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Medchemexpress/GSK583/HY-100339/5mg
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Medchemexpress/GSK583/HY-100339/5mg
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GSK583isahighlypotentandselectiveinhibitorofRIP2Kinase,withIC50of5nM.

CustomerValidation

  • JImmunol.2017May1;198(9):3729-3736.
Description

GSK583isahighlypotentandselectiveinhibitorofRIP2Kinase,withIC50of5nM.

IC50&Target

IC50:5nM(RIP2K)[1]

InVitro

GSK583(1μM)exhibitsexcellentselectivityinapanelof300kinases,includingp38αandVEGFR2.GSK583potentlyanddosedependentlyinhibitsMDP-stimulatedtumornecrosisfactor-alpha(TNFα)productionwithanIC50of8nM.GSK583demonstratesonlyamodestreductioninpotencywhenprofiledinasimilarMDP-inducedTNFαproductionassayinhumanwholeblood(IC50=237nM)andratwholeblood(IC50=133nM)[1].

InVivo

GSK583(0.1,1,and10mg/kg,p.o.)inhibitsserumKC(therodentorthologueofIL-8)levelsinratsinadose-dependentmanner,withanIC50derivedfromratbloodconcentrationsof50nM(or20ng/mL).Similarly,GSK583inhibitsserumKClevelsandrecruitmentofneutrophilsintotheperitonealcavityinmiceinadose-dependentmanner,withanIC50of37nM(15ng/mL)derivedfrommousebloodconcentration[1].

References
  • [1].HailePAetal.TheIdentificationandPharmacologicalCharacterizationof6-(tert-Butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine(GSK583),aHighlyPotentandSelectiveInhibitorofRIP2Kinase.JMedChem,2016May26,59(10):4867-80.

PreparingStockSolutions
ConcentrationVolumeMass1mg5mg10mg
1mM2.5097mL12.5486mL25.0972mL
5mM0.5019mL2.5097mL5.0195mL
10mM0.2510mL1.2549mL2.5097mL
Pleaserefertothesolubilityinformationtoselecttheappropriatesolvent.
KinaseAssay
[1]

AfluorescentpolarizationbasedbindingassayisdevelopedtoquantitateinteractionofnoveltestcompoundsattheATPbindingpocketofRIP2KbycompetitionwithafluorescentlylabeledATPcompetitiveligand.FulllengthFLAGHistaggedRIP2KispurifiedfromabaculovirusexpressionsystemandisusedatafinalassayconcentrationoftwicetheKDapparent.AfluorescentlabeledligandthatisreversIBLeandcompetitivewiththeinhibitorsisusedatafinalassayconcentrationof5nM.Boththeenzymeandligandarepreparedinsolutionsin50mMHEPESpH7.5,150mMNaCl,10mMMgCl2,1mMDTT,and1mMCHAPS.Testcompoundsarepreparedin100%DMSO,and100nLisdispensedtoindividualwellsofamultiwellplate.Next,5μLofRIP2Kisaddedtothetestcompoundsattwicethefinalassayconcentrationandincubatedatroomtemperaturefor10min.Followingtheincubation,5μLofthefluorescentlabeledligandsolutionisaddedtoeachreactionattwicethefinalassayconcentrationandincubatedatroomtemperatureforatleast10min.Finally,samplesarereadonaninstrumentcapableofmeasuringfluorescentpolarization.Testcompoundinhibitionisexpressedaspercent(%)inhibitionofinternalassaycontrols.Forconcentrationresponseexperiments,normalizeddataarefitusingthefollowingfourparameterlogisticequation:y=A+((B-C))/(1+(10x)/(10C)D),whereyisthe%activity(%inhibition)ataspecifiedcompoundconcentration,Aistheminimum%activity,Bisthemaximum%activity,C=log10(IC50),D=Hillslope,x=log10(compoundconcentration[M]),andpIC50=(−C).MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

AnimalAdmiNISTration
[1]

Mice[1]
FemaleC57Bl/6mice(forcytokineanalyses)ormaleBalb/cmice(forperitonealneutrophilanalyses)(n=10/treatmentgroup)aredosedorally15minpriortoMDPchallengewithvehicleorGSK583(0.1,1,or10mg/kg).Forperitonealneutrophilanalysis,micearesacrificedat4hpost-MDPchallenge(30μg,i.p.)andperitonealfluidiscollectedbylavage.PeritonealneutrophilsarequantifiedbyFACSanalysis.
Rat[1]
FemaleCrl:CD(SD)rats(n=8/treatmentgroup)aredosedorallywithvehicleorGSK58315minpriortoMDPchallenge(150μg/rat,IV).At2hpostMDPchallenge,ratsaresacrificedandterminalserumispreparedfrombloodcollectedviacardiacstick.Serumcytokinelevels(IL-6,IL-8orKC,IL-1β,andTNFα)arequantifiedbytheMSDplatform.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

References
  • [1].HailePAetal.TheIdentificationandPharmacologicalCharacterizationof6-(tert-Butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine(GSK583),aHighlyPotentandSelectiveInhibitorofRIP2Kinase.JMedChem,2016May26,59(10):4867-80.

MolecularWeight

398.45

Formula

C₂₀H₁₉FN₄O₂S

CASNo.

1346547-00-9

Storage
Powder-20°C3years
 4°C2years
Insolvent-80°C6months
 -20°C1month
Shipping

RoomtemperatureincontinentalUS;mayvaryelsewhere

Solvent&Solubility

DMSO:≥37mg/mL

*"<1 mg/ml"="" means="" slightly="" soluble="" or="" insoluble.="" "≥"="" means="" soluble,="" but="" saturation="">

Purity:98.07%