w ww.e l s e v i e r . c o m / l o c a t e / b j p
Original Article
Acetylcholinesterase inhibitory activity of a neurosteroidal alkaloid from the upside-down jellyfish Cassiopea andromeda venom
Gholamhossein Mohebbi
a, Iraj Nabipour
a,∗, Amir Vazirizadeh
b, Hossein Vatanpour
c, Maryam Farrokhnia
a, Ammar Maryamabadi
d, Afshar Bargahi
aaThePersianGulfMarineBiotechnologyResearchCenter,ThePersianGulfBiomedicalResearchCenter,BushehrUniversityofMedicalSciences,Bushehr,Iran
bDepartmentofMarineBiotechnology,ThePersianGulfResearchandStudiesCenter,ThePersianGulfUniversity,Bushehr,Iran
cDepartmentofPharmacologyandToxicology,FacultyofPharmacy,ShaheedBeheshtiMedicalSciencesUniversity,Tehran,Iran
dDepartmentofChemistry,FacultyofSciences,PersianGulfUniversity,Bushehr,Iran
a r t i c l e i n f o
Articlehistory:
Received4February2018 Accepted1June2018 Availableonline8July2018
Keywords:
Upside-downjellyfish Antiacetylcholinesteraseactivity AndrotoxinB
Alzheimer’sdisease Neurosteroidalalkaloid GABA-Areceptor
a b s t r a c t
Naturalcompounds frommarineorganisms havebeenrarely studiedfortheiracetylcholinesterase inhibitoryactivities.TheaimofthisstudywastoisolatenovelcompoundswithantiAChEactivityfrom thevenomofupside-downjellyfishCassiopeaandromedaForskål,1775.Thecompoundsofthefraction- atedvenomongelfiltrationchromatographywereidentifiedbyanalyzinggaschromatography–mass spectroscopydata.ThestructureoftheisolatedcompoundthatshowedthemostpotentantiAChEactiv- ityinadockingstudywaselucidatedbydifferentspectraldata,including1HNMRand13CNMR.Three compounds,includinganeurosteroidalalkaloidandrotoxinB,wereidentifiedfromtwovenomfractions.
Thisneurosteroidalalkaloidshowedstrongacetylcholinesteraseinhibitoryactivity(IC502.24±0.1M) comparedwiththereferencestandard,galantamine.Theresultsobtainedbyadockingstudydemon- stratedthatAndrotoxinBhadclosecontactwithtwoofthethreeaminoacidresiduesofthecatalytic triadofacetylcholinesterasegorgeandwasaccommodatedwithinaperipheralhydrophobicpocketcom- posedofnumerousaromaticsitechains.Inconclusion,theisolatedneurosteroidalalkaloidfromCassiopea andromedawasapotentantiAChEagentwithstrongbindingtoboththecatalyticandperipheralsites ofacetylcholinesterasethatcorrelatedwellwiththeexperimentaldata.Furtherstudiesarerequiredto determinewhetherandrotoxinBcouldbeapotentialtreatmentforAlzheimer’sdisease.
©2018SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Alzheimer’sdiseaseis a fatal progressive neurodegenerative disorder of the brain affecting 25 million people worldwide (Ferreira-Vieiraetal.,2016).Today,itiscalledthesilvertsunamiof the21stcentury(Sarkaretal.,2016).Theaccumulationofneu- rofibrillary tangles, the amyloid-beta plaques in the brain, the progressivelossofneuronsinthehippocampusandcortex,and theprogressivedeclineinmemory,thinking,andlanguageskills arecharacteristicsofthiscomplexdisease(Ferreira-Vieiraetal., 2016).
The degeneration of cholinergic neuronsresults in memory andattentiondeficitsofAlzheimer’sdisease(Ferreira-Vieiraetal., 2016). Three cholinesterase inhibitors, donepezil, galantamine, andrivastigmine,haveshownsomeeffectsonmildtomoderate
∗ Correspondingauthor.
E-mail:nabipourpg@bpums.ac.ir(I.Nabipour).
Alzheimer’sdisease(Birks,2006).Therefore,thesearchforeffective bioactivecompoundswithacetylcholinesterase(AChE)inhibitory activitiesfromnaturalsourcestocombatAlzheimer’sdiseaseisa hottopic.
Althougha largenumber of AChE inhibitorsfrom medicinal plants have been reported (Ahmed et al., 2013), natural com- pounds from marine organisms have been rarely studied for theirAChEinhibitoryactivities(Beedesseeetal.,2013).TheAChE inhibitoryactivityofplastoquinonesfromSargassumsagamianum (Choi et al., 2007); the acetone extracts of Padina gymnospora (Shanmuganathan et al.,2015); fucoxanthin,which isa marine carotenoid(Pengetal.,2011);avarol,whichisamarinesesquiter- penoidhydroquinone isolated fromDysidea avara (Tommonaro etal.,2016);extractsfromspongescollectedinMauritiuswaters (Beedesseeetal.,2013);andhalogenatedsesquiterpenesderived fromthePersianGulf(FarrokhniaandNabipour,2014)havebeen studied.
Cassiopeaandromeda(Forsskal1775;Cnidaria:Scyphozoa:Rhi- zostomea)isatypeofjellyfishthatlivesinshallow,tropicalinshore https://doi.org/10.1016/j.bjp.2018.06.002
0102-695X/©2018SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
creativecommons.org/licenses/by-nc-nd/4.0/).
30º
27º30
25º
45º 50º 55º
Iran
Nayband bay
N E S W
Persian Gulf
Cassiopea andromeda
Fig.1. Locationmapofthestudyarea(apopulationoftheCassiopeaandromeda jellyfishinNaybandBay,Bushehr-Iran).
marinewatersandisusuallyassociatedwithmangrovepopula- tions(Hollandetal.,2004).Thisjellyfishisalsocalledupside-down jellyfishbecause,unlikethestereotypicaljellyfish,itoccursinan invertedforminintertidalsandormudflatswiththeirtentacles orientedtowardthesky.AlthoughthegenusCassiopeahasaglobal distributionintheWesternAtlanticandIndo-Pacifictropicalareas, CassiopeawasnotobservedinthePersianGulfuntil2014.Thealien C.andromedahasbeensuggestedtoarriveatthePersianGulfinthe pellagicstagebytheballastwaterofships(Nabipouretal.,2015).
Currently;anestablishedpopulationofthisupside-downjellyfish ispresentinNaybandLagoon,Assalouyeh,Iran.
Cassiopeaandromedacaptures itspreythrough nematocysts.
SomeofthetoxiccharacteristicsofC.andromedahavebeeninves- tigated(Radwanetal.,2001;Ellitheyetal.,2014;Nabipouretal., 2017).Nematocystvenomofthisjellyfishexhibiteddermonecrotic, vasopermeability-producingproperties,andhemorrhagingeffects (Radwan et al., 2001; Nabipour et al., 2017). The methanolic extractofC.andromedaproducedpotentinhibitoryactivityagainst HIV-1protease(Ellitheyetal.,2014).Recently,weshowedthat theC. andromedavenom fractions inducedselective injury and cytochromeCreleaseinmitochondriaobtainedfrombreastade- nocarcinomapatients(Mirshamsietal.,2017).
Aspartofourresearchontheisolationofbioactivemolecules fromC.andromeda,theAChEinhibitoryactivitiesoftheextracted fractionsandcompoundsofthisupside-downjellyfishwereeval- uatedusingconventionalanddockingmethods.
Materialsandmethods Samplingandsamplepreparation
A number of thirty live specimens of Cassiopea andromeda Forskål,1775werecollectedbymeansofatrawlnet,fromtheNay- bandBayinthenorthernpart(27◦30S,52◦35E)ofBushehr,Iran, inspringof2014(Fig.1).Thebloommainlywaslocatedatindepths of0.5–1.5m,onamuddy-sandbottominLagoon,moreovertheir aggregationswereincludingabout4–5individualsineachm2and differentsizes,buttypicallymorerangesbetween6and15cmin diameter.TheidentitiesofthespecieswereverifiedbyProfessorB.
HollandfromtheUniversityofHawaii(Nabipouretal.,2015).
TheprojectwaspermittedbytheMedical EthicsCommittee of Bushehr University of Medical Sciences and Health Services, Bushehr,Iran.AllanimalexperimentswerebasedontheNational Ethical Guidelines for Animal Research in Iran (2005) under a projectlicense,whichwasapprovedbytheAnimalCareandUse CommitteeofBushehrUniversityofMedicalSciences,Iran,accord- ingtoProtocol:D/P/3758.
Theseparation ofthe tentacleswasperformed accordingto Bloom et al. (1998) method, with some modifications. Briefly, thetentacleswereexcisedmanuallyfromspecimens assoonas possible after capture by the trawl, directly placed into small
Fig.2. MicrographoftheCassiopeaandromedanematocystsalongthetentacles (blackarrows).
glasscontainersfilledwithaone-thirdportionofseawater,and transported in ice bagsto the PersianGulf Marine Biotechnol- ogyResearchCenterlaboratoriesinthePersianGulfBiomedical ResearchCenter,BushehrUniversityofMedicalSciences,Iran.At thattime,thetentacleswerehomogenizedbyaHomogenizer(IKA, Germany),keptat4◦Cfor twodaysfortheautolysisofthetis- suesandreleaseoftoxins(Burnettetal.,1992),andcentrifuged (Eppendorf,Germany)at12,000×gfor15minat4◦Ctoremove thesediments.Thefinalsupernatantwasfreeze-dried(Christ,UK) andkeptat−80◦Cuntiluse(Mustafaetal.,1995).Thenematocysts ofthetentacles(blackarrows)areshowninFig.2.
Proteinconcentrationandmolecularweightdetermination
Theproteinconcentrationofthelyophilizedcrudevenom(a sky bluish powder) was measured according to the Bradford method (Bradford, 1976) using an ultraviolet–visible (UV–Vis) spectrophotometer(CecilAquariusDoubleBeamSpectrophotome- ter,England).Bovineserumalbumin(BSA)(Sigma,Germany),was usedasthecalibrationstandardproteinatconcentrationsof(5, 10, 15,20,25mgml−1).Theprotein concentrationexpressedin unit of gml−1. Briefly, a total of 10lof freeze dried venom wassubjectedtosodiumdodecylsulfate-polyacrylamidegelelec- trophoresis(SDS-PAGE)at25◦C,accordingtotheLaemmlimethod (Laemmli,1970).
Resolvinggel(pH8):percentageofgel12.5%;3.1mlacrylamide:
bisacrylamide(30:0.8%w/v);3mlTris–ClpH:8.8;38lSDS20%;
1.3mldH2O;5lammoniumpersulfate(APS)10%;7.5mltetram- ethylethylenediamine(TEMED).
Stackinggel(pH6.8):percentageofstack6%;1mlacryl:bisacryl (30:0.8%w/v);630lTris–ClpH8.8,1M;25lSDS20%;3.6ml dH2O;24lAPS10%;and5ml TEMED)(Brinkman andBurnell, 2007).ProteinswerevisualizedusingCoomassie®BrilliantblueR 250(Sigma,Germany).Themolecularweightswereevaluatedby comparisontoBioRadmarkers(PrecisionPlusProtein®Standards, BioRad,USA) attherangesof 225,150, 102, 76.52,38,17, and 12kDa.AllotherchemicalswerepurchasedfromMerck(Darm- stadt,Germany).
AChEactivity
Thecrudevenomanditsfractionsweretestedfortheirability toinhibitAChEfromelectriceel(C2888–Electrophoruselectricus AChE,typeV,1000units/mgprotein,Sigma–Aldrich),accordingto theEllmankineticmethodmodifiedbyWoreketal.(1999).Hydrol- ysisratewasmonitoredbyverifyingtheformationoftheTNB−of DTNBat436nmduring3minatintervalsof15s,usingtheCecilUV- VISSpectrophotometer.Fortheblank,thebufferinsteadofenzyme
solutionwasused.Theenzymeactivitywasmeasuredusingthe equation:
Enzymeactivity(mol/l/min)=Sample(mE/min)−Blank(mE/min) 10.6
The percentage (%) inhibition was calculated as follows:
(E−S)/E×100,where“E”istheactivityoftheenzymewithouttest compoundsand“S”istheactivityofenzymewithtestcompounds.
Theexperimentwasdoneintriplicateandconcentrationsofthe samplesthatinhibitthehydrolysisofthesubstrateby50%(IC50) weredeterminedbylinearregressionanalysisbetweentheinhi- bitionpercentagesversustheextractconcentrationbytheExcel program.
Gelfiltrationchromatography
Sephadex G-200 (Pharmacia, USA) gels were poured into a 60cm×2.5cm diametercolumn. Elutionwascompleted witha 0.05Mphosphatebuffer(pH 7.2at4◦C),and theflow ratewas adjustedto0.5ml/min. About200mg ofthecrude venomof C.
andromedawasdissolvedinthephosphatebuffer.Aftercentrifuga- tionat12,000×gfor15minat4◦C,thesupernatantwasloadedon theSephadexG-200.Thetoxinswereelutedwiththephosphate buffer,the4mlfractionswerecollectedwithafractioncollector apparatus,andproteinabsorbancewasmeasuredat280nmbya UVspectrophotometer(Cecil7000series,England)(Othmanand Burnett,1990).
Gaschromatography–massspectroscopy(GC–MS)ofthefractions Thelyophilizedfractions(whitecolor),weresubjectedtothe 7890BAgilentGasChromatography–MassSpectroscopy.Electron ionization(EI)massspectra(scanrange,m/z50–500)wereobtained usingelectronswithenergyof70eVenergyandfilamentemission of0.5mA.TheGCseparationswereconductedusinganHP-5MSUI column(30m×0.25mmi.d.,filmthickness0.5m).Heliumwas usedasthecarriergas(flow:0.8mlmin−1)for EI.TheGCoven wastemperatureprogrammedat5◦Cmin−1from80◦Cafter3min sincethesampleinjectionandheldat250◦Cfor10min.Theinjec- tionportofthegaschromatograph,transferline,andionsourceof 5977MSDweremaintainedat240◦C,250◦C,and220◦C,respec- tively.The separatedcompounds wereidentified bycomparing themwiththecompounddatafromtheNationalInstituteofStan- dardsandTechnology(NISTMSdatabase,2014)library.Therelative percentamountofeachcomponentwasmeasuredbycomparing itsaveragepeakareatothetotalareas.
NMRanalysis
Thestructureofthepurifiedcompoundwiththemostpotent anticholinesteraseactivitydissolvedinDMSOwaselucidatedon thebasis oftheresult ofthe1HNMR (400MHz)and 13CNMR (100MHz)BrukerAvance400.
Computationaldetails
Thebindingaffinitiesbetweentheidentifiedstructures(inaddi- tiontogalantamineasthecontrolcompound)andtheactivesite of recombinant human AChE receptor were examined through dockingstudies usingthe AutoDock4.2.6 suites ((Morriset al., 2009).Dockinglogswereanalyzedinthegraphicaluserinterface oftheAutoDockTool(ADT)(Sanner,1999).Thecrystalstructureof rhAChEinthecomplexwith(−)-galantaminewasobtainedfrom theBrookhavenproteindatabankwithPDB accessioncodeey6 (Cheungetal.,2012).Beforestartingthedockingsimulations,(−)- galantaminewasseparatedfromthecomplex.Asthepositionsof
225 150
102
76 52
38
17 12
kDa M S
Fig.3. SDS-PAGE(12%polyacrylamidegelstainedwithCoomassiebrilliantblue) analysisofproteinstandardmarker(M),Cassiopeaandromedavenomsample(S).
thewatermoleculesinthecrystalstructureoftheenzymeswere unlikelytobeconserved,theywereexcludedbeforethedocking studies(Pilgeretal.,2001).H-atomswereaddedtothehAChEpro- teinforcorrecttautomericstatesofaminoacidresidues,andthe non-polarhydrogenswerecombined.TheKollmanUnitedAtom chargesandatomicsolvationparameterswereassignedtothepro- teinandtheGasteigerchargetotheligands.Allstructureswere convertedtoPDBQTformatinADTasrequiredintheAutoDock calculations.Thesizeofthegridplottedforspecifyingthesearch spacewassetto70×70×70 ˚A3andcenteredonthepredictedcav- itieswithadefaultgridpointspacingof0.375 ˚A.Pre-calculatedgrid maps,whichstoregridsofinteractionenergybasedontheinter- actionoftheligandatomwiththereceptortarget,wereobtained usingAutoGrid.ItcomputesthevanderWaalsenergiesateachsin- glegridpoint.Additionally,theLamarckiangeneticalgorithm(GA) wasusedwithapopulationsizeof150dockingsand100GAruns.
Fivemillionenergyevaluationswereusedinthedocking,andthe crossoverandmutationrateswererunwithdefaultsettings.The obtainedlowestdockedenergyconformationoftheligandinthe mostpopulatedclusteramongallthe100GArunsusedforfurther studies.TheoutputsfromAutoDockandimagesweregenerated withPyMOL(DeLano,2002),andAccelerysViewerLite5.0a(2002).
Resultsanddiscussion
TheproteinconcentrationandSDS-PAGEanalysis
Theproteincontentofthecrudevenom,quantifiedbytheBrad- fordmethod,wasfoundtobe17.5mgml−1.TheSDS-PAGEanalysis with12%acrylamidegel,revealedatleastelevenprominentpro- teinbands,withmolecularmassesrangingfrom12to225kDa.The mainbandswerelocatedattheapproximatemolecularweightsof 12,17,28,34,38,43,50,53,76,102and225kDa(Fig.3).Anumber ofminorbandswerealsoobservedaroundtheseregions.
AChEactivity
Experimental data based on Ellman’s spectroscopic method showedthatthecrudevenomandfractionBexhibitedproperAChE inhibitoryactivities.TheIC50ofthecrudevenom,fractionsAandB were4.81±0.25,7.03±0.012and2.24±0.1(M)at37◦C,respec- tively.
3
2.5
2
1.5
1
0.5
0
0 10 20 30
ATX-A
ATX-B C.andromeda GelFiltration (Sephadex G-200)
40 50 60 70 80
fraction number
abs orbtion at 280 nm
Fig.4. GelfiltrationchromatogramofCassiopeaandromedacrudevenomona SephadexG-200(60×2.5cm).Atotalof200mgofdissolvedcrudevenomin3ml 0.15MNaCl(pH7.2)wasappliedandelutedwiththesamebuffer.FlowRatewas 0.5ml/min.
Gelfiltrationchromatography
Thecrude venom was fractionatedusing gelfiltrationchro- matography.AsshowninFig.4,thechromatogramofC.andromeda crudevenomonSephadexG-200revealedtwofractions(faandfb) withabsorbanceat280nm.ThefawascalledAndrotoxinA(ATXA) andfbwascalledAndrotoxinB(ATXB).Thefractionswerecollected on4mlvolumes,freezedried,andstoredat−70◦C.
GC–MSofthecrudevenomandfractions
The analysis of the ATXA dissolved in methanol: hex- ane (3:2) detected two compounds (ATX A1, A2) using the GC–MS(seesupportinginformation).Theirpatternswereconsis- tentwithATXA1(1):1,2-dicarboxy-3-(4-chlorophenyl)-2,3(1H)- dihydropyrido(1,2-a)benzimidazole; (C19H15ClN2O4); MW: 370., ATXA2 (2): 1,5-diphenyl-1,2-dihydro-3H-1,2,4-triazole-3-thione
#;(C14H11N3S);MW:253.ThepatternofATXB(3) wasconsis- tentwith1H-androst-16-eno[17,16-g]indol-3-ol,acetate(ester), (3á,5à)-;(C27H35NO2);MW:405.
Moleculardocking
Table1showstheestimatedbindingenergyofthethreeidenti- fiedcompoundsA1,A2,andB.ATXBwasthemostactivecompound againstrhAChEincomparisonwithandrotoxinsA1andA2andeven thecontrolmoleculeofgalantamine.
The involved residues in the close interaction are listed in Table2.
Theinvestigatedcompoundswereinclosecontactwithtwoof thethreeaminoacids(Ser203andHis447)ofthecatalytictriadof AChEthroughthevanderWaalsandelectrostaticinteractions.
ATXBwasaccommodatedwithintheperipheralsitecomposed by Asp74, Tyr72, Tyr124, Trp286, Phe295, Phe297, Tyr337 and Tyr341(Fig.5).
Theaminonitrogenofthiscompoundformedahydrogenbond withthehydroxyl groupofTyr341.Therewereseveralvander WaalsinteractionsamongcycloalkaneringsofATXB,Tyr124and Tyr337, in additionto the pi-interaction witharomatic ring of Trp286.ClosecontactsofATXBwithquaternaryammoniumbind- ingsiteresidues(suchasTrp86)andesteraticsite(Gly120and121) werefound.
NMRspectroscopy
The1Hand13CNMRspectraoftheATXBdissolvedinDMSO wererecordedonaBrukerAvance400.HNMR(400MHz,DMSO):
␦0.96(3H,s,CH3),1.12(3H,s,CH3),1.43–1.89(16H,m,aliphatic), 2.13(3H,s,OCH3),2.75(3H,m,aliphatic),3.01(2H,m,COCH2),5.28 (1H,d,allylic),6.46(1H,d,allylic),6.63(1H,d,aromatic),7.34(1H,d, aromatic).CNMR(100MHz,DMSO):␦20.0,20.9,21.7,24.2,27.7, 28.5,29.0,33.2,33.3,35.7,36.0,36.6,37.7,40.9,43.0,48.9,50.3, 74.7,102.7,122.6,125.4,127.1,127.8,130.6,137.0,150.2,170.3 (seesupportinginformation).
In this study, we found that the venom of C. andromeda containedATXB, thestructure of which waselucidatedas 1H- androst-16-eno[17,16-g]indol-3-ol, acetate (ester), (3á,5à)-.This steroidalalkaloidshoweda strongAChEinhibitoryactivity.This compoundisthesecondisolatedmarine-derivedalkaloidsteroid withinhibitionagainstAChE.
Morethan35alkaloidshavebeenexaminedasinhibitinglig- andsofAChE(Moragaetal.,2016).Thecholinesterase-inhibiting activitiesofsteroidalalkaloidsfromFritillariaimperialis(Atta-Ur- Rahman et al., 2002), Sarcococca saligna (Zaheer-Ul-Haq et al., 2003),Sarcococcahokeriana(Choudharyetal.,2005), andHolar- rhenapubescens(Cheenprachaetal.,2016)havebeenreported.
Although marine organisms including jellyfishes have been thesubjectofinvestigationstodeterminenovelcompoundswith acetylcholine-inhibitingactivity,onereportwasconductedinthe scientificliteratureonmarine-derivedsteroidalkaloidswithAChE- inhibiting activity (Langjae et al., 2007). Langjae et al. (2007) isolated a newstigmastane-type steroidal alkaloid, a 4-acetoxy plakinamine,fromthespongeCorticiumsp.Thecomparisonofthis marine-derivednon-pregnane-typesteroidwithacloselyrelated steroidalalkaloidisolatedfromSarcococcashowedhighpotencyin itsAChE-inhibitingactivity.Onthecontrary,thismarine-derived steroidexpressedanunusualmixed-competitivemodeofenzyme inhibitionintheinhibitingmodeofmostSarcococcaalkaloids.
Zaheer-Ul-Haqetal.(2003)investigatedtheAChEinteractions withfifteensteroidalalkaloidsisolatedfromS.saligna.Theyfound thattheligandsbindtothearomaticgorgeoftheirsteroidback- boneareaoftheenzymethroughthehydrophobicsix-membraned ringA.Theseinteractionsreduceflexibilityatthegorgewithout externallyalteringtheenzymestructure.Accordingtotheirdock- ingstudies,theAChE inhibitoryactivitiesoftheligands mainly exhibitedanoncompetitivecharacter(Choudharyetal.,2005).
Inthepresentstudy,weselectedtherhAChEforthemolecular dockingstudybecauseofitsmoreaccurateresultsthanTorpedo AChE(Cheungetal.,2012).ATXBwasrevealedtobeactiveagainst rhAChE,and itsbindingenergy wasgreaterthanthat ofgalan- tamine.
ThemostremarkablefeatureofAChEisadeepandnarrowgorge.
Thecatalyticsite ofAChEis locatedatthis gorgeknownasthe catalytictriad,whichiscomposedofHis447,Glu334,andSer203.
Thispartisconsideredcrucialbecauseofitsenzymaticactivity,
Table1
TheresultsofdockingstudyforgalantamineandthecrudevenomandisolatedcompoundsfromCassiopeaandromedavenomfractions.
Molecule Bindingenergy(kcal/mol) EstimatedinhibitionConstant,Ki(T=298.15K) ExperimentalIC50(M)
ATXA 7.03±0.012
ATXA1 −6.11 33.03M
ATXA2 −7.84 1.79M
ATXB −12.31 951.28pM 2.24±0.1
Crudevenom – – 4.81±0.25
Galantamine −9.34 141.68nM 6.1±0.13
Table2
TheResiduesoftheisolatedcompoundsfromCassiopeaandromedavenomfractionsandgalantamineindockingstudywithAChEenzyme.
Molecules Residues
A ATXA1 Phe297,Trp286,Phe338,Tyr341,Tyr337,His447,Ser125,Gly120,Gly121,Gly122,Asp74,Ser203,Trp86.
ATXA2 Asp74,Phe297,Ser203,Val294,His447,Tyr337,Tyr341,Phe338,Trp286,Gly121,Gly122,Tyr124.
B ATXB Trp286,Tyr124,Ser125,Asp74,Trp86,Tyr341,Tyr337,Ser203,Phe338,His447,Gly448,Ile451,Gly120,Gly121,Gly122,Phe297,Glu202,Gly448.
Galantamine Tyr124,Ser125,Gly126,Ala127,Leu130,Phe297,Trp86,Tyr133,Ser203,His447,Phe338,Glu202,Gly448,Ile451,Tyr449,Gly120,Gly121,Gly122.
Fig.5.Dockigoftheisolatedcompounds(ATXA1,A2andB)fromCassiopeaandromedavenomfractionsandgalantamine(asthestandardmolecule),withAChEenzyme.
inwhich acetylcholine ishydrolyzedtoacetic acidand choline (Sussmanetal.,1991).ATXBproducedaclosecontactwithtwo ofthethreeaminoacidresiduesofthecatalytictriad(Ser203and His447)throughvanderWaalsandelectrostaticinteractionsinthe dockingstudy.
Asecondorperipheralsitecomposedofnumerousaromatic sitechainsthatextendbeyondTyr337atthecatalytic/peripheral siteinterfacetotheentranceofgorgecontributestotheAChEcat- alyticefficiency(Taraetal.,1998;Szegletesetal.,1999).Thissite includesAsp74,Tyr72,Tyr124,Trp286,Phe295,Phe297,Tyr337, and Tyr341;Asp74is responsible for acetylcholinerecognition.
ATXBwasaccommodatedwithinthishydrophobicpocket,andthe aminonitrogenofthiscompoundformedahydrogenbondwith thehydroxylgroupofTyr341.SeveralvanderWaalsinteractions wereobservedamongthecycloalkaneringsofATXB,Tyr124,and Tyr337 asidefrom thepi-interaction withthearomatic ring of
Trp286.Thus,thelargehydrophobicskeletonofATXBresultedin hydrophobicinteractionswiththeactivesitesofAChE.Thebinding ofthiscompoundtoboththeperipheralandcatalyticsitesinvolved extensiveprotein–ligandcontacts.Thenon-classicalCH···Ohydro- genbondinteractionbetweenthehydroxylgroupofAsp74andCH ofthecycloalkaneringofATXBcouldbeinvolved.Furthermore, theoccurrenceoftheclosecontactofATXBwiththequaternary ammoniumbindingsiteresidues(e.g.,Trp86)andtheesteraticsite (Gly120and121)confirmedthatthiscompoundlocalizedinthe activedomainoftheenzyme.
ThebiologicalactivitiesofATXBhavenotbeenstudiedyet.How- ever,androstanol,itscloselyrelatedsteroid,hasbeenknownasa neurosteroid(Kaminskietal.,2006).Neurosteroidsaredefinedas aclassofendogenoussteroidssynthesizedwithinthebrainand modulateneuronsandglialactivitybyrapidnon-genomicactions (Reddy,2010;Tvrdei ´candPoljak,2016).
TheC19 16-unsaturatedsteroid androstanol (5␣-androst-16- en-3␣-ol)hasbehavioralandsocialresponsesinhumansthrough itsneuroactivities(Reddy,2010;Tvrdei ´c andPoljak,2016).It is regarded asa potent steroidal pheromone. However,its mech- anismofactionremains unknown(Gowerand Ruparelia,1993;
Grammeretal.,2005).Inexaminingthesub-acutetoxicityofthe crudevenomofC.andromeda,wefoundthatthesexualactivitiesof theexperimentalratsincreasedseveralminutesaftertheinjections (Mohebbietal.,2016).Thisinterestingfindingimpliesthepresence ofapheromone-likeactivityoftheATXBcontentofthevenom.
Thebioregulatoryfunctionsofsteroidalmoleculesinthephylum Cnidaria,includingtheScyphozoaclass(“true”jellyfishes),arenot elucidated.Theidentifiedsexsteroidsincnidariantissuesnotonly havea defensiverolebut arealsolikelytoserve anendocrine- likesignalingroleinregulatinggametogenesisand/orspawning (Tarrant,2005).Theconcentrationsofthesesexsteroidsarevari- ableindifferentseasonsinbothsexesintheAnthozoaclass(e.g., coralsandanemones)ofthecnidarians.Polarsteroidshavebeen speculatedtoserveaschemicalcuesorwater-bornepheromones totrigger coral spawning(Tarrant,2005; Tarrant, 2007).These speculationspromptthesuggestionthatthepresenceofATXBin thevenomof C.andromedamightplay a roleas awater-borne pheromoneasidefromitsdefensiverole.
Theprototypicneurosteroidsarepositiveallostericmodulators ofGABA-Areceptorswithdiversepharmacologicaleffects, such asanti-seizureandanti-anxietyactivities(Reddy,2010).Kaminski etal.(2006)showedthatandrostenolactsasaGABA-Areceptor modulatorandthatitsactivityiscomparablewiththoseofother protypicneurosteroids.ATXBisreducedatthe5-and3-positions oftheA-ring,and thesubstituents atthesepositionsareinthe
␣-configuration.Thepotentneurosteroidsthatarepositivemod- ulatorsof theGABA-Areceptorhave similarstereochemistryat thesecritical positions.The 3-epimerof androstenoldoesnot havemodulatoryeffectsontheGABA-Areceptors(Kaminskietal., 2006).InthestructuralhomologybetweenATXBandandrostenol, theneurosteroid-likeactivityofATXBasaGABA-Areceptormodu- latorshouldbeinvestigated.Therefore,furtherstudiesarerequired todeterminewhetherATXBisapotentialmodulatorofGABA-A.
Thepositiveanswertothisquestionwillopennewfrontiersinthe treatmentofepilepsy.
Conclusions
The isolated neurosteroidal alkaloid androtoxin B, from C.
andromedawasapotentantiAChEagentwithstrongbindingto boththecatalyticandperipheralsitesofAChEthatcorrelatedwell withtheexperimentaldata.Furtherstudiesarerequiredtodeter- minewhetherATXBcouldbeapotentialtreatmentforAlzheimer’s disease.
Authorcontributions
Conceivedanddesignedtheexperiments:GMandIN.Performed theexperiments:GM,IN,AV,AM,ABandHV.Analyzedthedata:
GM,INandMF.Contributedreagents/materials/analysistools:GM, AM,AVandIN.Wrotethepaper:GM,INandMF.Alltheauthors havereadthefinalmanuscriptandapprovedthesubmission.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.
Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.
Righttoprivacyandinformedconsent.Theauthorsdeclarethat nopatientdataappearinthisarticle.
Conflictsofinterest
The authors declared no potential conflicts of interest with respecttotheresearch,authorship,andpublicationofthisarticle.
Acknowledgments
Authors gratefully acknowledge the help and financial sup- port provided by Bushehr University of Medical Sciences and HealthServices,Bushehr-Iran.Theprojectwaspartlysupported by Iran National ScienceFoundation (Research Chair AwardN.
95/INSF/44913).
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2018.06.002.
References
Ahmed,F.,Ghalib,R.M.,Sasikala,P.,Ahmed,K.K.M.,2013.Cholinesteraseinhibitors frombotanicals.Pharmacogn.Rev.7,121–130.
Atta-Ur-Rahman,Akhtar, M.N.,Choudhary,M.I.,Tsuda, Y.,Sener,B.,Khalid,A., Parvez,M.,2002.NewsteroidalalkaloidsfromFritillariaimperialisandtheir cholinesteraseinhibitingactivities.Chem.Pharm.Bull.50,1013–1016.
Beedessee,G.,Ramanjooloo,A.,Surnam-Boodhun,R.,vanSoest,R.W.,Marie,D.E., 2013.Acetylcholinesterase-inhibitoryactivitiesoftheextractsfromsponges collectedinmauritiuswaters.Chem.Biodivers.10,442–451.
Birks,J.,2006.CholinesteraseinhibitorsforAlzheimer’sdisease.CochraneDatabase Syst.Rev.1,http://dx.doi.org/10.1002/14651858,CD005593.
Bloom,D.A.,Burnett,J.W.,Alderslade,P.,1998.Partialpurificationofboxjellyfish (Chironexfleckeri)nematocystvenomisolatedatthebeachside.Toxicon36, 1075–1085.
Bradford,M.M.,1976.Arapidandsensitivemethodforthequantitationofmicro- gramquantitiesofproteinutilizingtheprincipleofprotein-dyebinding.Anal.
Biochem.72,248–254.
Brinkman,D.,Burnell,J.,2007.Identification,cloningandsequencingoftwomajor venomproteinsfromtheboxjellyfish,Chironexfleckeri.Toxicon50,850–860.
Burnett,J.W.,Long,K.O.,Rubinstein,H.M.,1992.Beachsidepreparationofjellyfish nematocysttentacles.Toxicon30,794–796.
Cheenpracha, S., Jitonnom, J., Komek, M., Ritthiwigrom, T., Laphookhieo, S., 2016.Acetylcholinesteraseinhibitoryactivityandmoleculardockingstudyof steroidalalkaloidsfromHolarrhenapubescensbarks.Steroids108,92–98.
Cheung, J., Rudolph, M.J., Burshteyn, F., Cassidy, M.S., Gary, E.N., Love, J., Franklin, M.C.,Height, J.J., 2012.Structures ofhuman acetylcholinesterase in complex with pharmacologically important ligands. J. Med. Chem. 55, 10282–10286.
Choi,B.W.,Ryu,G.,Park,S.H.,Kim,E.S.,Shin,J.,Roh,S.S.,Shin,H.C.,Lee,B.H.,2007.
AnticholinesteraseactivityofplastoquinonesfromSargassumsagamianum:lead compoundsforAlzheimer’sdiseasetherapy.Phytother.Res.21,423–426.
Choudhary,M.I.,Devkota,K.P.,Nawaz,S.A.,Ranjit,R.,Atta-ur-Rahman,v.,2005.
Cholinesteraseinhibitorypregnane-typesteroidalalkaloidsfromSarcococca hookeriana.Steroids70,295–303.
DeLano,W.L.,2002.ThePyMOLMolecularGraphicsSystem.DeLanoScientificLLC, SanCarlos,CA.
Ellithey,M.S.,Lall,N.,Hussein,A.A.,Meyer,D.,2014.CytotoxicandHIV-1enzyme inhibitoryactivitiesofRedSeamarineorganisms.BMCComplement.Altern.
Med.14,http://dx.doi.org/10.1186/1472-6882-14-77.
Farrokhnia,M.,Nabipour,I.,2014.Marinenaturalproductsasacetylcholinesterase inhibitor:comparativequantummechanicsandmoleculardockingstudy.Curr.
Comput.Aided.Drug.Des.10,83–95.
Ferreira-Vieira,T.H.,Guimaraes,I.M.,Silva,F.R.,Ribeiro,F.M.,2016.Alzheimer’sdis- ease:targetingthecholinergicsystem.Curr.Neuropharmacol.14,101–115.
Gower,D.B.,Ruparelia,B.A.,1993.Olfactioninhumanswithspecialreferenceto odorous16-androstenes:theiroccurrence,perceptionandpossiblesocial,psy- chologicalandsexualimpact.J.Endocrinol.137,167–187.
Grammer,K.,Fink,B.,Neave,N.,2005.Humanpheromonesandsexualattraction.
Eur.J.Obstet.Gynecol.Reprod.Biol.118,135–142.
Holland,B.S.,Dawson,M.N.,Crow,G.L.,Hofmann,D.K.,2004.Globalphylogeography ofCassiopea(Scyphozoa:Rhizostomeae):molecularevidenceforcrypticspecies
andmultipleinvasionsoftheHawaiianIslands.Mar.Biol.145,1119–1128.
Kaminski,R.M., Marini,H., Ortinski, P.I.,Vicini,S.,Rogawski, M.A.,2006. The pheromoneandrostenol(5␣-androst-16-en-3␣-ol)isaneurosteroidpositive modulatorofGABAAreceptors.J.Pharmacol.Exp.Ther.317,694–703.
Laemmli,U.K.,1970.Cleavageofstructuralproteinsduringtheassemblyofthehead ofbacteriophageT4.Nature227,680–685.
Langjae,R.,Bussarawit,S.,Yuenyongsawad,S.,Ingkaninan,K.,Plubrukarn,A.,2007.
Acetylcholinesterase-inhibitingsteroidalalkaloidfromthespongeCorticiumsp.
Steroids72,682–685.
Mirshamsi,M.R.,Omranipour,R.,Vazirizadeh,A.,Fakhri,A.,Zangeneh,F.,Mohebbi, G.H., Seyedian, R., Pourahmad, J., 2017. Persian Gulf jellyfish (Cassiopea andromeda)venomfractionsinduceselectiveinjuryandCytochromeCrelease inmitochondriaobtainedfrombreastadenocarcinomapatients.AsianPac.J.
CancerPrev.18,277–286.
Mohebbi,G.H.,Farzadinia,P.,Vatanpour,H.,Hashemi,A.,Vazirizadeh,A.,khazaei,Z., Akbarzadeh,S.,Mirshamsi,M.,Nabipour,I.,2016.Afirstrecordonpopulationof thealienvenomousjellyfish,Cassiopeaandromeda(Forsskål,1775)(Cnidaria:
scyphozoa:Rhizostomea)intheNaybandLagoonfromBushehr-Iran(Persian Gulf).Ent.Appl.Sci.Lett.3,65–71.
Moraga,F.,Hormazábal,E.,Venthur,H.,Quiroz,A.,Mutis,A.,2016.Alkaloidsdiscov- eryasnaturalacetylcholinesteraseinhibitors,fromnaturetomoleculardocking.
Rev.Farmacol.9,16–25.
Morris,G.M.,Huey,R.,Lindstrom,W.,Sanner,M.F.,Belew,R.K.,Goodsell,D.S.,Olson, A.J.,2009.AutoDock4andAutoDockTools4:automateddockingwithselective receptorflexibility.J.Comput.Chem.30,2785–2791.
Mustafa,M.R.,White,E.,Hongo,K.,Othman,I.,Orchard,C.H.,1995.Themechanism underlyingthecardiotoxiceffectofthetoxinfromthejellyfishChironexfleckeri.
Toxicol.Appl.Pharmacol.133,196–206.
Nabipour,I.,Mohebbi,G.H.,Vatanpour,H.,Vazirizadeh,A.,2017.Hematological parametersontheeffectofthejellyfishvenomCassiopeaandromedainanimal models.DataBrief11,517–521.
Nabipour,I.,Moradi,M.,Mohebbi,G.H.,2015.Afirstrecordonpopulationofthealien venomousjellyfish,Cassiopeaandromeda(Forsskål,1775)(Cnidaria:scyphozoa:
Rhizostomea)intheNaybandLagoonfromBushehr-Iran(PersianGulf).J.Chem.
Pharm.Res.7,1710–1713.
Othman,I.,Burnett,J.W.,1990.TechniquesapplicableforpurifyingChironexfleckeri (box-jellyfish)venom.Toxicon28,821–835.
Peng,J.,Yuan,J.P.,Wu,C.F.,Wang,J.H.,2011.Fucoxanthin,amarinecarotenoid presentinbrownseaweedsanddiatoms:metabolismandbioactivitiesrelevant tohumanhealth.Mar.Drugs9,1806–1828.
Pilger,C.,Bartolucci,C.,Lamba,D.,Tropsha,A.,Fels,G.,2001.Accurateprediction oftheboundconformationofgalanthamineintheactivesiteofTorpedocali- fornicaacetylcholinesteraseusingmoleculardocking.J.Mol.Graph.Model.19, 288–296.
Radwan,F.F.,Burnett,J.W.,Bloom,D.A.,Coliano,T.,Eldefrawi,M.E.,Erderly,H., Aurelian,L.,Torres,M.,Heimer-delaCotera,E.P.,2001.Acomparisonofthe toxinologicalcharacteristicsoftwoCassiopeaandAureliaspecies.Toxicon39, 245–257.
Reddy,D.S.,2010.Neurosteroids:endogenousroleinthehumanbrainandthera- peuticpotentials.Prog.Brain.Res.186,113–137.
Sanner,M.F.,1999.Python:aprogramminglanguageforsoftwareintegrationand development.J.Mol.Graph.Model.17,57–61.
Sarkar,A.,Irwin,M.,Singh,A.,Riccetti,M.,Singh,A.,2016.Alzheimer’sdisease:the silvertsunamiofthe21stcentury.Neural.Regen.Res.11,693–697.
Shanmuganathan,B.,SheejaMalar,D.,Sathya,S.,PandimaDevi,K.,2015.Anti- aggregation potential of Padinagymnospora against the toxicAlzheimer’s beta-amyloidpeptide25-35andcholinesteraseinhibitorypropertyofitsbioac- tivecompounds.PLOSONE10,e0141708.
Sussman,J.L.,Harel,M.,Frolow,F.,Oefner,C.,Goldman,A.,Toker,L.,Silman,I.,1991.
AtomicstructureofacetylcholinesterasefromTorpedocalifornica:aprototypic acetylcholine-bindingprotein.Science253,872–879.
Szegletes, T., Mallender, W.D., Thomas, P.J., Rosenberry, T.L., 1999. Substrate binding to the peripheralsite of acetylcholinesterase initiates enzymatic catalysis,Substrateinhibitionarisesasasecondaryeffect.Biochemistry38, 122–133.
Tara,S.,Elcock,A.H.,Kirchhoff,P.D.,Briggs,J.M.,Radic,Z.,Taylor,P.,McCammon,J.A., 1998.Rapidbindingofacationicactivesiteinhibitortowildtypeandmutant mouseacetylcholinesterase:Browniandynamicssimulationincludingdiffusion intheactivesitegorge.Biopolymers46,465–474.
Tarrant,A.M.,2005.Endocrine-likesignalinginCnidarians:currentunderstanding andimplicationsforecophysiology.Integr.Comp.Biol.45,201–214.
Tarrant,A.M.,2007.Hormonalsignalingincnidarians:doweunderstandthepath- wayswellenoughtoknowwhethertheyarebeingdisrupted?Ecotoxicology16, 5–13.
Tommonaro,G.,García-Font,N.,Vitale,R.M.,Pejin,B.,Iodice,C.,Ca ˜nadas,S.,Marco- Contelles,J.,Oset-Gasque,M.J.,2016.AvarolderivativesascompetitiveAChE inhibitors,non-hepatotoxicandneuroprotectiveagentsforAlzheimer’sdisease.
Eur.J.Med.Chem.122,326–338.
Tvrdei ´c,A.,Poljak,L.,2016.Neurosteroids,GABAAreceptorsandneurosteroidbased drugs:arewewitnessingthedawnofthenewpsychiatricdrugs?E.O.M.2, 49–60.
ViewrLite5.0,VersionJul11,2002.AccelrysInc.:http://www.accelrys.com.
Worek,F.,Mast,U.,Kiderlen,D.,Diepold,C.,Eyer,P.,1999.Improveddetermination ofacetylcholinesteraseactivityinhumanwholeblood.Clin.Chem.Acta288, 73–90.
Zaheer-Ul-Haq,Z.U.,Wellenzohn,B.,Liedl,K.R.,Rode,B.M.,2003.Moleculardocking studiesofnaturalcholinesterase-inhibitingsteroidalalkaloidsfromSarcococca saligna.J.Med.Chem.46,5087–5090.
