PD98059isaMEKinhibitorwithIC₅₀of5μM,alsosuppressesTCDDbindingtothearylhydrocarbonreceptor(AHR)withIC₅₀of4μM.

IC50:5μM(MEK)^[1]

ConcentrationsofPD98059of≤20μMarenotcytotoxictoculturedMCF10A,MCF10A-Neo,andMCF10A-NeoTcells.However,PD98059isweaklycytostatictoallthreelinesatconcentrationsof≥10μM.TreatmentofMCF10A-NeoandMCF10A-NeoTcultureswithconcentrationsofPD98059upto20μMfor2-22hrdoesnotalterthetotalERKcontent.However,treatmentwithPD98059doesresultinconcentration-dependentreductionsintheduallyphosphorylatedformsofERK1andERK2.Within2hrofa10-μMtreatment,phosphorylatedERKcontentsarereduced~74%and~86%inMCF10A-NeoandMCF10A-NeoTcultures,respectively(IC₅₀=1μM).Within22hroftreatment,phosphorylatedERKformsarealmostcompletelyeliminatedinbothcelllines^[1].PD98059(PD098059)preventstheactivationofMAPKK1byRaforMEKkinaseinvitroatconcentrations(IC₅₀=2-7μM).PD98059inhibitsboththeactivationandphosphorylationofMAPKK1invitrobyeitherc-RaforMEKkinasewithIC₅₀valuesof4±2μM.IncubationofSwiss3T3cellswithPD98059(50μM)suppressedby80-90%theactivationofMAPKKinducedbyeachagoNIST,buttheactivationofc-Rafisenhanced2-3-fold^[2].

ThetreatmentofmicewithPD98059significantlyreducesthelevelofp-ERK1/2.Moreover,asignificantincreaseinthephospho-p38expressionisobservedinZymosan-treatedmiceat18hafterZymosanadministrationcomparedtothesham-operatedmice.ThetreatmentwithPD98059significantlyreducesthep38expression^[3].RepeatedtreatmentwithPD98059attenuatesmechanicalallodyniameasuredbythevonFreytestthree(18.0g±0.8,n=10)andseven(20.21g±0.67,n=26)daysafterCCIincomparisontothevehicle-treatedCCI-exposedrats(15.1g±1.3,n=7and14.21g±0.44,n=28,respectively).RepeatedinjectionofPD98059diminishesthermalhyperalgesia,asisevaluatedbythecoldplatetest,three(17.5s±2.1,n=10)andseven(25.54s±1.03,n=26)daysfollowingCCIcomparedtovehicle-treatedCCI-exposedrats(11.5s±1.8,n=7and11.4s±0.88,n=28,respectively)^[4].

• [1].ReinersJJJr,etal.PD98059isanequipotentantagonistofthearylhydrocarbonreceptorandinhibitorofmitogen-activatedproteinkinasekinase.MolPharmacol.1998Mar;53(3):438-45.

  [2].AlessiDR,etal.PD098059isaspecificinhibitoroftheactivationofmitogen-activatedproteinkinasekinaseinvitroandinvivo.JBiolChem,1995,270(46),27489-27494.

  [3].DiPaolaR,etal.PD98059,aspecificMAPkinaseinhibitor,attenuatesmultipleorgandysfunctionsyndrome/failure(MODS)inducedbyzymosaninmice.PharmacolRes.2010Feb;61(2):175-87.

  [4].RojewskaE,etal.PD98059InfluencesImmuneFactorsandEnhancesOpioidAnalgesiainModelofNeuropathy.PLoSOne.2015Oct1;10(10):e0138583.

Kinasereactionsareperformedin50μLreactionvolumesandcontain50mMTris,pH7.4,10mMMgCl₂,2mMEGTA,10μMATP(containing1μCiof3000Ci/mmol[γ-³²P]ATP),7.6μgofGST-MEK1,7.2μgofGST-ERK1,and20μgofMBP.PD98059andotherflavonoidsareaddedtothereactionsmixturesimmediatelyaftertheadditionofGST-MEK1butbeforetheadditionofGST-ERK1andATP.ControlreactionscontainERK1andMBPbutnoMEK.Reactionmixturesareincubatedat30°Cfor15minbeforebeingstoppedbytheadditionofLaemmli’sSDSsamplebuffer.ProteinsareseparatedonSDS-15%polyacrylamidegels.Aftervacuumdryingofthegel,rADIoactivityisdetectedbyautoradiographyonX-rayfilmorphosphoimagingusingaBioRadGS-525MolecularImager^[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

PD98059isdissolvedinDMSOandstored,andthendilutedwithappropriatemedia(DMSO<0.1%) before="">^[1].

TheMCF10A,MCF10A-Neo,andMCF10A-NeoTcelllinesareused.SubconfluentculturesaretreatedwithPD98059(0-100μM).ViABIlityofcellsaftertreatmentisassessedbyabilitytoexcludetrypanblue.CulturesearmarkedforRNAisolationarewashedtwicewithphosphate-bufferedsaline(2.7mMKCl,1.5mMKH₂PO₄,137mMNaCl,8mMNa₂HPO₄,pH7.2)atharvestingandstoredat-80°C^[1].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

PD98059ispreparedinnon-pyrogenicsaline(0.9%NaCl)(Mice)^[3].
PD98059isdissolvedin75%DMSO(Rat)^[4].

Mice^[3]
MaleCDmice(20-22g)arerandomlyallocatedintothefollowinggroups:1.Zymosan+DMSOgroup.Micearetreatedintraperitoneally(i.p.)withZymosan(500mg/kg,sUSPendedinsalinesolution)andwiththevehicleforPD98059(10%DMSO,v/v)i.p.1and6hafterZymosanadministration(N=10).2.PD98059group.IdenticaltotheZymosan+DMSOgroupbutareadministeredPD98059(10mg/kg,i.p.bolus)at1and6hafterZymosan(N=10)insteadofDMSO.3.Sham+DMSOgroup.IdenticaltotheZymosan+DMSOgroupbutareadministeredsalinesolutioninsteadofZymosan(N=10).4.Sham+PD98059group.IdenticaltoSham+DMSOgroup,exceptfortheadministrationofPD98059(10mg/kgi.p.bolus)1and6haftersalineadministration(N=10).
Rat^[4]
Therats(maleWistar,300-350g)areused.ThePD98059(2.5μg/5μL,i.t.)issingleorrepeatedpreemptivelyadministered16hand1hbeforeCCIandthenoncedailyfor7days.TheVehicle-treatedCCI-exposedratsreceive75%DMSOaccordingtothesameschedule.Thereisnosignificantdifferenceinpainbehaviorbetweenno-treatedandV(DMSO)-treatedCCI-exposedrats.ThismethodofPD98059orvehicleadministrationisusedthroughoutthestudyandisreferredtointhetextas“repeatedadministration”.Atday7^thafterCCI30minafterPD98059administrationtactileallodyniaismeasuredusingvonFreytestandthermalhyperalgesiaisconductedusingcoldplatetest.Additionally,atday7^thafterCCIthevehicle-treatedandPD98059-treatedratsreceiveasinglei.t.vehicle,Morphine(2.5μg/5μL)orBuprenorphine(2.5μg/5μL)injection30minafterPD98059,andthen30minlaterthevonFreyand/orcoldplatetestsarerepeated.Sincethedoseofmorphine2.5μg/5μLinnaiveratsproducesmaximalanalgesiceffectintail-flicktest.LowerdoseofMorphineareusedforco-administrationexperiments,sothatobservingthepossIBLeenhancementofopioideffectiveness.Thevehicle-treatedandPD98059-treatednaiverats(uninjuredrats)receiveasinglei.t.vehicle,Morphine(0.5μg/5μL)orBuprenorphine(2.5μg/5μL)injection30minafterPD98059,andthen30minlaterthetailflicktestisperformed.MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

• [1].ReinersJJJr,etal.PD98059isanequipotentantagonistofthearylhydrocarbonreceptorandinhibitorofmitogen-activatedproteinkinasekinase.MolPharmacol.1998Mar;53(3):438-45.

  [2].AlessiDR,etal.PD098059isaspecificinhibitoroftheactivationofmitogen-activatedproteinkinasekinaseinvitroandinvivo.JBiolChem,1995,270(46),27489-27494.

  [3].DiPaolaR,etal.PD98059,aspecificMAPkinaseinhibitor,attenuatesmultipleorgandysfunctionsyndrome/failure(MODS)inducedbyzymosaninmice.PharmacolRes.2010Feb;61(2):175-87.

  [4].RojewskaE,etal.PD98059InfluencesImmuneFactorsandEnhancesOpioidAnalgesiainModelofNeuropathy.PLoSOne.2015Oct1;10(10):e0138583.

267.28

C₁₆H₁₃NO₃

167869-21-8

RoomtemperatureincontinentalUS;mayvaryelsewhere

DMSO:16mg/mL;H₂O:<0.01="">

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