K-604-dihydrochloride-DataSheet-生命科學試劑-MedChemExpress

Hotline:400-820-3792Inhibitors?ScreeningLibraries?Proteinswww.MedChemEK-604dihydrochlorideCat.No.:HY-100400ACASNo.:217094-32-1分?式:C??H??Cl?N?OS?分?量:575.64作?靶點:Acyltransferase作?通路:MetabolicEnzyme/Protease儲存?式:4°C,sealedstorage,awayfrommoisture*Insolvent:-80°C,6months;-20°C,1month(sealed

storage,awayfrommoisture)溶解性數據體外實驗H2O:100mg/mL(173.72mM;Needultrasonic)DMSO:62.5mg/mL(108.57mM;Needultrasonic)MassSolvent1mg5mg10mgConcentration制備儲備液1mM1.7372mL8.6860mL17.3720mL5mM0.3474mL1.7372mL3.4744mL10mM0.1737mL0.8686mL1.7372mL請根據產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，避免反復凍融造成的產品失效。儲備液的保存?式和期限：-80°C,6months;-20°C,1month(sealedstorage,awayfrommoisture)。-80°C儲存時，請在6個?內使?，-20°C儲存時，請在1個?內使?。體內實驗請根據您的實驗動物和給藥?式選擇適當的溶解?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：(為保證實驗結果的可靠性，澄的儲備液可以根據儲存條件，適當保存；體內實驗的?作液，建議您現?現配，當天使?；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沉淀、析出現象，可以通過加熱和/或超聲的?式助溶)1.請依序添加每種溶劑：10%DMSO>>40%PEG300>>5%Tween-80>>45%salineSolubility:≥2.25mg/mL(3.91mM);Clearsolution2.請依序添加每種溶劑：10%DMSO>>90%(20%SBE-β-CDinsaline)1/3MasterofBioactiveMolecules—您?邊的抑制劑?師www.MedChemESolubility:≥2.25mg/mL(3.91mM);Clearsolution3.請依序添加每種溶劑：10%DMSO>>90%cornoilSolubility:≥2.25mg/mL(3.91mM);Clearsolution4.請依序添加每種溶劑：PBSSolubility:100mg/mL(173.72mM);Clearsolution;Needultrasonicandwarmingandheatto60°CBIOLOGICALACTIVITY?物活性K-604dihydrochloride?種有效的選擇性酰輔酶A：膽醇酰轉移酶1(ACAT-1)抑制劑，IC50為0.45±0.06μM。IC50&TargetACAT-1ACAT-20.45μM(IC50)102.85μM(IC50)體外研究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].體內研究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?邊的抑制劑?師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