ShK(StichodactylahelianthusNeurotoxin)hasbeenisolatedfromthevenomoftheCarribeanseaanemoneStoichactishelianthus.ShKinhibitsvoltage-dependentpotassiumchannels.ItblocksKv1.3(KCNA3)potentlyandalsoKv1.1(KCNA1),Kv1.4(KCNA4)andKv1.6(KCNA6)respectivelywithaKdof11pM,16pM,312pMand165pM.Interestingly,itwasalsodemonstratedthatShKpotentlyinhibitsthehKv3.2bchannelwithanIC50valueofapproximately0.6nM.
Description:
AAsequence:Arg-Ser-Cys3-Ile-Asp-Thr-Ile-Pro-Lys-Ser-Arg-Cys12-Thr-Ala-Phe-Gln-Cys17-Lys-His-Ser-Met-Lys-Tyr-Arg-Leu-Ser-Phe-Cys28-Arg-Lys-Thr-Cys32-Gly-Thr-Cys35-OH
Disulfidebonds:Cys3-Cys35,Cys12-Cys28andCys17-Cys32
Length(aa):35
Formula:C169H274N54O48S7
MolecularWeight:4054.85Da
Appearance:Whitelyophilizedsolid
Solubility:waterandsalinebuffer
CASnumber:165168-50-3
Source:Synthetic
Purityrate:>97%
Reference:
DurablepharmacologicalresponsesfromthepeptideShK-186,aspecificKv1.3channelinhibitorthatsuppressesTcellmediatorsofautoimmunedisease
TheKv1.3channelisarecognizedtargetforpharmaceuticaldevelopmenttotreatautoimmunediseasesandorganrejection.ShK-186,aspecificpeptideinhibitorofKv1.3,hasshownpromiseinanimalmodelsofmultiplesclerosisandrheumatoidarthritis.Here,wedescribethepharmacokinetic-pharmacodynamicrelationshipforShK-186inratsandmonkeys.ThepharmacokineticprofileofShK-186wasevaluatedwithavalidatedhigh-performanceliquidchromatography-tandemmassspectrometrymethodtomeasurethepeptide’sconcentrationinplasma.Theseresultswerecomparedwithsingle-photonemissioncomputedtomography/computedtomographydatacollectedwithan¹¹¹In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid-conjugateofShK-186toassesswhole-bloodpharmacokineticparametersaswellasthepeptide’sabsorption,distribution,andexcretion.AnalysisofthesedatasupportamodelwhereinShK-186isabsorbedslowlyfromtheinjectionsite,resultinginbloodconcentrationsabovetheKv1.3channel-blockingIC₅₀valueforupto7daysinmonkeys.PharmacodynamicstudiesonhumanperipheralbloodmononuclearcellsshowedthatbriefexposuretoShK-186resultedinsustainedsuppressionofcytokineresponsesandmaycontributetoprolongeddrugeffects.Indelayed-typehypersensitivity,chronicrelapsing-remittingexperimentalautoimmuneencephalomyelitis,andpristane-inducedarthritisratmodels,asingledoseofShK-186every2to5dayswasaseffectiveasdailyadmiNISTration.ShK-186’sslowdistributionfromtheinjectionsiteanditslongresidencetimeontheKv1.3channelcontributetotheprolongedtherapeuticeffectofShK-186inanimalmodelsofautoimmunedisease.
TarchaEJ.,etal.(2012)DurablepharmacologicalresponsesfromthepeptideShK-186,aspecificKv1.3channelinhibitorthatsuppressesTcellmediatorsofautoimmunedisease.JPharmacolExpTher.PMID: 22637724
ThebeneficialeffectofblockingKv1.3inthepsoriasiformSCIDmousemodel
TheKv1.3channelisimportantintheactivationandfunctionofeffectormemoryTcells.Recently,specificblockersoftheKv1.3channelhavebeendevelopedasapotentialtherapeuticoptionfordiverseautoimmunediseases.Inpsoriaticlesions,mostlymphocytesarememoryeffectorTcells.TheaimofthepresentstudywastodetecttheexpressionofKv1.3channelsinthesecellsinpsoriaticlesionsaswellasinhumanpsoriasiformskingraftsusingtheseverecombinedimmunodeficient(SCID)mousemodel.HistologicalandimmunohistochemicalstainingforKv1.3expressionandvariousinflammatoryMarkerswasperformedinsectionsobtainedfromsixpsoriaticpatientsand18beige-SCIDmicewithpsoriasiformhumanskingrafts.SixgraftedmiceweretreatedwithStichodactylahelianthusneurotoxin(ShK),aknownKv1.3blocker.TheresultsshowedanincreasednumberofKv1.3+cellsinthepsoriaticskinaswellasinthepsoriasiformskingraftsascomparedwithnormalskinandnormalskingrafts.InjectionsofShKshowedamarkedtherapeuticeffectinthreeofsixpsoriasiformskingrafts.AsignificantlydecreasednumberofKv1.3+cellswasobservedintheresponderscomparedwiththecontrolgrafts.Thispilotstudy,althoughperformedinasmallnumberofmice,revealsthepossIBLebeneficialeffectofKv1.3blockersinpsoriasispatients.
GilharA.,etal.(2011)ThebeneficialeffectofblockingKv1.3inthepsoriasiformSCIDmousemodel.JInvestDermatol.PMID: 20739949
Modelingthebindingofthreetoxinstothevoltage-gatedpotassiumchannel(Kv1.3)
Theconductionpropertiesofthevoltage-gatedpotassiumchannelKv1.3anditsmodesofinteractionwithseveralpolypeptidevenomsareexaminedusingBrowniandynamicssimulationsandmoleculardynamicscalculations.Employinganopen-statehomologymodelofKv1.3,wefirstdeterminecurrent-voltageandcurrent-concentrationcurvesandascertainthatsimulatedresultsaccordwithexperimentalmeasurements.Wetheninvestigate,usingamoleculardockingmethodandmoleculardynamicssimulations,thecomplexesformedbetweentheKv1.3channelandseveralKv-specificpolypeptidetoxinsthatareknowntointerferewiththeconductingmechanismsofseveralclassesofvoltage-gatedK(+)channels.ThedepthsofpotentialofmeanforceencounteredbycharyBDotoxin,α-KTx3.7(alsoknownasOSK1)andShKare,respectively,-19,-27,and-25kT.Thedissociationconstantscalculatedfromtheprofilesofpotentialofmeanforcecorrespondcloselytotheexperimentallydeterminedvalues.Wepinpointtheresiduesinthetoxinsandthechannelthatarecriticalfortheformationofthestablevenom-channelcomplexes.
ChenR.,etal.(2011)Modelingthebindingofthreetoxinstothevoltage-gatedpotassiumchannel(Kv1.3).BiophysJ.PMID: 22261053
BlockadeofT-lymphocyteKCa3.1andKv1.3channelsasnovelimmunosuppressionstrategytopreventkidneyallograftrejection
Currently,thereisanunmetclinicalneedfornovelimmunosuppressiveagentsforlong-termpreventionofkidneytransplantrejectionasalternativestothenephrotoxiccalcineurininhibitorcyclosporine(CsA).RecentstudieshaveshownthatK(+)channelshaveacrucialroleinT-lymphocyteactivity.WeinvestigatedwhethercombinedblockadeoftheT-cellK(+)channelsK(Ca)3.1andK(v)1.3,bothofwhichregulatecalciumsignalingduringlymphocyteactivation,iseffectiveinpreventionofrejectionofkidneyallograftsfromFisherratstoLewisrats.AllrecipientswereinitiallytreatedwithCsA(5mg/kgd)for7days.Inratswithintactallograftfunction,treatmentwascontinuedfor10dayswitheitherCsA(5mg/kgd),oracombinationofTRAM-34(K(Ca)3.1inhibitor;120mg/kgd)plusStichodactylahelianthustoxin(ShK,K(v)1.3inhibitor;80microg/kg3timesdaily),orvehiclealone.Kidneysectionswerestainedwithperiodicacid-Schifforhematoxylin-eosinandhistochemicallyformarkersofmacrophages(CD68),T-lymphocytes(CD43),orcytotoxicT-cells(CD8).OurresultsshowedthattreatmentwithTRAM-34andShKreducedtotalinterstitialmononuclearcellinfiltration(-42%)andthenumberofCD43+T-cells(-32%),cytotoxicCD8+T-cells(-32%),andCD68+macrophages(-26%)inallograftswhencomparedtovehicletreatmentalone.EfficacyofTRAM-34/ShKtreatmentwascomparablewiththatofCsA.Inaddition,novisibleorgandamageorotherdiscernibleadverseeffectswereobservedwiththistreatment.Thus,selectiveblockadeofT-lymphocyteK(Ca)3.1andK(v)1.3channelsmayrepresentanovelalternativetherapyforpreventionofkidneyallograftrejection.
GrgicI.,etal.(2009)BlockadeofT-lymphocyteKCa3.1andKv1.3channelsasnovelimmunosuppressionstrategytopreventkidneyallograftrejection.TransplantProc.PMID: 19715983
MolecularmechanismoftheseaanemonetoxinShKrecognizingtheKv1.3channelexploredbydockingandmoleculardynamicsimulations
ComputationalmethodsareemployedtosimulatetheinteractionoftheseaanemonetoxinShKincomplexwiththevoltage-gatedpotassiumchannelKv1.3frommice.Alloftheavailable20structuresofShKintheProteinDataBankwereconsideredforimprovingtheperformanceoftherigidproteindockingofZDOCK.ThetrADItionalandnovelbindingmodeswereobtainedamongalargenumberofpredictedcomplexesbyusingclusteringanalysis,screeningwithexpertknowledge,energyminimization,andmoleculardynamicsimulations.Thequalityandvalidityoftheresultingcomplexeswerefurtherevaluatedtoidentifyafavorablecomplexstructureby500psmoleculardynamicsimulationsandthechangeofbindingfreeenergieswithacomputationalalaninescanningtechnique.ThenovelandreasonableShK-Kv1.3complexstructurewasfoundtobedifferentfromthetraditionalmodelbyusingtheLys22residuetoblockthechannelpore.FromtheresultingstructureoftheShK-Kv1.3complex,ShKmainlyassociatesthechanneloutervestibulewithitssecondhelicalsegment.StructuralanalysisfirstrevealedthattheLys22residuesidechainoftheShKpeptidejusthangsbetweenCandDchainsoftheKv1.3channelinsteadofphysicallyblockingthechannelpore.TheobviouslossoftheShKSer20AlaandTyr23AlamutantbindingABIlitytotheKv1.3channeliscausedbytheconformationalchange.ThefivehydrogenbondsbetweenArg24inShKandH404(A)andD402(D)inKv1.3makeArg24themostcrucialforitsbindingtotheKv1.3channel.BesidesthedetailedinteractionbetweenShKandKv1.3attheatomlevel,thesignificantconformationalchangeinducedbytheinteractionbetweentheShKpeptideandtheKv1.3channel,accompaniedbythegradualdecreaseofbindingfreeenergies,stronglyimpliesthatthebindingoftheShKpeptidetowardtheKv1.3channelisadynamicprocessofconformationalrearrangementandenergystabilization.AllofthesecanacceleratethedevelopmentofShKstructure-basedimmunosuppressants.
JinL,WuY.(2007)MolecularmechanismoftheseaanemonetoxinShKrecognizingtheKv1.3channelexploredbydockingandmoleculardynamicsimulations.JChemInfModel. PMID: 17718553
K+channelblockers:noveltoolstoinhibitTcellactivationleadingtospecificimmunosuppression
DuringthelasttwodecadessincetheidentificationandcharacterizationofTcellpotassiumchannelsgreatadvanceshavebeenmadeintheunderstandingoftheroleofthesechannelsinTcellfunctions,especiallyinantigen-inducedactivation.TheirlimitedtissuedistributionandtherecentdiscoverythatdifferentTcellsubtypescarryingoutdistinctimmunefunctionsshowspecificexpressionlevelsofthesechannelshavemadeTcellpotassiumchannelsattractivetargetsforimmunomodulatorydrugs.Manytoxinsofvariousanimalspeciesandastructurallydiversearrayofsmallmoleculesinhibitingthesechannelswithvaryingaffinityandselectivitywerefoundandtheirsuccessfuluseinimmunosuppressioninvivowasalsodemonstrated.Betterunderstandingofthetopologicaldifferencesbetweenpotassiumchannelpores,detailedknowledgeoftoxinandsmall-moleculestructuresandtheidentificationofthebindingsitesofblockingcompoundsmakeitpossibletoimprovetheselectivityandaffinityoftheleadcompoundsbyintroducingmodificationsbasedonstructuralinformation.Inthisreviewthebasicpropertiesandphysiologicalrolesofthevoltage-gatedKv1.3andtheCa2+-activatedIKCa1potassiumchannelsarediscussedalongwithanoverviewofcompoundsinhibitingthesechannelsandapproachesaimingatproducingmoreefficientmodulatorsofimmunefunctionsforthetreatmentofdiseaseslikesclerosismultiplexandtypeIdiabetes.
PanyiG,etal.(2006)K+channelblockers:noveltoolstoinhibitTcellactivationleadingtospecificimmunosuppression.CurrPharmDes. PMID: 16787250
Stichodactylahelianthuspeptide,apharmacologicaltoolforstudyingKv3.2channels
Voltage-gatedpotassium(Kv)channelsregulatemanyphysiologicalfunctionsandrepresentimportanttherapeutictargetsinthetreatmentofseveralclinicaldisorders.Althoughsomeofthesechannelshavebeenwell-characterized,thestudyofothers,suchasKv3channels,hasbeenhinderedbecauseoflimitedpharmacologicaltools.ThecurrentstudywasinitiatedtoidentifypotentblockersoftheKv3.2channel.Chinesehamsterovary(CHO)-K1cellsstablyexpressinghumanKv3.2b(CHO-K1.hKv3.2b)wereestablishedandcharacterized.Stichodactylahelianthuspeptide(ShK),isolatedfromS.helianthusvenomandaknownhigh-affinityblockerofKv1.1andKv1.3channels,wasfoundtopotentlyinhibit86Rb+effluxfromCHO-K1.hKv3.2b(IC50approximately0.6nM).InelectrophysiologicalrecordingsofKv3.2bchannelsexpressedinXenopuslaevisoocytesorinplanarpatch-clampstudies,ShKinhibitedhKv3.2bchannelswithIC50valuesofapproximately0.3and6nM,respectively.DespitethepresenceofKv3.2proteininhumanpancreaticbetacells,ShKhasnoeffectontheKvcurrentofthesecells,suggestingthatitisunlikelythathomotetramericKv3.2channelscontributesignificantlytothedelayedrectifiercurrentofinsulin-secretingcells.InmousecorticalGABAergicfast-spikinginterneurons,however,applicationofShKproducedeffectsconsistentwiththeblockadeofKv3channels(i.e.,anincreaseinactionpotentialhalf-width,adecreaseintheamplitudeoftheactionpotentialafterhyperpolarization,andadecreaseinmaximalfiringfrequencyinresponsetodepolarizingcurrentinjections).Takentogether,theseresultsindicatethatShKisapotentinhibitorofKv3.2channelsandmayserveasausefulpharmacologicalprobeforstudyingthesechannelsinnativepreparations.
YanL.,etal.(2005)Stichodactylahelianthuspeptide,apharmacologicaltoolforstudyingKv3.2channels.MolPharmacol.PMID: 15709110
TargetingeffectormemoryTcellswithaselectivepeptideinhibitorofKv1.3channelsfortherapyofautoimmunediseases
Thevoltage-gatedKv1.3K(+)channelisanoveltargetforimmunomodulationofautoreactiveeffectormemoryT(T(EM))cellsthatplayamajorroleinthepathogenesisofautoimmunediseases.WedescribethecharacterizationofthenovelpeptideShK(L5)thatcontainsl-phosphotyrosinelinkedviaanine-atomhydrophiliclinkertotheNterminusoftheShKpeptidefromtheseaanemoneStichodactylahelianthus.ShK(L5)isahighlyspecificKv1.3blockerthatexhibits100-foldselectivityforKv1.3(K(d)=69pM)overKv1.1andgreaterthan250-foldselectivityoverallotherchannelstested.ShK(L5)suppressestheproliferationofhumanandratT(EM)cellsandinhibitsinterleukin-2productionatpicomolarconcentrations.NaiveandcentralmemoryhumanTcellsareinitially60-foldlesssensitivethanT(EM)cellstoShK(L5)andthenbecomeresistanttothepeptideduringactivationbyup-regulatingthecalcium-activatedK(Ca)3.1channel.ShK(L5)doesnotexhibitinvitrocytotoxicityonmammaliancelllinesandisnegativeintheAmestest.Itisstableinplasmaandwhenadministeredoncedailybysubcutaneousinjection(10mug/kg)attains“steadystate”bloodlevelsofapproximately300pM.ThisregimendoesnotcausecardiactoxicityassessedbycontinuousEKGmonitoringanddoesnotalterclinicalchemistryandhematologicalparametersafter2-weektherapy.ShK(L5)preventsandtreatsexperimentalautoimmuneencephalomyelitisandsuppressesdelayedtypehypersensitivityinrats.ShK(L5)mightproveusefulfortherapyofautoimmunedisorders.
BeetonC.,etal.(2005)TargetingeffectormemoryTcellswithaselectivepeptideinhibitorofKv1.3channelsfortherapyofautoimmunediseases.MolPharmacol. PMID: 15665253
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
SolutionstructureofShKtoxin,anovelpotassiumchannelinhibitorfromaseaanemone
AnessentialbindingsurfaceforShKtoxininteractionwithratbrainpotassiumchannels.
An“Alascan”analysisofShKtoxin,a35-residuebasicpeptidepossessingthreedisulfidebonds,identifiessevensidechainswhichinfluencebindingtobraindelayedrectifierpotassiumchannels.Additionalanalogsweresynthesizedandtestedtofurtherdeciphertherolesoftheseresidues,particularlyTyr23.Theinhibitoryeffectsoftheseanalogson125I-labeleddendrotoxinbindingtoratbrainmembranesshowedthatreplacementofTyr23withAladrasticallyloweredtheaffinityofthetoxinfortheKv1.2channels.AlasubstitutionofPhe27reducedpotencymorethan15-fold.MonosubstitutedAlaanalogsforIle7,Ser20,orLys30eachdisplayed5-foldreductionsinpotency.Thus,aromaticityatposition23isimportantforeffectivedelayedrectifierbrainKchannelbinding.Incontrast,thearomaticresidueatposition27wasnotcritical,sincecyclohexylalaninesubstitutionincreasedaffinity.ThesolutionstructureofShKtoxinclustersIle7,Arg11,Ser20,Lys22,Tyr23,andPhe27incloseproximity,formingthepotassiumchannelbindingsurfaceofthetoxin.WeproposeanessentialbindingsurfaceonthetoxininwhichLys22andTyr23aremajorcontributors,throughionicandaromatic(hydrophobic)interactions,withthepotassiumchannel.
PenningtonMW.,etal.(1996)AnessentialbindingsurfaceforShKtoxininteractionwithratbrainpotassiumchannels.Biochemistry. PMID: 8987971
CharacterizationofapotassiumchanneltoxinfromtheCaribbeanSeaanemoneStichodactylahelianthus
Apeptidetoxin,ShK,thatblocksvoltage-dependentpotassiumchannelswasisolatedfromthewholebodyextractoftheCaribbeanseaanemoneStichodactylahelianthus.ItcompeteswithdendrotoxinIandalpha-dendrotoxinforbindingtosynaptosomalmembranesofratbrain,facilitiesacetylcholinereleaseatanavianneuromuscularjunctionandsuppressesK+currentsinratdorsalrootganglionneuronesinculture.ItsaminoacidsequenceisR1SCIDTIPKS10RCTAFQCKHS20MKYRLSFCRK30TCGTC35.ThereisnohomologywithotherK+channel-blockingpeptides,exceptforBgKfromtheseaanemoneBunodosomagranulifera.ShKandBgKappeartobeinadifferentstructuralclassfromothertoxinsaffectingK+channels.
Castaneda,O.,etal.(1995)CharacterizationofapotassiumchanneltoxinfromtheCaribbeanSeaanemoneStichodactylahelianthus,Toxicon. PMID: 7660365
