Reactionmixturesareincubatedfor25minat37°Candterminatedbyadding150μLofChloroform/Methanol(2:1).Theorganicphaseisdriedunderastreamofnitrogen.Thelipidsareresuspendedwith150μLofChloroform/Methanol(2:1)anddevelopedbythinlayerchromatography(TLC)usinghexane/diethylether/aceticacid(75:25:1).Radioactivitiesofcholesterol[14C]oleatearedeterminedusingaBAS2500imageanalyzer[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.CellAssay[2]ThemouseneuroblastomacelllineN2aisculturedinDMEM/Opti-MEM(50:50)with10%fetalbovineserum(FBS)at37°Cwith5%CO2inahumidifiedincubator.Cellsareincubatedfor24hwiththeACAT1-specificinhibitorK604(0.1,0.5and1µM)orisotype-nonspecificACATinhibitorCI-1011.Primarycorticalneuronsareisolatedfrommousebrainsatpostnatalday0-3.Corticalneuronsareplatedin6wellplatesat350,000cells/wellandgrownin2mL/wellNeurobasalAwith1×B27,0.5mML-glutamine,and5ng/mLfibroblastgrowthfactor.Halfofthemediaisreplacedwithfreshmediaonceevery7days.After14-21daysinculture,cellsareusedforindividualexperiments[2].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.AnimalHamsters[1]Administration[1]MaleF1Bhamsters(n=30)at8weeksoldareusedforanimalexperiments.Theanimalroomiscontrolledat23±3°Candrelativehumidityof50±20%.AnimalsarefedaCE-2chowdiet,followedbysupplementationwithCE-2containing0.3%cholesteroland10%coconutoilfor10weeks.Duringfatloading,K-604isadministeredorallyat1,3,10,or30mg/kg/day(n=6foreachdosegroup).Thecontrolgroup(n=6)receiveanaqueoussolutionof0.5%methylcelluloseinsteadofK-604.Tapwaterisgivenadlibitum.Bodyweightismeasuredatthetimeofdrugadministration.Bloodissampledfordeterminationofplasmacholesterollevelsusingacommerciallyavailablekit(CholesterolE-Test).MCEhasnotindependentlyconf