DorsomorphindihydrochlorideisapotentandselectiveAMPKinhibitor,thatiscompetitivewithATP,withK_iof109±16nMintheabsenceofAMP.

Ki:109±16nM(AMPK)^[1]

HT1080cellsaretreatedwith10μMDorsomorphinfor2hunder2DGstress.ImmunoblotanalysisrevealsthatphosphorylationlevelsofthecatalyticαsubunitofAMPKareincreasedbyexposureofHT1080cellsto2DG,whereasbothbasaland2DG-inducedphosphorylationlevelsareclearlyreducedwhenDorsomorphinisadded.MeasurementsofcellularkinaseactivityusinganELISA-basedassaysystemconfirmedthatDorsomorphindoesreducetheendogenousAMPKactivityregardlessofcellcultureconditions^[2].

AdmiNISTrationofDorsomorphinover24hleadstoa60%increaseintotalserumironconcentrations.Dorsomorphintreatmentisthereforeeffectiveinreducingbasallevelsofhepcidinexpressionandincreasingserumironconcentrationsinadultmice^[3].

• [1].ZhouG,etal.RoleofAMP-activatedproteinkinaseinmechanismofmetforminaction.JClinInvest.2001Oct;108(8):1167-74.

  [2].SaitoS,etal.CompoundCpreventstheunfoldedproteinresponseduringglucosedeprivationthroughamechanismindependentofAMPKandBMPsignaling.PLoSOne.2012;7(9):e45845.

  [3].YuPB,etal.DorsomorphininhibitsBMPsignalsrequiredforembryogenesisandironmetabolism.NatChemBiol.2008Jan;4(1):33-41.

  [4].ZhangDY,etal.Roleofautophagyanditsmolecularmechanismsinmiceintestinaltractaftersevereburn.JTraumaAcuteCareSurg.2017Oct;83(4):716-724.

HT1080cellsareseededin24-wellplates(2×10⁴cellsperwell)andtreatedwithDorsomorphininthepresenceorabsenceofglucoseor10mM2DGfor2h.HT1080cellsthatoverexpressedthewild-typeanddominantnegativeAMPKα1arepreparedbytransfectingplasmidDNA(pAMPKα1-wt,pAMPKα1-D168AandpcFlagasacontrol)in6-wellplates,seedingin24-wellplateandtreatingwithUPRinhibitors.Cellsarelysedwithcelllysisbuffer(20mMTris-HCl,pH7.5,250mMNaCl,10%glycerol,0.5%NP-40,1mMEDTA,1mMEGTA,0.2mMPMSF,1μg/mLpepstatin,0.5μg/mLleupeptin,5mMNaF,2mMNa₃Vo₄,2mMβ-glycerophosphate,1mMDTT).RelativeAMPKkinaseactivity(mean±SDofduplicatedeterminations)tocontrolsample(vehicleorpcFlagundernormalgrowthconditions)isdeterminedusingtheCycLexAMPKkinaseassaykit^[2].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

DorsomorphinisdissolvedinDMSO(10mM)andstored,andthendilutedwithappropriatemedia(DMSO0.5%)beforeuse^[2].

HeLaand786-OcellsaretreatedwithvariousconcentrationsofDorsomorphin(0,0.3,1,3,10μM),VersipelostatinandPhenformininthepresenceorabsenceof10mM2DGor1μg/mLofTunicamycinasastressorfor30hin96-wellplates.Forthecombinationstudy,786-OcellsaretreatedwithvariousconcentrationsofUPRinhibitorsinthepresenceorabsenceof10mM2DGfor24h.Themediumisthenreplacedwithfreshgrowthmedium,andcellsareculturedforafurther15h.Subsequently,MTTisaddedtotheculturemedium,andtheabsorbanceofeachwellisdetermined.FortheviABIlityassayunderglucose-withdrawalconditions,HT1080cellsaretreatedwithvariousconcentrationsofDorsomorphinandphenforminin12-wellplatesinthepresenceorabsenceofglucosefor18h,seededin96-wellplateswithgrowthmedium,andthenculturedforafurther48hbeforeMTTisadded.Relativecellsurvival(mean±SDofquadruplicatedeterminations)iscalculatedbysettingeachcontrolabsorbancefromuntreatedcellsas100%.Theeffectsofdrugcombinationsatconcentrationsproducing80%cellgrowthinhibition(IC₈₀)areanalyzedusingtheisobologrammethod^[2].MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

DorsomorphinispreparedasastocksolutioninDMSO^[3].

Mice^[3]
12-week-oldC57BL/6miceraisedonastandarddietareinjectedviathetailveinwith0.2g/kgofDextranor0.2g/kgofiron-dextranUSP.Dextranisinjectedwithvehicleonly,whereasiron-dextranisinjectedwitheithervehicleorDorsomorphin(10mg/kg).1hafterinjection,micearekilledandliversegmentsarecollectedin500μLofSDS-lysisbufferandmechanicallyhomogenized.20μLofliverextractsareresolvedbySDS-PAGEandimmunoblotted.TotalRNAisharvestedusingTrizolfrommechanicallyhomogenizedmouselivers(6hafterinjectionwithasingleintraperitonealdoseofDorsomorphin(10mg/kg)orDMSO).MCEhasnotindependentlyconfirmedtheaccuracyofthesemethods.Theyareforreferenceonly.

• [1].ZhouG,etal.RoleofAMP-activatedproteinkinaseinmechanismofmetforminaction.JClinInvest.2001Oct;108(8):1167-74.

  [2].SaitoS,etal.CompoundCpreventstheunfoldedproteinresponseduringglucosedeprivationthroughamechanismindependentofAMPKandBMPsignaling.PLoSOne.2012;7(9):e45845.

  [3].YuPB,etal.DorsomorphininhibitsBMPsignalsrequiredforembryogenesisandironmetabolism.NatChemBiol.2008Jan;4(1):33-41.

  [4].ZhangDY,etal.Roleofautophagyanditsmolecularmechanismsinmiceintestinaltractaftersevereburn.JTraumaAcuteCareSurg.2017Oct;83(4):716-724.

472.41

C₂₄H₂₇Cl₂N₅O

1219168-18-9

RoomtemperatureincontinentalUS;mayvaryelsewhere

H₂O:≥48mg/mL;DMSO:5.2mg/mL(Needultrasonic)

Dorsomorphindihydrochlorideisdissolvedinsaline^[4].

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

• [1].ZhouG,etal.RoleofAMP-activatedproteinkinaseinmechanismofmetforminaction.JClinInvest.2001Oct;108(8):1167-74.

  [2].SaitoS,etal.CompoundCpreventstheunfoldedproteinresponseduringglucosedeprivationthroughamechanismindependentofAMPKandBMPsignaling.PLoSOne.2012;7(9):e45845.

  [3].YuPB,etal.DorsomorphininhibitsBMPsignalsrequiredforembryogenesisandironmetabolism.NatChemBiol.2008Jan;4(1):33-41.

  [4].ZhangDY,etal.Roleofautophagyanditsmolecularmechanismsinmiceintestinaltractaftersevereburn.JTraumaAcuteCareSurg.2017Oct;83(4):716-724.