SelectiveantagoNISTofthealpha(1A)-adrenoceptor
AdTx1(rho-Da1a–ρ-Da1a)isa65amino-acidpeptideoriginallyisolatedfromthevenomofthegreenmamba,anAfricansnake(Dendroaspisangusticep).AdTx1isstABIlizedbyfourdisulfidebridgesandbelongstothefamilyofthethree-finger-foldpeptide.AdTx1hassubnanomolaraffinity(K(i)=0.35nM)andhighspecificityforthehumanalpha(1A)-adrenoceptorsubtypeandis1000timesmorepotentonthissubtypethanonotheradrenoceptorsubtypes.AdTx1isapotentrelaxantofsmoothmuscles.
Description:
AAsequence:LTC3VTSKSIFGITTEDC17PDGQNLC24FKRRHYVVPKIYDSTRGC42AATC46PIPENYDSIHC57C58KTDKC63NE
Disulfidebonds:Cys3-Cys24,Cys17-Cys42,Cys46-Cys57,Cys58-Cys63
Length(aa):65
Formula:C310H481N87O100S8
MolecularWeight(average): 7283.29Da
Appearance:<hitelyophilizedsolid
Solubility: waterandsalinebuffer
CASnumber: notavailable
Source:synthetic
Purityrate: >98%
Reference:
IsolationandpharmacologicalcharacterizationofAdTx1,anaturalpeptidedisplayingspecificinsurmountableantagonismofthea1A-adrenoceptor
Abstract:
Venomsarearichsourceofligandsforionchannels,butverylittleisknownabouttheircapacitytomodulateG-proteincoupledreceptor(GPCR)activity.WedevelopedastrategytoidentifynoveltoxinstargetingGPCRs.Westudiedtheinteractionsofmambavenomfractionswithalpha(1)-adrenoceptorsinbindingexperimentswith(3)H-prazosin.Theactivepeptide(AdTx1)wassequencedbyEdmandegradationandmassspectrometryfragmentation.Itssynthetichomologuewaspharmacologicallycharacterizedbybindingexperimentsusingclonedreceptorsandbyfunctionalexperimentsonrabbitisolatedprostaticsmoothmuscle.AdTx1,a65amino-acidpeptidestabilizedbyfourdisulphidebridges,belongstothethree-finger-foldpeptidefamily.Ithassubnanomolaraffinity(K(i)=0.35nM)andhighspecificityforthehumanalpha(1A)-adrenoceptorsubtype.Weshowedhighselectivityandaffinity(K(d)=0.6nM)ofrADIo-labelledAdTx1indirectbindingexperimentsandrevealedaslowassociationconstant(k(on)=6x10(6).M(-1).min(-1))withanunusuallystablealpha(1A)-adrenoceptor/AdTx1complex(t(1/2diss)=3.6h).AdTx1displayedpotentinsurmountableantagonismofphenylephrine’sactionsinvitro(rabbitisolatedprostaticmuscle)atconcentrationsof10to100nM.AdTx1isthemostspecificandselectivepeptideinhibitorforthealpha(1A)-adrenoceptoridentifiedtodate.Itdisplaysinsurmountableantagonism,actingasapotentrelaxantofsmoothmuscle.Itspeptidicnaturecanbeexploitedtodevelopnewtools,asaradio-labelled-AdTx1orafluoro-labelled-AdTx1.IdentificationofAdTx1thusoffersnewperspectivesfordevelopingnewdrugsfortreatingbenignprostatichyperplasia.
Gprotein-coupledreceptors,anunexploitedanimaltoxintargets:Explorationofgreenmambavenomfornoveldrugcandidatesactiveagainstadrenoceptors
Abstract:
AtatimewhenpharmaceuticalcompaniesarehavingtroublefindingnewlowMWdrugsandwhenBIOLOGicsarebecomingmorecommon,animalvenomscouldconstituteanunderexploitedsourceofnoveldrugcandidates.WelookedforidentifyingnovelanimaltoxinsactiveagainstGprotein-coupledreceptors(GPCR),themostfrequentlyexploitedclassoftreatmenttargets,withtheaimtodevelopnovelresearchtoolsanddrugcandidates.Screeningofgreenmamba(Dendroaspisangusticeps)venomagainstadrenoceptorsidentifiedtwonovelvenompeptides.ρ-Da1ashownanaffinityof0.35 nMfortheα1a-ARwhileρ-Da1bdisplayedaffinitiesbetween14and73 nMforthethreeα2-ARs.Thesetwovenompeptideshavesequencessimilartothoseofmuscarinictoxinsandbelongtothethree-finger-foldproteinfamily.α1a-ARistheprimarytargetforthetreatmentofprostatehypertrophy.Invitroandinvivotestsdemonstratedthatρ-Da1areducedprostaticmuscletoneasefficientlyastamsulosin(anantagonistpresentlyused),butwithfewercardiovascularsideeffects.α2-ARsaretheprototypeofGPCRsnotcurrentlyusedastreatmenttargetsduetoalackofspecificligands.Blockageofthesereceptorsincreasesintestinalmotility,whichmaybecompromisedbyaBDominalsurgeryandreducesorthosterichypotension.Invitroandinvivotestsdemonstratedthatρ-Da1bantagonizesα2-ARsinsmoothmusclesandincreasedheartrateandbloodcatecholamineconcentrations.TheseresultshighlightpossIBLeexploitationofρ-Da1aandρ-Da1binimportantpathologies.
Crystallizationofrecombinantgreenmambaρ-Da1atoxinduringalyophilizationprocedureanditsstructuredetermination
MaïgaA.,etal.(2013)Crystallizationofrecombinantgreenmambaρ-Da1atoxinduringalyophilizationprocedureanditsstructuredetermination.ActaCrystallogrSectFStructBiolCrystCommun.PMID:23722859
Abstract:
ρ-Da1atoxinfromeasterngreenmamba(Dendroaspisangusticeps)venomisapolypeptideof65aminoacidswithastrongaffinityfortheG-protein-coupledα(1A)-adrenoceptor.Thisneurotoxinhasbeencrystallizedfromresolubilizedlyophilizedpowder,butthebestcrystalsgrewspontaneouslyduringlyophilization.ThecrystalsbelongedtothetrigonalspacegroupP3(1)21,withunit-cellparametersa=b=37.37,c=66.05Å,anddiffractedto1.95Åresolution.Thestructuresolvedbymolecularreplacementshowedstrongsimilaritiestogreenmambamuscarinictoxins.
Effectsofρ-Da1aapeptidicα(1)(A)-adrenoceptorantagonistinhumanisolatedprostaticadenomaandanaesthetizedrats
Abstract
ρ-Da1a,a65amino-acidpeptide,hassubnanomolaraffinityandhighselectivityforthehumanα(1)(A)-adrenoceptorsubtype.Thepurposeofthisstudywastocharacterizethepharmacologicaleffectsofρ-Da1aonprostaticfunction,bothinvivoandinvitro.ρ-Da1awastestedasanantagonistofadrenaline-inducedeffectsonCOScellstransfectedwiththehumanα(1)(A)-adrenoceptoraswellasonhumanisolatedprostaticadenomaobtainedfrompatientssufferingfrombenignprostatichyperplasia.Moreover,wecomparedtheeffectsofρ-Da1aandtamsulosinonphenylephrine(PHE)-inducedincreasesinintra-urethral(IUP)andarterialpressures(AP)inanaesthetizedrats,followingi.v.orp.o.administration.OnCOScellsexpressinghumanα(1)(A)-adrenoceptorsandonhumanprostaticstrips,ρ-Da1ainhibitedadrenaline-andnoradrenaline-inducedeffects.Inanaesthetizedrats,ρ-Da1aandtamsulosinadministeredi.v.30 minbeforePHEsignificantlyantagonizedtheeffectsofPHEonIUP.ThepK(B)valuesfortamsulosinandρ-Da1aforthiseffectweresimilar.WithregardstoAP,ρ-Da1aonlyreducedtheeffectofPHEonAPatthelowestdosetested(10 μg·kg(-1)),whereastamsulosinsignificantlyreducedPHEeffectsatdosesbetween10and150 μg·kg(-1).ρ-Da1aexhibitedarelevanteffectonIUPandasmalleffectonAP.Incontrast,tamsulosinantagonizedtheeffectsofPHEonbothIUPandAP.Weconcludethatρ-Da1aismoreuroselectivethantamsulosin.ρ-Da1aisthemostselectivepeptidicantagonistforα(1A)-adenoceptorsidentifiedtodateandcouldbeanewtreatmentforvariousurologicaldiseases.
Orthostericbindingofρ-Da1a,anaturalpeptideofsnakevenominteractingselectivelywiththeα1A-adrenoceptor
Abstract:
ρ-Da1aisathree-fingerfoldtoxinfromgreenmambavenomthatishighlyselectivefortheα1A-adrenoceptor.Thistoxinhasatypicalpharmacologicalproperties,includingincompleteinhibitionof(3)H-prazosinor(125)I-HEATbindingandinsurmountableantagonistaction.Weaimedtoclarifyitsmodeofactionattheα1A-adrenoceptor.Theaffinity(pKi9.26)andselectivityofρ-Da1afortheα1A-adrenoceptorwereconfirmedbycomparingbindingtohumanadrenoceptorsexpressedineukaryoticcells.Equilibriumandkineticbindingexperimentswereusedtodemonstratethatρ-Da1a,prazosinandHEATcompeteattheα1A-adrenoceptor.ρ-Da1adidnotaffectthedissociationkineticsof(3)H-prazosinor(125)I-HEAT,andtheIC50ofρ-Da1a,determinedbycompetitionexperiments,increasedlinearlywiththeconcentrationofradioligandsused,whiletheresidualbindingbyρ-Da1aremainedstable.Theeffectofρ-Da1aonagonist-stimulatedCa(2+)releasewasinsurmountableinthepresenceofphenethylamine-orimidazoline-typeagonists.Tenmutationsintheorthostericbindingpocketoftheα1A-adrenoceptorwereevaluatedforalterationsinρ-Da1aaffinity.TheD106(3.32)AandtheS188(5.42)A/S192(5.46)Areceptormutationsreducedtoxinaffinitymoderately(6and7.6times,respectively),whiletheF86(2.64)A,F288(6.51)AandF312(7.39)Amutationsdiminisheditdramaticallyby18-to93-fold.Inaddition,residueF86(2.64)wasidentifiedasakeyinteractionpointfor(125)I-HEAT,asthevariantF86(2.64)Ainduceda23-foldreductioninHEATaffinity.UnliketheM1muscarinicacetylcholinereceptortoxinMT7,ρ-Da1ainteractswiththehumanα1A-adrenoceptororthostericpocketandsharesreceptorinteractionpointswithantagonist(F86(2.64),F288(6.51)andF312(7.39))andagonist(F288(6.51)andF312(7.39))ligands.Itsselectivityfortheα1A-adrenoceptormayresult,atleastpartly,fromitsinteractionwiththeresidueF86(2.64),whichappearstobeimportantalsoforHEATbinding.
Polypharmacologyprofilesandphylogeneticanalysisofthree-fingertoxinsfrommambavenom:caseofaminergictoxins
Compositionofmamba’svenomisquiteatypicalandcharacterizedbythepresenceofalargediversityofthree-fingerfoldtoxins(3FTx)interactingwithvariousenzymes,receptorsandionchannels.Inparticular,3FTxfrommambasdisplaytheuniquepropertytointeractwithclassAGPCRs,sometimeswithahighaffinityandselectivity.Ascreeningoffiveofthesetoxins(MT1,MT3,MT7,ρ-Da1aandρ-Da1b)on29differentsubtypesofbioaminergicreceptors,usingcompetitionbindingexperiments,highlightsthediversityoftheirpharmacologicalprofiles.Thesetoxinsmaydisplayeitherabsoluteselectivityforonereceptorsubtypeorapolypharmacologicalpropertyforvariousbioaminergicreceptors.Nevertheless,adrenoceptoristhemainreceptorfamilytargetedbythesetoxins.FurThermore,anewreceptortargetwasidentifiedfor3FTxandtoxinsingeneral,theρ-Da1binteractingcompetitivelywiththehumandopamineD3receptorinthemicromolarrange.ThisresultexpandsthediversityofGPCRstargetedbytoxinsandmoregenerallyhighlightsthemultipotentinteractingpropertyof3FTx.Phylogenicanalyzesofthesetoxinsshowthatmuscarinic,adrenergicanddopaminergictoxinsmaybepooledinonefamilycalledaminergictoxins,thisfamilycomingprobablyfromaspecificradiationofligandspresentinmambavenoms.
