bioactive peptide from marine sources.docx

7/30/2019 Bioactive peptide from marine sources.docx

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42 A.Aneiros,A.Garateix/J.Chromatogr.B803(2004)4153biodiversity,theseaoffersanenormousresourcefornovelcompounds[1],andithasbeenclassifiedasthelargestre-maining reservoir of natural molecules tobe evaluated fordrugactivity[2].

A very different kind of substances havebeen obtainedfrom marine organisms among other reasonsbecause theyare

living

in

a

very

exigent,

competitive,

and

aggressive

sur-

rounding very different in many aspects from the terres-trial environment, a situation that demands theproductionofquitespecificandpotentactivemolecules.

The discovery of the bioregulatory role of differentendogenouspeptidesintheorganismaswellastheunder-standingofthemolecularmechanisms ofactionofsomenewbioactivepeptidesobtainedfromnaturalsourcesonspecificcellular targets, contributed to considerpeptides also aspromising lead drug candidates. Recently marinepeptideshaveopenedanewperspective forpharmaceuticaldevelop-ments[3].Cyclicandlinearpeptidesdiscoveredfromma-rineanimalshaveincreasedourknowledgeaboutnewpotentcytotoxic,antimicrobial, ionchannelsspecificblockers,andmanyotherpropertieswithnovelchemicalstructures asso-ciated to original mechanisms ofpharmacological activity.Thesefactsintroducemarinepeptidesasanewchoicefortheobtainmentofleadcompoundsforbiomedicalresearch.Thisnonexhaustivereviewisanintenttosystematize someoftherecentandnovelinformationinthisfield.

2. Cyclicpeptidesanddepsipeptides2.1. Bioactivepeptidesfromsponges

Sponges are a large and diverse group of colonial or-ganismsthatconstitutethephylumPoriferawiththousandsofdifferentspeciesextensivelydistributed fromsuperficialwatersneartheseashoresuptodeepwatersoftheocean.Spongeshavebeentraditionally knownasasourceofnovelbioactivemetabolites liketerpenoids,alkaloids,macrolides,polyethers, nucleoside derivatives and many other organiccompounds.SyntheticanaloguesoftheC-nucleosides Spon-gouridineandSpongothymidine isolatedfromaCaribbeanspongeledlatertothedevelopmentofCytosineArabinoside, an anticancer compound [4]. More recently, the attentionhasbeen directed also to the search ofbioactivepeptidesfromsponges,beingactuallyawellestablished sectorintheresearchofmarinenaturalproducts.

Activepeptidesfromspongesmostofthemwithuniqueunprecedent structures in comparison with these kind ofcompoundsfromothersourcesareoftencyclicorlinearpep-tidescontainingunusualaminoacidswhichareeitherrarein terrestrial and microbial systems or even totally novel,andalsofrequentlycontaininguncommoncondensationbe-tweenaminoacids.

One of the first novelpeptides isolated from spongeswere the cell growth inhibitory tetradecapeptide Discoder-mins[5].Areviewofcytotoxicpeptidesfromsponges[6]

Fig.1.BasicstructureofGeodiamolidesAFpeptideswhereXandRrepresentdifferentatomsorradicals.describessomeofthediscoveriesinthisfieldatthattime.DiscoderminsAHandthestructuralrelatedPolydiscamideA, obtained from sponges of the genusDiscodermia are agroup of cytotoxicpeptides containing 1314 known andrareaminoacidsasachain,withamacrocyclicringconsti-tutedbylactonizationofathreonineunitwiththecarboxyterminal.TheyareallcytotoxicandweretestedagainstP388murineleukemiacells,A549humanlungcelllineorbytheinhibitionofthedevelopmentofstarfishembryoswithIC50valuesfrom0.02to20 g/ml.DiscoderminA,wasfurtherstudiedrecently[7]showingapermeabilizingeffectontheplasmamembraneofvascularsmoothmusclecellsandtis-sues.

Geodiamolides AG (Fig. 1) initially isolated from theCaribbean sponge Geodia sp. and the similarpeptide Jas-pamidearealsoagroupofcytotoxicpeptidesinwhichthreeaminoacidsformacyclicpeptidewithacommonpolyke-tideunit.ArenastatinA[8]acyclicdepsipeptidefromDy-sidiaarenariaisaextremepotentcytotoxiccompoundwithaIC50 of5pg/mlagainstKBcells.

Phakellistatins[9]areagroupofprolinerichcyclichep-tapeptides. Phakellistatin 2 showedpotent activity againstP388murineleukemiacells(ED50=0.34 g/ml),butalsoagainstdifferentmelanomacelllines.

TheonellamidesAFarealsoagroupofcytotoxiccom-poundsisolatedbyMatsunagaetal.[10]withnovelaminoacid residues and complexbicyclic macrocyclic rings. Asitwasdemonstratedlater,itissuggestedthattheoriginofsomeofthesepeptideswouldbeassociatedwithamicrobialsourceorwithsymbiontswithinthesponges,becauseofthesimilarity of some of these molecules to marine microbialmetabolites.

Newmembershavebeenaddedlatelytothelistofbioac-tivepeptides from sponges and many of them were eval-uated as cytotoxic exhibiting antitumor and antimicrobialactivities.

A novel peptide, Polydiscamide A, and its deriva-tives wereproposed as antibacterial and antitumor agents[11]. Discobahamin A and B are twobioactive antifungalpeptides evaluated as inhibitors of the growth of Candida


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A.Aneiros,A.Garateix/J.Chromatogr.B803(2004)4153 43albicans, isolated from the Bahamian deep water marinespongeDiscodermiasp.[12].

The depsipeptides Halicylindramides AC [13] and DandE[14]withantifungalandcytotoxicproperties(againstP388),wereobtainedfromtheJapanesemarinespongeHali-chondria cylindrata. Halicylindramides AC are tetrade-capeptides

with

an

N-terminus

blocked

by

a

formyl

group

andtheC-terminuslactonizedwithathreonineresidue.Hal-icylindramide Disatridecapeptide alsowithantifungalandcytotoxicproperties,whileHalicylindramide EisatruncatedlinearpeptidewithaC-terminalamide.

Threenewantifungalcyclicpeptideswithunprecedentedamino acids, Microsclerodermins CE were isolated fromtwospeciesofsponges,Theonellasp.andMicrosclerodermasp.fromthePhilippines [15].Furtherantifungalcyclicpep-tides from sponges are the Aciculitins AC [16] and theTheonegramide[17].OtherexamplesaretheHaligramides AandB,twonewcytotoxichexapeptidesfromthespongeHaliclona nigra [18], and Mozamides A and B from atheonellidspongecollectedinMozambique [19].

AbioassayguidedfractionationoftwospeciesofmarinespongesfromthePacificOcean[20],ledtotheisolationofdifferent new cytotoxicpeptides like the cyclic depsipep-tidesGeodiamolides HNandP.Geodiamolides JNandPrepresented the first examples in the Geodiamolide familyinwhichaserineresiduehasbeenincorporated.

Other cytotoxic and antiproliferative peptides fromspongesareHemiasterlin [21],Milnamide A[22]andJas-plakinolide[23].

HIV-inhibitorypeptidesfromspongesconstitutearecentdiscovery. The cyclic depsipeptides Papuamides AD [24]isolated from sponges of the genus Theonella, containinganumberofunusualaminoacidsarealsothefirstmarine-derivedpeptides reported to contain 3-hydroxyleucine andhomoproline residues, and thepreviously underscribed 2,3-dihydroxy-2,6,8-trimethyldeca-(4Z,6E)-dienoic acid mo-ietyN-linkedtoaterminalglycineresidue.PapuamidesAand B inhibited the infection of human T-lymphoblastoidcellsby HIV-1 sub(RF) in vitro with an EC50 of approx-imately 4ng/ml. Another anti-HIV candidate from thesponge Sidonops microspinosa is the Microspinosamide [25] a new cyclic depsipeptide incorporating 13 aminoacid residues, that is the first naturally occurringpeptidecontaining abetahydroxy-p-bromophenylalanine residue.Microspinosamide inhibitedthecytopathiceffectofHIV-1infectioninanXTT-basedinvitroassay.

Other novelpeptides, Keramamides BD were also iso-latedfromspongesofthesamepreviously mentionedgenusTheonella [26] as well as Orbiculamide A [27] cytotoxicagainst P388 murine leukemia cells (IC50 = 4.7ng/ml).Other activepeptides also isolated from members of thesame genus are the Swinholide A, Bistheonellides, On-namide A, Theonellamide F, Theonellapeptolides, andCyclotheonamides. Cyclotheonamides AandB[28]arepo-tent antithrombin cyclicpeptides which strongly inhibitedvariousproteinases,particularly thrombin.

On the other hand, newpolypeptides from sponges act-ing on membranes of excitable cells havebeen also use-ful to discover new targets forphysiological mechanisms.Multiple-steppurificationprocedureledtothenewdimericpeptideMapacalcine(Mr19,064)fromClionavastifica[29]which selectivelyblocked a non-L-type new calcium con-ductance

characterized

in

mouse

duodenal

myocytes

with

an

IC50 value of approximately 0.2 M. Mapacalcine is sug-gestedasaspecificinhibitorofanewtypeofcalciumcur-rent,firstidentifiedinduodenalmyocytes.

The origin and role of bioactive peptides inside thesponges, in many cases are unclear [30]. Many of thesecompoundshavepotentactivitiesnotalwaysclearlyrelatedto their in situ role. Examples are antitumorals, antivirals,immunosuppressive and antimicrobial agents, as well asneurotoxins,hepatotoxin,andcardiacstimulants.Addition-allyotherstudiesindicatethatdifferentcompoundsisolatedfrom marine macroorganisms like sponges and tunicatesmaybeproduced from dietary or by microorganisms likebacteria or symbioticblue-green algae [31]. This fact il-lustrates the need for further research into the symbioticassociation within these macroorganisms and also suggesttoorientatemoreintensivelythesearchofthesecompoundsalsodirectlytothemarinemicroorganisms.

Theisolationandstructureelucidationofthefirstbioac-tivepeptidesisolateddirectlyfromamarinebacteriumwasreportedin1991byMcKee[32].Someotherpeptideshavebeen isolated from marinebacteria. However up to nowthereisnocomparisonwiththeamountofbioactivepeptidesisolatedfrommarinemacroorganisms,andthebiologicallyactivepeptides from marine microorganisms. Thepoten-tial of marine microorganisms for the obtainment of leadmoleculesisclearlyimmenseduetootherreasons,e.g.thefeasibility to cultivate in laboratory conditions and to ma-nipulatetheseinitialsourcesofbioactivenaturalproducts.

Other examples ofbioactivepeptides isolated from mi-croorganismslivinginassociationwithspongesarethebi-cyclicglycopeptideTheopalauamide[33]andthecytotoxichalogenatedhexapeptideCyclocinamideAwithapotentinvitroselectiveactivityagainstColon-38tumorcells[34].2.2. BioactivepeptidesfromAscidians

Cyclic and linearpeptides and depsipeptides with novelstructuresandbiologicalfunctionshavebeendiscoveredalsoinascidiansusuallycalledtunicates.Didemninsareafam-ilyofdepsipeptideswithantitumor,antiviralandimmuno-supressive activitiesprimarily isolated from the CaribbeantunicateTrididemnumsolidum,butlaterobtainedfromotherspecies of the same genus [35]. Didemnin B (Fig. 2) wasthemostprominentmemberofthefamilywiththemostpo-tent antitumor activity (in vitro L1210 IC50 = 2.5ng/ml)[36]anditwasthefirstmarinenaturalproductevaluatedinhumanclinicaltrials.

Inanotherstudy,DidemninBtogetherwithsomeofthemorepromisingmarineantitumorcandidateswasevaluated


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44 A.Aneiros,A.Garateix/J.Chromatogr.B803(2004)4153

Fig.3.Dolastatin10fromtheseahare(mollusk)Dolabellaauricularia.2.3. BioactivecyclicpeptidesfromMollusks

Cytotoxic cyclicpeptides havebeen also found in mol-lusks.Dolastatinsareagroupofcyclicandlinearpeptides

Fig.2.DidemnimBfromthetunicateTrididemnumsolidum.asanantiproliferative agentagainsthumanprostaticcancercelllines[37].Atconcentration levelsofpmol/mlDidemninB was more effective in the inhibition ofprostate cancercellproliferation thanTaxol.Neurotoxic sideeffectforthiscompoundwasalsodetectedandinthissensesomecautionintheuseofitintheclinicwassuggested.

Didemnim A and B also show antiviral activity againstdifferentkindsofviruslikeherpessimplex,parainfluenza,anddenguevirus,amongothers[38].

Different new members of the family, also cyclic dep-sipeptides related to Didemnins havebeen later isolatedfromextractsofthistunicate.SomeofthemareDidemninsX, Y, M,Nordidemnin N, and Epididemnin A, as wellas twenty-two new Didemnin analogues were preparedsemisynthetically and theirbiological activities evaluated,includingcytotoxicityandantiviralandimmunosuppressive properties [39]. Lissoclinamides constitute another largefamilyofcyclicpeptides[40]isolatedfromascidians(Lis-soclinumpatella)showingrelevantantineoplastic aswellasotherpharmacologicalproperties against human fibroblastandbladder carcinoma cell lines. Some members of thefamily contain an oxazoline ring, oneproline, one valine,twophenylalanine residues, and thiazole and/or thiazolinerings[41].

Another recently discovered cytotoxic peptide froman ascidian is Styelin D [42], a 32-residue C-terminally amidated antimicrobial peptide, isolated fromblood cells(hemocytes) of the ascidian Styela clava. The moleculecontains extensive post-translational modifications, andnovelandunusualaminoacids.StyelinDexhibitedactivityagainst Gram-negative and Gram-positivebacteria, that isretained in high salinity (200mMNaCl). This author in-dicates that these kind ofpeptide antibiotics from marineorganisms couldbe useful for the development of topicalmicrobicides in conditions of physiological or elevatedNaClconcentrations.

TamandarinsAandBarealsotwocytotoxicdepsipeptides recently discovered from a marine ascidian of the familyDidemnidae [43]. The cytotoxicity of Tamandarin A wasevaluatedagainstvarioushumancancercelllinesandshowntobeslightlymorepotentthanDidemnin.

isolated from the marine mollusk Dolabella auricularia,withprominentcellgrowthsuppressingactivity.Frommol-lusksithasbeenalsoisolatedanotherprominentfamilyofpeptides,inthiscasehighlycompactandstablelinearpep-tides,knownasconotoxinsbutwithspecificactionsonionchannels and membrane receptors of excitable cells, as itwillbeshownlaterinSection3.1.Dolabella auricularia is a mollusk devoid of shell and

for this reason initially appears to lack defenses againstpredators,but this is only apreliminary supposition. Ac-cumulatedevidencessupportthefactthatOpisthobranchiamolluskshavedevelopedverypowerfulchemicaldefenses[44].TheearliestinformationabouttheisolationofsomeofthesecompoundswasreportedbyPettitin1981[45].Thepentapeptide Dolastatin 10 (Fig. 3) when isolated in 1987[46]wasreportedasthemostactiveanticancernaturalsub-stanceatthattimewithaED50of4.6105 g/mlagainsttheP388cellline.AnotherstudyshowedthatDolastatin10inhibitstubulinpolymerizationandtubulindependentGTPhydrolysis[47].Dolastatin10hasbeenevaluatedwithpromisingperspec-tivesinaphaseIclinicalstudyinpatientswithsolidtumors.Subsequentlyitsnoticeableantitumoractivitywaswelldoc-umentedinvariousinvitroandinvivotumormodels[48].MorethanadozenofDolastatinshavebeenisolateduptonow. The study of the cytotoxic mechanism of action ofmembersofthefamilyofDolastatinsexhibitparticularities.Recent studies have shown for example that the depsipep-tide Dolastatin 11 arrests cells at cytokinesisby causing arapid and massive rearrangement of the cellular actin fila-mentnetworkandinduceshyperpolymerizationofpurifiedactin [49]. The effects of Dolastatin 11 were most similartothoseofthesponge-deriveddepsipeptideJasplakinolide,butDolastatin11wasaboutthree-foldmorecytotoxicthanJasplakinolideinthecellsstudied.

AnothernewcytotoxiccyclichexapeptidefromamarinemolluskistheKeenamideA,isolatedfromPleurobranchusforskalii [50]. Keenamide A exhibited significant activityagainst the P-388, A-549, MEL-20, and HT-29 tumor celllines.

Newclassesofanticancerdrugscandidates,isolatedfrommarineorganisms,havebeenshowntopossesspowerfulcy-totoxicactivityagainstmultipletumortypes.Accordingtostructuralcharacteristics,cytotoxiccompoundsfrommarine


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A.Aneiros,A.Garateix/J.Chromatogr.B803(2004)4153 45organisms include very different kind of metabolites likemacrolides, terpenes, nitrogen including compounds likecomplex alkaloids, and many others. However among themost active of them are the cyclicpeptides and depsipep-tidesorlinearpeptidesbearinguncommonaminoacidsiso-latedfromsponges,tunicates,andmollusks.InconformitywithSchmitzetal.[51]themostactivecytotoxins(ED5010kDa)orsmallmoleculessuchasbiologicallyactiveamines.

According to Cruz et al. [60] the list of macromolecu-lartargetsofthesepeptidesincludessixtypesofionchan-nels and receptors: acetylcholine receptors ( -, A- and-conotoxins),NMDAreceptors(conantokins),sodium( -,O- and -conotoxins) calcium ( -conotoxins) andpotas-

sium (k-conotoxins) channels, vasopressin (conopressins)andneurotensin(contulateins)receptors(Table1).However,thediscoveryofnewconopeptideshavingspecificstructuralandbiologicalfeaturesisenhancing.

The -conopeptides represent thebest structurally stud-iedclassandtheyactasantagonistsofthenicotinicacetyl-cholinereceptors(AchRs)[61,62].AccordingtoAriasand


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46 A.Aneiros,A.Garateix/J.Chromatogr.B803(2004)4153Table1TypesofConuspeptidesandmoleculartargetsMoleculartarget Cysteinearrangement Class Modeofaction PrototypicalexampleAcetylcholinereceptors CCCC -Conotoxins Competitiveinhibitor -GI

CCCCCC A-Conotoxins Competitiveinhibitor A-EIVACCCCCC -Conotoxins Noncompetitiveinhibitor -PIIIE

Sodiumchannel CCCCCC -Conotoxins Blockcurrent -PIIIACCCCCC O-Conotoxins Blockcurrent O-MrVIBCCCCCC -Conotoxins DelayinactivationofNa+channel(siteVI) -TxVIA

Calciumchannel CCCCCC -Conotoxins blockcurrentofspecificsubtypes -MVIIAPotassiumchannels CCCCCC -Conotoxins Blocker A-SVIANMDAreceptor Linear Conantokins Inhibitor Conantokin-GVasopressinreceptors CC Conopressin Agonist Conopresin-SNeurotensinreceptors Linear Contulateins Agonist Contulakin-GBlanton[63]theseneurotoxinsarecapabletodiscriminatebetweenmuscleandneuronaltypeAchRsandevenamongdistinct AchR subtypes and the binding toxin-receptorshows specific characteristics It hasbeenpostulated thatthesepeptidescouldbeofinterestinthetreatmentofanx-iety, Parkinsons disease,pain, hypertension, cancer andalsoasmusclerelaxants[61].

Conantokinsareaspecificclassoflinearpeptideswhichinhibit N-methyl-d-aspartate (NMDA) receptors This kindofreceptorsplaysanimportantroleinthepathophysiologyofcentralnervoussystem(CNS)disorders.Allconantokinsarelinearpeptidesexcepttheconantokin isolatedfromC.ra-diathuswhichhasathreeaminoaciddisulfidebridgedloopneartheC-terminus [61].AsitwasindicatedbyOlivera[57]thesepeptideswereoriginallypurifiedfollowingtheirchar-acteristictoinduceasleep-like stateinmiceunder2weeksofage,whereasinmiceolderthan3weeksahyperactivity isobserved.TheyrepresentanovelclassofNMDAreceptorantagoniststhatcanmodulateCNSexcitability.Theyofferaninterestingalternative inthetreatmentofepilepsy,pain,strokeandParkinsonsdisease[61].

Differenttypesofconotoxinsactonthevoltage-sensitivesodiumchannels.Thebeststudiedofthesecompoundsare-conotoxins which act as selectively blockers on verte-

brateskeletalmusclesubtypesofvoltage-dependent sodiumchannel [58]. -Conotoxins which act on neuromuscularsodiumchannelsmightbeusefultotreatneuromusculardis-orders[61]. -Conotoxin-GmVIA,purifiedfromConusglo-riamaris,inducesconvulsive-like contractions wheninjectedintolandsnailsbuthasnodetectable effectsinmammals.Itactsspecifically onmolluskansodiumchannels[64].Itslowsdowntheinactivation processofthiscurrentandinducesac-tionpotentialbroadening. O-Conotoxins includeafamilyofpeptideswhichblocksodiumcurrentsofvoltage-sensitive sodiumchannels. O-Conotoxins MrVIAandMrVIBiso-latedfromC.marmoreuspotently blockthesodiumconduc-tanceinAplysianeurons.Althoughthesepeptidesexhibitapharmacologicalactionwhichissimilarofthe -conotoxins,theyarestructurally unrelated[65].

Specifically -conotoxins, selective for calcium chan-nels,werepurifiedusingtheirabilitytoproduceashaking

behaviorinmiceasitwasdescribedbyOlivera[66].Theyinclude -conotoxins GVIA and MVIIA from C.geogra-phus andC. magus, respectively, among others. Four new

-toxins were isolated from the venom of C. catus andtheirpotencies in differentbioassays were compared [67].Becauseofthetherapeuticalpotentialofthistypeofneuro-toxins in the management of severepain they are of greatinterest. The syntheticpeptide SNX-111 corresponds tothe sequence of an -conopeptide MVIIA obtained fromthe venom of Conus magus and it acts as a highlypotentand selective antagonist ofN-type calcium channels [68].The team ofNadasdi et al., synthesized and developed astructure-activity study of analogs to SNX-111. NeurexCorporation presented a patent (No. 5364842) [69] for-conopeptides or derivatives (SNX-111 or SNX-185) to

beusedaspotentanalgesics.From the venom of Conuspurpurascens the first conesnail toxin thatblockspotassium channels ( -conotoxin

PVIIA) was isolated. The overall fold of -conotoxin issimilar to that of calcium channel-blocking -conotoxinsbut differs in this aspect frompotassium channel-blockingtoxinsfromseaanemones,scorpionsandsnakes[70].

Inaddition,therehavebeenisolatedfewnewconotoxinswhichtargetatdifferentsites.Forinstance,Serotoninrecep-torsareanothermoleculartargetforconotoxinsandspecif-ically -conotoxin GVIIIA selectively inhibits the 5-HT3receptor[71].3.2. Seaanemonelinearpolypeptides3.2.1. Channeltoxins

Sessileanimalslikeseaanemoneshavedevelopedalongtheevolutionspecificchemicalmechanismsforthecomplexinteractionsinwhichtheyparticipateinthemarineenviron-ment.Thetoxicityoftheseanimalsisinpartassociatedtothepresenceofnematocysts,aspecializedstingingorganell,characteristicoftheCoelenteratePhyllum.

Fromnematocystrichtissues,secretionsaswellasdiffer-entpartsorthewholebodyfromseaanemoneshavebeenobtaineddifferentcompoundsofproteinicnatureincludingpore-forming toxins or cytolisins [7274],phospholipases


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A.Aneiros,A.Garateix/J.Chromatogr.B803(2004)4153 47[75],Na+ channel toxins [7678], K+ channel inhibitors[7983], other neurotoxins [84] and evenproteinase in-hibitors[85,86].Nevertheless,theexactoriginofbothneu-rotoxinsandcytolysinsisnotquiteclearandotherssourcesbesides nematocysts seem to contribute to the toxicity ofseaanemones[74].

Differentauthorshavesuggestedthatitispossiblethatthecytolisinsandthetoxinsthattargetionicchannelstogetheractsynergistically [87].

Accordingtothemolecularweight(MW)andsomephar-macologicalproperties, sea anemone sodiumpolypeptidetoxinswereearlierclassifiedinfourgroups[88]:1. Basicpolypeptides withMW3000.2. Polypeptides in the MW range between 4000 and

6000. These two groups include toxins that act on thevoltage-activated Na+channel.

3. PolypeptidesintheMWrangebetween6000and7000.Thisgrouppossesproteinaseinhibitingactivityandtheyshow great homology with otherproteinase inhibitorsisolatedfrommammalianspecies.

4. Polypeptides in the MW range 10000. These com-poundsshowmainlycytolyticactivity.Ithasnotbeencorroborated subsequently ifallthefour

groups ofpolypeptides arepresent in each sea anemonespecie.

Morerecently,adifferenttypeofpolypeptideneurotoxinsactingonthevoltage-dependent K+ channelswasreported[7983].

The studies on sea anemones toxins were startedbyShapiro[89]andBressandBress[76]whoisolatedtoxinsfromCondylactisgigantea andAnemonia sulcata, respec-tively.Thesestudiesledtothediscoveryofnewcompoundscapabletoactatthelevelofvoltageactivatedsodiumchan-nels. After that time many otherspolypeptide toxins havebeen isolated and chemically characterized from differentseaanemonespecieswhichsimilarlytotoxinsfromA.sul-cata and C.gigantea inhibit theNa+ channel inactivation[90,91].

There are two more recent classification of the sodiumchanneltoxins,oneofthemtakeintoaccounttheirsequencehomologiesandthesecondoneisaccordingtothespecificbindingsiteandtotheirspecificphysiologicaleffectinthesodiumchannel.

AccordingtosequencehomologiessodiumchanneltoxinscanbeclassifiedastypesIandII[78]:

TypeItoxinsarerepresentedbyATXI,ATXIIandATXVfromA. sulcata, AftI and AftII fromAnthopleura fus-coviridis, ApA and ApB fromA.xantogrammica, ApCfromA. elegantissima [90,92] and BgII and BgIII fromBunodosomagranuliferaamongothers[93,94].

TypeIItoxinsincludeShIfromS.helianthusandtoxinsfromHeteractissp.Thesetwotypesoftoxinsarecrosslinkedbythreedisul-

fidebridges,havemolecularweightsabout5kDaandshowthesameelectrophysiologicalactivity.

Othertoxinshavebeenisolatedwhichdonotfallnearlyintooneoftheseclasses.

InFig.4,differenttypeIseaanemonetoxinsaredepicted.Asitcanbeseenaconsiderablesimilarityexistintheaminoacidsequenceamongthem.

Voltage-dependentsodiumchannelsconstituteanimpor-tant target for different groups of neurotoxins which havecontributed to the structural and functional characteriza-tion of this channel [95]. At least six different neurotoxinreceptor sites have been described for the mammalianNa+ channels and sea anemonepeptide neurotoxins and

-scorpiontoxinssharereceptorsite3onsodiumchannels[96,97], which involves the extracellular loop IVS3S4and additional unidentified contacts in the IS5S6, andIVS5S6 loops of the ionic channel [98]. Their main ef-fect is to delay sodium channel inactivation which leadstoneurotoxic(repetitivefiring)andcardiotoxic(arrhytmia)effect.

In general, sea anemone toxins arebasicproteins thatbind across to the IVS3S4 loop through electrostatic,hydrogen-bonding, or Van der Waals interactions [98].Many of these toxins induce apositive inotropic effect indifferent mammalian heartpreparations and an increase intheactionpotential(AP)duration,thatcanbeexplainedbymodulation viaNa+/Ca2+ exchange that results from theincreasedcellularNa+load[99].

Chemical modification studies of sea anemone toxinswere carried outby several laboratories to determine therole of different amino acids in the channelbinding butdo not exist a general conclusion about the role of eachresidue, however different residues that seem toplay arole in the interaction with toxin receptor such as Arg-12,Arg-14,Lys-49andLys-37wereidentified[92,100,101].Ingeneral,basicaminoacidsoftoxinsandacidicresiduesofthedomainsIandIVofthesodiumchannelalphasubunithavebeenimplicatedintoxinbinding.

1 10 20 30 40 50 %id.BgII GASCRCDSDGPTSRGNTLTGTLWLIG -CPSGWHNCRGSGPFIGYCCKQ--- 100BgIII GASCRCDSDGPTSRGDTLTGTLWLIG -RCPSGWHNCRGSGPFIGYCCKQ--- 97.9ApA GVSCLCDSDGPSVRGNTLSGTLWLYPSGCPSGWHNCKAHGPTIGWCCKQ--- 72.9ATXII GVPCLCDSDGPSVRGNTLSGIIWLAG--CPSGWHNCKKHGPTIGWCCKQ--- 70.8

Fig.4.Comparisonofthesequencesofsomeanemonetoxins:BgIIandBgIIIfromBunodosomagranuliferaApAfromAnthopleuraxanthogrammicaandATXIIfromAnemoniasulcata.Identicalaminoacidsareindicatedwithablackbackground,homologousaminoacidsareindicatedwithagraybackground,dashesrepresentgaps.AttherightcolumnthepercentageofidentityincomparisontoBgII(modifiedfromGoudetetal.[93]).


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48 A.Aneiros,A.Garateix/J.Chromatogr.B803(2004)4153Concerning to the K+ channels more recently a new

type ofblocking toxins from different sea anemones wasobtainedsuchasBgKfromB.granulifera[79],ShKfromStichodactyla helianthus [80], AsKS and AsKC fromA.sulcata[81],HmKfromHeteractismagnifica[82]andAeKfromActinia equina [83] which are structurally differentincomparisonwithotherK+ channeltoxinsisolatedfromsnake,scorpionandbeevenoms.Theseseaanemonetoxinsare formedby about 3537 amino acids and theypresentthree disulfidebridge [87]. They inhibit K+ channelsbybinding to a receptor site in the external vestibule of thechanneloccludingphysicallytheporeandthusblockingtheioniccurrent[102].Becauseoftheiractionmechanismandtheir specificities sea anemone toxins and scorpion toxinshavebeenusedtoidentifytheporeregionandtomeasurethe external mouth of the channelpore [102]. A commoncharacteristic ofbothseaanemoneandscorpionpotassiumchannelblockersisthepresenceofafunctionaldiadformedbylysineandahydrophobicresidue[103,104].Thisdiadoftoxinsresiduesdeterminants forthepotassiumchannelin-teractionsconsistinaLys-25andTyr-26forBgK,whereasfor ShK the diad is composedby a Lys-22 and Tyr-23[104,105].

Both BgK and ShK compete with dendrotoxin-I (fromsnakevenom)forbindingtoratbrainsynaptosomalmem-branes,andsuppressK+currentsinratdorsalrootganglionneurons in culture [79,80]. BgKblocks K+ currents insnail neurons [106] and Kv1 channels inXenopus oocytesalmost equipotently at nanomolar concentrations [87]. Incontrast, ShKblocks different Kv1 channels at nano andsubnanomolar concentrations [87,103]. Even at 100nM,BgK, and ShK did not affect the Kv3.1 channel [87].Additionally, they potently blocked at nanomolar con-centrations the intermediate conductance Ca2+-activatedpotassium channel IKCa1, a well-recognized therapeutictargetpresent in erythrocytes, human T lymphocytes andcolon [107]. In addition ShK is apotentblocker of Kv1.3potassium channel in Jurkat T-lymphocytes [108]. As theKv1.3 channel hasbeen implicated in the T-lymphocyteproliferation and lymphokineproduction,blockers of thischannelareofinteresttooaspotentialimmunosuppressants [109].

FromA.sulcatatheblooddepressing substancesBDS-1andBDS-IIarethefirstspecificblockersfortheKv3.4potas-siumchannel[110].

HmK,thepotassiumchanneltoxinfromtheseaanemoneH. magnifica [82] was reported to inhibit thebinding of[125I]- -dendrotoxin (a ligand for voltage-gated K+ chan-nel)toratbrainsynaptosomal membranes withakIofabout1nMblocksK+currentsthroughKv1.2channelsexpressedinamammaliancelllineandfacilitatesacetylcholine releaseattheavianneuromuscularjunctions.3.2.2. Cytolisins

According to their molecular weight sea anemones cy-tolyticpolypeptidescanbeclassifiedinfourgroups[74]:

About58kDapeptideswithantihistamineactivity. About20kDapore-formingproteinsinhibitedbysphin-

gomyelin. About 3040kDa cytolysins with or withoutphospholi-

paseA2 activity. Aputativegroupofproteinsrepresentedatpresentsolely

byan80kDaMetridiumsenilecytolysin.Group 1: Cytolysinsbelonging to this group havebeen

found in Tealia felina [111], Radianthus macrodactylus[112]andCondylactisaurantiaca[111,112].Thecytolysinsofthisgroupingeneralarenotinhibitedbysphingomyelinand are less hemolytically active than the groups II andIII cytolysins. Cytolysins isolated from T. felina and R.macrodactylusshowantihistamineactivity.

The structural characteristics of this group of cytolysinsisnotwellknown.IncomparisonwithgroupIIcytolysinstheypossesscysteineresiduesandlacktryptophan.Group2:Thisisthebeststudiedgroupofseaanemone

cytolysins.Theyareknownasactinoporins[77]takingintoaccountthattheirsourceareseaanemones(Actinaria)andtheirabilitytoformporesinnaturalandmodellipidmem-brane.Thisporeformationincludesatleasttwosteps:thefirstoneinvolvesthebindingofasolubletoxinmonomertothelipidbilayer,thisprocessisfast,itisconcludedinfewseconds and is irreversible [113,114]. The second step in-volvessloweroligomerizationintheplaneofthemembrane,which eventually leads to the formation of the functionalpore[115,116].

Theyareallmonomeric,cysteinlessproteinswithisoelec-tricpointvaluesmostlyabovenine.Threeorfourmoleculesoligomerise in thepresence of a lipid membrane to formcation-selectiveporesofabout2nmhydrodynamicdiameter[117,118].

Hemolysis hasbeen mainly used to characterize the ac-tivityofactinoporins[119].

Actinoporinsareverypotenttoxinsthataffectalmostallkind of eukaryotic cells. They use sphyngomyelin, a lipidthatisubiquitousinanimalcells,asalowaffinityreceptor[120].

The most studied actinoporins are Equinatoxins II, IVand V fromA. equina [121], Sticholysin I and II [73]from S. helianthus and HMgIII from H. magnifica [72].Theprimarystructureoftheseactinoporinsischaracterizedby a conservedputativeN-terminal amphiphilic -helix, atriptophan-rich stretch, and an RGD-motif [74]. Thebind-ing of thesepolypeptides to the membrane is associatedwithapartialconformationalchangeofthetoxinwithoutasignificantchangeinitssecondarystructure.

Theyarehighlylethalinmammalsandthisactivityisat-tributedtocardiorespiratoryarrestandcoronaryvasospasm.Theyarealsohighlycytotoxicandlytictoavarietyofcellsandtheirvesicularorganelles.Theyalsoproducesignificantdegranulationofgranulocytesandplatelets.Group3:Thisgroupincludeproteinswithphospholipase

A2 likeactivity.


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A.Aneiros,A.Garateix/J.Chromatogr.B803(2004)4153 49ThevenomoftheseaanemoneAiptasiapallidapossesses

hemolyticactivitywhichisproducedbyatleastthreesyn-ergisticvenomproteins.Oneoftheseproteinsisaphospho-lipaseA2 whichexistsintwoforms, and [75].

Phospholipase activity was also reported in Up1, fromthe crude extract of the sea anemone Urciniapiscivora[122,123].

Up1

is

apotent

hemolysin

on

erythrocytes

of

rat, guineapig, dog, and humans causing numerous holesandbreaks on membranes as indicatedby scanning elec-tron microscopy. Its hemolytic activity is not inhibitedbycholesterol [122]. According to Cline [123] itproduces apotentpositiveinotropiceffectoftheleftatriaoftheheartof rat and guineapig with little or no increase in heartrate in vivo. It alsoproduces ileal contractions similar toacetylcholine and such action was inhibitedby atropine.Intravenousadministration ofUp1producedafallinbloodpressureinnormotensive ratspartially reducedbyatropine.

Group4:Thisgroupisrepresentedbymetridiolysin, an80kDa haemolytic and lethalprotein fromM. senile. It isacholesterolinhibitablecytolysin[124].Thetoxinexhibitspreferenceforthelysisofhorseanddogerythrocytes,whichwascorrelatedwithcholesterolcontentsinerythrocytemem-branes[125].Accordingtoastudy[126]thetoxindoesnotshowhighspecificity forinteractions withlipids,anditwasconcludedthatthistoxiniscapableofformingchannelsatlowtoxinconcentrations andlargerporesathighconcentra-tionswhichresultsinlipidbilayermembranedestruction. 3.3. IsolationoftoxinsfromConusandseaanemones

Peptidetoxinswithspecificactiononionchannelsfrommarineanimalslikecoelenterates (seaanemones)andmol-lusks(Conus)aremainlylinearbasicpeptides.Forthatrea-son the isolationprocedure has to include steps forbasicpeptidepurification.Sometimeswhenatoxicacidicpeptideisdetected,astepofanionexchangechromatography isin-cludedintheprotocoltoseparatebasicfromacidictoxins.

The generalprocedure used to fractionate the complexmixtureofdifferentpeptidetoxinscontainedincrudeConusvenoms,comprisegelfiltrationandreverse-phase HPLCasitwasusedtopurifythepotentsleeperpeptideConotoxinGVwhichcontainsfiveresiduesof -carboxyglutamate inachainof17aminoacids,isolatedfromthefishhuntingma-rine snail Conusgeographus [127,128]. The same generalprocedurewasrecentlyappliedbythesameresearchgroupto isolate the newpeptide mr10a fromConus marmoreus,withpotent antinociceptive properties [129]. Inbrief, thecrudevenomwassizefractionatedusingSephadexG25withaceticacidaselutionbuffer,andafterlyophilization theac-tivefractionwasfractionatedonaC18HPLCcolumnwithalineargradientofacetonitrileintrifluoroacetic acid,witha further rechromatography in the same column to elimi-natecontaminants andafinalpurification steponaVydacprotein-SCX(strongcationexchange)column.

On the other hand, isolation methods of sea anemonesodium channel toxins used since thebeginning, included

combinations of traditional chromatographic methods forproteingroupseparationassizeexclusionandionexchangechromatography.AnearlynowclassicalproceduredescribedbyBressetal.[130]forionchanneltoxinsisolationfromA.sulcata,comprisealcoholicextractionofthecrudetoxicmaterial from freshly collected sea anemones,batch-wiseadsorption

of

the

toxins

on

acation

exchanger

followed

by

gel filtration on Sephadex G-50, then ion-exchange chro-matographyonQAESephadexA25andSPSephadexC25anddesaltingonSephadexG25.

Averysimilarprocedurewasdescribedfortheisolationof toxins from the sea anemone Radianthuspaumotensis[131].

SequencestudiesofATXI,ATXIIandATXVtoxinspre-viously isolated fromA. sulcata [130] indicated that thesetoxins were notpureproteins, each having microhetero-geneitiesinthesequence.Thisfactsledtothediscoveryofsea anemone isotoxins, sometimes with only one differentamino acid in a specificposition of the chainbut with adramatic influence in thepharmacological activity in con-trast.AdescriptionindetailsoftheseisolationproceduresisgivenbyBressetal.[132]whichindicatedthatthesep-arationoftheisotoxinscouldnotbeachievedevenwiththeuseofveryprecisegelfiltrationandionexchangechromato-graphic techniques. The use of a RP C-18 HPLC columnenabled the resolution of the sea anemone toxins fromA.sulcataintodifferentisotoxins.

Thepresenceofisotoxinsinseaanemonevenomsbecamelaterawellknownfact.

Even though that thepresence of some toxins in typi-calstructuresdenominatednematocystsfromcoelenteratesis well documented in the literature [133,134], thepreciselocalizationofseaanemonetoxinsinsidetheanimalisnotalwaysclearoranotheroriginnotassociatedwithnemato-cystshasbeensuggested.Thus,otherdifferentcrudestart-ingextractshavebeenusedlikeseaanemonesecretions[79]and mainly the whole animalbody extract [99], which isthemostfrequentlyusedinitialsource.Wholebodyextractsareverycomplexmixturesofalargenumberofmoleculescontainingnotonlytoxinsbutalsomanyothercomponentsfrom tissues and sea water salts. This has tobe taken intoaccountfortheisolationprocedure,whichhasitsownpe-culiaritiesincomparisonwiththesameprocessfromcrudesnake,scorpion,andbeevenomsasasourceoftoxins.

The method described above with slight variants hasbeen later used for the obtainment of other sodium andsubsequently forpotassium channel sea anemone toxins.Some of the modifications or additions later included arethe use of tentacle ion exchangers like Fractogel, Bio-Gel,orTSKHPLCcationexchangers,theemploymentofthe research-grade Serdolit AD-2 as an initial additionalalternativeofnormalpressurehydrophobicadsorptionstepand the repeated use of the same chromatographic step orrechromatographytoimprovetheseparation,andtheexten-siveuseofallthepossibilitiesofRP-HPLCchromatography[79,81,135].


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50 A.Aneiros,A.Garateix/J.Chromatogr.B803(2004)41533.4. Isolationofcytolysins

Purification of cytolytic toxins or cytolysins from seaanemones aqueous extracts commonly include a first gelfiltration step (usually Sephadex G50 or similar) followedby cation exchange chromatography (CM-32 cellulose orother

equivalent

exchanger)

as

essential

combination

to

achievetheresolution ofthecomplexcrudematerials.Asanexample, thisprotocol was usedby Kem and Dunn [136],andLanioetal.[137]toisolateproteincytolysinsfromthesea anemone S. helianthus. Sticholysins I and II (StI andStII)purifiedby Lanio et al. [137]by thisprocedure, aretwo cysteinless polypeptides containing a highproportionof nonpolar amino acids, with a highlybasic isoelectric pointandmolecularweightof19392and19283(2)Da,respectively[120].Similarprocedureshavebeenalsousedfordifferentauthorstopurifyothermembersofthefamilyofseaanemonecytolisins referredbefore.

4. PeptidesandproteinsfromseaweedsBiologically activepeptides andproteins havebeen iso-

latednotonlyfrommarineanimals,butalsofromseaweeds.Inascreeningofmedicinalpotentialities ofseaweedwerediscovereddifferentproteinsandpeptidesexhibitingbioac-tivesproperties[138].

Althoughinaminorscaleincomparisonwithmarineani-mals,someusefulproteinslikelectinsandphycobiliproteins(PBP)havebeenreportedfromseaweeds.

Lectins are glycoproteins with aggregation and recogni-tion functions in the organism thatposses them, and theyhaveprovedtobeusefulinthefieldofclinicaldiagnosisandotherhealthapplications.Lectinshavebeenisolatedbeforefromprimitivemarineorganismslikesponges[139].

From the red marine alga Bryothamnion triquetrum amemberofanewstructuralfamilyoflectinswasisolated.It contains 91 amino acids and two disulfidebonds. TheprimarystructureoftheB.triquetrumlectindoesnotshowamino acid sequence similarity with knownplant and ani-mallectinstructures[140].

Mitogenicandantineoplastic isoagglutinin glycoproteins havebeenequallydescribedfromtheredalgaSolieriaro-busta [141]. A mitogenic hexapeptide with the sequenceGlu-Asp-Arg-Leu-Lys-Pro denominatedSECMA1waspu-rifiedfromthealgaUlvasp.whichisactiveonhumanfore-skinfibroblasts[142].

In another field of application, a novel class of non-toxicbioactiveoligopeptides, foundinmarineredalgaeandcyanobacteria [143], wereproposed tobe useful forboththe diagnosis and therapy of certainbrain and central ner-vous system disorders resulting from malfunctions of sys-temscontrolled bytheneurotransmitter -aminobutyric acid(GABA). Several of these oligopeptides were more activein competing for specific super(3)H-muscimol binding tothemammalian GABAsub(A)receptorsthanGABAitself,

demonstratingtheirpotentialasanewclassofpotentGABAanalogs.

Asitwasshownbefore,anorigenassociatedwithsym-bionts from some new metabolites isolated from marinemacroorganismsissuspected.Thisisalsothecaseincyclicpeptidesisolatedfromsomealgae.Ceratospongamidesaretwo

isomers

of

anew

bioactive

thiazole-containing

cyclic

heptapeptide, cis,cis-Ceratospongamide and trans,trans-Ceratospongamide. They were isolated from the marinered alga (Rhodophyta) Ceratodictyon spongiosum con-taining the symbiotic sponge Sigmadocia symbiotica.trans,trans-Ceratospongamide exhibitspotent inhibition ofsecretoryphospholipase (sPLA) expression in a cell-basedmodel for antiinflammation (ED50 = 32nM), whereas thecis,cisisomerisinactive[144].

On the other hand,phycobiliproteinsplay an importantrole in thephotosyntheticprocess ofbluegreen, red andcryptophyte algae. Light is efficiently collectedbyphyco-biliproteins assembled into macromolecular structures, thephycobilisomes (PBS). Phycobiliproteins are oligomericproteins,builtupfromtwochromophore-bearingpolypep-tides.Theyincludethreemaingroupsofcompounds:phy-coerythrins (PEs) with a redpigmentjoined to theproteinmolecule,phycocianinswithabluepigmentandalophyco-cianins, absorbing them all at different wavelength of thespectrum [145,146]. Phycobiliproteins are spontaneouslyfluorescentinvivoandinvitromolecules.Thispropertyisspeciallyusedinthefieldofbiotechnologicalapplications[147]wheretheyareusedinawidevarietyofbiomedicaldiagnostic systems mainly in immunochemical methods.Phycoerythrin is the most used phycobiliprotein [148],specificallyinfluorescentimmunoassays[149],fluorescentimmunohistochemistry[150]andothermethodologies.

Phycobiliproteins have also antioxidantproperties [151]withapplicationsincosmetology,andinthefoodindustry.4.1. Isolationofphycobiliproteins

Isolationmethodsandmolecularcharacterizationofphy-cobiliproteinsfromseaweedsaredescribedintheliterature[152,153]. Because of its applications,phycoerythrin hasbeenthemoststudiedphycobiliprotein. Onetypicalproce-dure[154]fortheisolationoftheseproteinsandinpartic-ular ofphycoerythrins includes a combination of differentstepsofdensitygradientcentrifugation,columnchromatog-raphyandelectrophoresis.Inbrief,thefirststepistheiso-lation of an intactphycobilisome fraction from the crudeseaweedextractcontainingphycobiliproteinpigmentsbyamethod [155] including disruption of cells and a discon-tinuoussucrosegradientcentrifugation.PBSdissociationislater achieved, and another step of sucrose density gradi-ent centrifugation is used for the obtainment of two maincolored fractions, ablue-colored one (fraction A), and theotherpink-violet(fractionB).FractionsAandBaresubmit-tedseparatelytochromatographyoncolumnsoftheanionicDEAE-celluloseDE52developedwithalineargradientof


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A.Aneiros,A.Garateix/J.Chromatogr.B803(2004)4153 51anappropriatebuffercontainingmercaptoethanol. Thepig-ment complexes from each major column arepooled andthen loaded onto linear sucrose density gradients and cen-trifuged.Purefractionsofphycoerythrin,asjudgedbytheirelectrophoretic composition arefurtherpurifiedbycentrifu-gationonalinearsucrosedensitygradient.

Anotherpurification

procedure

in

alarge

preparative

level

usingalsoananionicchromatographic columnofDEAEcel-lulose,andsodiumdodecylsulfatepoly-acrylamidegelelec-trophoresis(SDS-PAGE)isusedtoobtainB-phycoerythrin andR-phycocyaninfromamicroalga[156].AnalternativeproceduretopurifyR-phycoerythrin fromaMediterraneanredalgae[157]proposetheuseofhydroxyapatite producedin the laboratory that canbe usedbatch-wise with largeamountsofextractsandthereforecanbescaledup.

5. ConcludingremarksPeptidiccompoundsanalyzedhereareobtainedfromvery

different marine organisms exhibiting different chemicalstructuresanddisplaying alargevarietyofpharmacological effectsonspecifictargets.Theyhavebeenproducedalongofmillionsofyearsofevolutioninresponseofthenecessitytoevolveinaverycompetitive environment.Becauseofthepeculiarities ofthelifeinthesea,manyofthesemoleculescanbefoundonlyinasinglesource.

Fromanotherside,thesecompoundsseemtobeveryuse-ful andpromising forbiomedical research to clarify manynormalandpathologicalmechanismofactioninthehumanbodyaswellasinthedesignofveryspecificandpotentnewpharmaceuticals forawidevarietyofdiseases.A multidisciplinary and cooperative effort with the useof more sensitive and fast methods in the analysis of thestructureofpeptides,e.g.exactdescriptionofthemolecularweightandthesequence,aswellasinthepharmacological evaluation,willspeedupdrastically thediscoveryofnovelactivepeptidesfrommarinesources.References

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