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Smartox/（Dap22）-ShK是一种高选择性Kv1.3阻断剂/13SHD001-00010/0.01mg
(Dap22)-ShK(ShKDap22)peptideisasyntheticderivativeofthewell-knownShKtoxin#08SHK001(Stichodactylahelianthusneurotoxin)isolatedfromthevenomoftheCarribeanseaanemoneStoichactishelianthus.Wild-typeShKblockspotentlyKv1.3(KCNA3),Kv1.1(KCNA1),Kv1.4(KCNA4),andKv1.6(KCNA6)respectivelywithaKdof11pM,16pM,312pMand165pM.In(Dap22)-ShK,lysine22hasbeenreplacedbyadiaminopropionicacid(Dap)residuethatgreatlyimprovestheselectivityofthepeptideforthevoltage-gatedpotassiumchannelKv1.3(IC50around23pM)againstKv1.1(1.8nM),Kv1.4(37nM)andKv1.6(10nM)channels.Thehighselectivityof(Dap22)-ShkisachievedthankstothestrongbindingbetweentheDapandHis404/Asp386residuesofKv1.3channel.(Dap22)-ShKinhibitsTcellproliferationinducedbyanti-CD3atsubnanomolarconcentrations.
Description:
AAsequence:Arg-Ser-Cys3-Ile-Asp-Thr-Ile-Pro-Lys-Ser-Arg-Cys12-Thr-Ala-Phe-Gln-Cys17-Lys-His-Ser-Met-Dap-Tyr-Arg-Leu-Ser-Phe-Cys28-Arg-Lys-Thr-Cys32-Gly-Thr-Cys35-OH
Disulfidebonds:Cys3-Cys35,Cys12-Cys28andCys17-Cys32
Length(aa):35
Formula:C166H268N54O48S7
MolecularWeight:4012.8Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:220384-25-8
Source:Synthetic
Purityrate:>97%
Reference:
HypoxiamodulatesearlyeventsinTcellreceptor-mediatedactivationinhumanTlymphocytesviaKv1.3channels
Tlymphocytesareexposedtohypoxiaduringtheirdevelopmentandwhentheymigratetohypoxicpathologicalsites.AlthoughithasbeenshownthathypoxiainhibitsKv1.3channelsandproliferationinhumanTcells,themechanismsbywhichhypoxiaregulatesTcellactivationarenotfullyunderstood.HereinwetestthehypothesisthathypoxicinhibitionofKv1.3channelsinducesmembranedepolarization,thusmodulatingtheincreaseincytoplasmicCa2+thatoccursduringactivation.HypoxiacausesmembranedepolarizationinhumanCD3+Tcells,asmeasuredbyfluorescence-activatedcellsorting(FACS)withthevoltage-sensitivedyeDiBAC4(3).SimilardepolarizationisproducedbytheselectiveKv1.3channelblockersShK-Dap22andmargatoxin.FurThermore,pre-exposuretosuchblockerspreventsanyfurtherdepolarizationbyhypoxia.SincemembranedepolarizationisunfavourabletotheinfluxofCa2+throughtheCRACchannels(necessarytodrivemanyeventsinTcellactivationsuchascytokineproductionandproliferation),theeffectofhypoxiaonTcellreceptor-mediatedincreaseincytoplasmicCa2+wasdeterminedusingfura-2.HypoxiadepressestheincreaseinCa2+inducedbyanti-CD3/CD28antibodiesinapproximately50%oflymphocytes.Intheremainingcells,hypoxiaeitherdidnotelicitanychangeorproducedasmallincreaseincytoplasmicCa2+.Similareffectswereobservedinrestingandpre-activatedCD3+cellsandweremimickedbyShK-Dap22.TheseeffectsappeartobemediatedsolelybyKv1.3channels,aswefindnoinfluenceofhypoxiaonIKCa1andCRACchannels.OurfindingsindicatethathypoxiamodulatesCa2+homeostasisinTcellsviaKv1.3channelinhibitionandmembranedepolarization.
RobbinsJR.,etal.(2005)HypoxiamodulatesearlyeventsinTcellreceptor-mediatedactivationinhumanTlymphocytesviaKv1.3channels.JPhysiol.PMID: 15677684
PotassiumchannelblockadebytheseaanemonetoxinShKforthetreatmentofmultiplesclerosisandotherautoimmunediseases
Expressionofthetwolymphocytepotassiumchannels,thevoltage-gatedchannelKv1.3andthecalciumactivatedchannelIKCa1,changesduringdifferentiationofhumanTcells.WhileIKCa1isthefunctionallydominantchannelinnaiveand“early”memoryTcells,Kv1.3iscrucialfortheactivationofterminallydifferentiatedeffectormemory(TEM)Tcells.BecauseoftheinvolvementofTEMcellsinautoimmuneprocesses,Kv1.3isregardedasapromisingtargetforthetreatmentofT-cellmediatedautoimmunediseasessuchasmultiplesclerosisandthepreventionofchronictransplantrejection.ShK,a35-residuepolypeptidetoxinfromtheseaanemone,Stichodactylahelianthus,blocksKv1.3atlowpicomolarconcentrations.ShKadoptsacentralhelix-kink-helixfold,andalanine-scanningandothermutagenesisstudieshavedefineditschannel-bindingsurface.ModelshavebeendevelopedofhowthistoxineffectsK+-channelblockadeandhowitsdockingconfigurationmightdifferinShK-Dap22,whichcontainsasinglesidechainsubstitutionthatconfersspecificityforKv1.3blockade.ShK,ShK-Dap22andtheKv1.3blockingscorpiontoxinkaliotoxinhavebeenshowntopreventandtreatexperimentalautoimmuneencephalomyelitisinrats,amodelformultiplesclerosis.AfluoresceinatedanalogofShK,ShK-F6CA,hasbeendeveloped,whichallowsthedetectionofactivatedTEMcellsinhumanandanimalbloodsamplesbyflowcytometryandthevisualizationofKv1.3channeldistributioninlivingcells.ShKanditsanalogsarecurrentlyundergoingfurtherevaluationasleadsinthedevelopmentofnewbiopharmaceuticalsforthetreatmentofmultiplesclerosisandotherT-cellmediatedautoimmunedisorders.
NortonRS.,etal.(2004)PotassiumchannelblockadebytheseaanemonetoxinShKforthetreatmentofmultiplesclerosisandotherautoimmunediseases.CurrMedChem. PMID: 15578998
SubstitutionofasingleresidueinStichodactylahelianthuspeptide,ShK-Dap22,revealsanovelpharmacologicalprofile
ShK,apeptideisolatedfromStichodactylahelianthusvenom,blocksthevoltage-gatedpotassiumchannels,K(v)1.1andK(v)1.3,withsimilarhighaffinity.ShK-Dap(22),asyntheticderivativeinwhichadiaminopropionicacidresiduehasbeensubstitutedatpositionLys(22),hasbeenreportedtobeaselectiveK(v)1.3inhibitorandtoblockthischannelwithequivalentpotencyasShK[Kalmanetal.(1998)J.Biol.Chem.273,32697-32707].Inthisstudy,alargebodyofevidenceispresentedwhichindicatesthatthepotenciesofwild-typeShKpeptideforbothK(v)1.3andK(v)1.1channelshavebeenpreviouslyunderestimated.Therefore,theaffinityofShK-Dap(22)forbothchannelsappearstobeca.10(2)-10(4)-foldweakerthanShK.ShK-Dap(22)doesdisplayca.20-foldselectivityforhumanK(v)1.3vsK(v)1.1whenmeasuredbythewhole-cellvoltageclampmethodbutnotinequilibriumbindingassays.ShK-Dap(22)haslowaffinityforK(v)1.2channels,butheteromultimericK(v)1.1-K(v)1.2channelsformareceptorwithca.200-foldhigheraffinityforShK-Dap(22)thanK(v)1.1homomultimers.Infact,K(v)1.1-K(v)1.2channelsbindShK-Dap(22)withonlyca.10-foldlesspotencythanShKandrevealanovelpharmacologynotpredictedfromthehomomultimersofK(v)1.1orK(v)1.2.TheconcentrationsofShK-Dap(22)neededtoinhibithumanTcellactivationwereca.10(3)-foldhigherthanthoseofShK,ingoodcorrelationwiththerelativeaffinitiesofthesepeptidesforinhibitingK(v)1.3channels.Allofthesedata,takentogether,suggestthatShK-Dap(22)willnothavethesameinvivoimmunosuppressantefficacyofotherK(v)1.3blockers,suchasmargatoxinorShK.Moreover,ShK-Dap(22)mayhaveundesiredsideeffectsduetoitsinteractionwithheteromultimericK(v)1.1-K(v)1.2channels,suchasthosepresentinbrainand/orperipheraltissues.
MiddletonRE.,etal.(2003)SubstitutionofasingleresidueinStichodactylahelianthuspeptide,ShK-Dap22,revealsanovelpharmacologicalprofile.Biochemistry. PMID: 14622016
MutatingacriticallysineinShKtoxinaltersitsbindingconfigurationinthepore-vestibuleregionofthevoltage-gatedpotassiumchannel,Kv1.3
Thevoltage-gatedpotassiumchannelinTlymphocytes,Kv1.3,animportanttargetforimmunosuppressants,isblockedbypicomolarconcentrationsofthepolypeptideShKtoxinanditsanalogueShK-Dap22.ShK-Dap22showsincreasedselectivityforKv1.3,andourgoalwastodeterminethemolecularbasisforthisselectivitybyprobingtheinteractionsofShKandShK-Dap22withtheporeandvestibuleofKv1.3.Thefreeenergiesofinteractionsbetweentoxinandchannelresiduesweremeasuredusingmutantcycleanalyses.Thesedata,interpretedasapproximatedistancerestraints,guidedmoleculardynamicssimulationsinwhichthetoxinsweredockedwithamodelofKv1.3basedonthecrystalstructureofthebacterialK(+)-channelKcsA.Despitethesimilartertiarystructuresofthetwoligands,themutantcycledataimplythattheymakedifferentcontactswithKv1.3,andtheycanbedockedwiththechannelinconfigurationsthatareconsistentwiththemutantcycledataforeachtoxinbutquitedistinctfromoneanother.ShKbindstoKv1.3withLys22occupyingthenegativelychargedporeofthechannel,whereastheequivalentresidueinShK-Dap22interactswithresiduesfurtheroutinthevestibule,producingasignificantchangeintoxinorientation.TheincreasedselectivityofShK-Dap22isachievedbystronginteractionsofDap22withHis404andAsp386onKv1.3,withonlyweakinteractionsbetweenthechannelporeandthetoxin.PotentandspecificblockadeofKv1.3apparentlyoccurswithoutinsertionofapositivelychargedresidueintothechannelpore.Moreover,thefindingthatasingleresiduesubstitutionaltersthebindingconfigurationemphasizestheneedtoobtainconsistentdatafrommultiplemutantcycleexperimentsinattemptstodefineproteininteractionsurfacesusingthesedata.
LaniganMD.,etal.(2002)MutatingacriticallysineinShKtoxinaltersitsbindingconfigurationinthepore-vestibuleregionofthevoltage-gatedpotassiumchannel,Kv1.3.Biochemistry. PMID: 12356296
SelectiveblockadeofTlymphocyteK(+)channelsamelioratesexperimentalautoimmuneencephalomyelitis,amodelformultiplesclerosis
Adoptivetransferexperimentalautoimmuneencephalomyelitis(AT-EAE),adiseaseresemblingmultiplesclerosis,isinducedinratsbymyelinbasicprotein(MBP)-activatedCD4(+)Tlymphocytes.Bypatch-clampanalysis,encephalitogenicratTcellsstimulatedrepeatedlyinvitroexpressedauniquechannelphenotype(“chronicallyactivated”)withlargenumbersofKv1.3voltage-gatedchannels(approximately1500percell)andsmallnumbersofIKCa1Ca(2+)-activatedK(+)channels(approximately50-120percell).Incontrast,restingTcellsdisplayed0-10Kv1.3and10-20IKCa1channelspercell(“quiescent”phenotype),whereasTcellsstimulatedonceortwiceexpressedapproximately200Kv1.3andapproximately350IKCa1channelspercell(“acutelyactivated”phenotype).Consistentwiththeirchannelphenotype,[(3)H]thymidineincorporationbyMBP-stimulatedchronicallyactivatedTcellswassuppressedbythepeptideShK,ablockerofKv1.3andIKCa1,andbyananalog(ShK-Dap(22))engineeredtobehighlyspecificforKv1.3,butnotbyaselectiveIKCa1blocker(TRAM-34).ThecombinationofShK-Dap(22)andTRAM-34enhancedthesuppressionofMBP-stimulatedTcellproliferation.Basedontheseinvitroresults,weassessedtheefficacyofK(+)channelblockersinAT-EAE.SpecificandsimultaneousblockadeoftheTcellchannelsbyShKorbyacombinationofShK-Dap(22)plusTRAM-34preventedlethalAT-EAE.BlockadeofKv1.3alonewithShK-Dap(22),butnotofIKCa1withTRAM-34,wasalsoeffective.WhenadmiNISTeredaftertheonsetofsymptoms,ShKorthecombinationofShK-Dap(22)plusTRAM-34greatlyamelioratedtheclinicalcourseofbothmoderateandsevereAT-EAE.WeconcludethatselectivetargetingofKv1.3,aloneorwithIKCa1,mayprovideaneffectivenewmodeoftherapyformultiplesclerosis.
BeetonC.,etal.(2001)SelectiveblockadeofTlymphocyteK(+)channelsamelioratesexperimentalautoimmuneencephalomyelitis,amodelformultiplesclerosis.ProcNatlAcadSciUSA. PMID: 11717451
ShK-Dap22,apotentKv1.3-specificimmunosuppressivepolypeptide
Thevoltage-gatedpotassiumchannelinTlymphocytes,Kv1.3,isanimportantmoleculartargetforimmunosuppressiveagents.Astructurallydefinedpolypeptide,ShK,fromtheseaanemoneStichodactylahelianthusinhibitedKv1.3potentlyandalsoblockedKv1.1,Kv1.4,andKv1.6atsubnanomolarconcentrations.UsingmutantcycleanalysisinconjunctionwithcomplementarymutagenesisofShKandKv1.3,andutilizingthestructureofShK,wedeterminedalikelydockingconfigurationforthispeptideinthechannel.Baseduponthistopologicalinformation,wereplacedthecriticalLys22inShKwiththepositivelycharged,non-naturalaminoaciddiaminopropionicacid(ShK-Dap22)andgeneratedahighlyselectiveandpotentblockeroftheT-lymphocytechannel.ShK-Dap22,atsubnanomolarconcentrations,suppressedanti-CD3inducedhumanT-lymphocyte[3H]thymidineincorporationinvitro.Toxicitywiththismutantpeptidewaslowinarodentmodel,withamedianparalyticdoseofapproximately200mg/kgbodyweightfollowingintravenousadministration.TheoverallstructureofShK-Dap22insolution,asdeterminedfromNMRdata,issimilartothatofnativeShKtoxin,buttherearesomedifferencesintheresiduesinvolvedinpotassiumchannelbinding.Basedontheseresults,weproposethatShK-Dap22orastructuralanaloguemayhaveuseasanimmunosuppressantforthepreventionofgraftrejectionandforthetreatmentofautoimmunediseases.
KalmanK,etal.(1998)ShK-Dap22,apotentKv1.3-specificimmunosuppressivepolypeptide.JBiolChem. PMID: 9830012
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