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Publié par | biomed |
Publié le | 01 janvier 2011 |
Nombre de lectures | 6 |
Langue | English |
Poids de l'ouvrage | 6 Mo |
Extrait
Moneriz
etal
.
MalariaJournal
2011,
10
:82
http://www.malariajournal.com/content/10/1/82
RESEARCH
OpenAccess
Parasitostaticeffectofmaslinicacid.I.Growth
arrestof
Plasmodiumfalciparum
intraerythrocytic
stages
CarlosMoneriz
1,5
,PatriciaMarín-García
1,2
,AndrésGarcía-Granados
3
,JoséMBautista
1,4
,AmaliaDiez
1,4
and
AntonioPuyet
1,4*
Abstract
Background:
Naturalproductshaveplayedanimportantroleasleadsforthedevelopmentofnewdrugsagainst
malaria.Recentstudieshaveshownthatmaslinicacid(MA),anaturaltriterpeneobtainedfromolivepomace,
whichdisplaysmultiplebiologicalandantimicrobialactivities,alsoexertsinhibitoryeffectsonthedevelopmentof
someApicomplexan,including
Eimeria,Toxoplasma
and
Neospora
.ToascertainifMAdisplaysanti-malarialactivity,
themainobjectiveofthisstudywastoassestheeffectofMAon
Plasmodiumfalciparum
-infectederythrocytes
in
vitro
.
Methods:
Synchronized
P.falciparum
-infectederythrocytecultureswereincubatedunderdifferentconditionswith
MA,andcomparedtochloroquineandatovaquonetreatedcultures.Theeffectsonparasitegrowthwere
determinedbymonitoringtheparasitaemiaandtheaccumulationofthedifferentinfectivestagesvisualizedinthin
bloodsmears.
Results:
MAinhibitsthegrowthof
P.falciparum
Dd2and3D7strainsininfectederythrocytesin,dose-dependent
manner,leadingtotheaccumulationofimmatureformsatIC
50
concentrations,whilehigherdosesproducednon-
viableparasitecells.MA-treatedinfected-erythrocytecultureswerecomparedtothosetreatedwithchloroquineor
atovaquone,showingsignificantdifferencesinthepatternofaccumulationofparasiticstages.TransientMA
treatmentatdifferentparasitestagesshowedthatthecompoundtargetedintra-erythrocyticprocessesfromearly-
ringtoschizontstage.TheseresultsindicatethatMAhasaparasitostaticeffect,whichdoesnotinactivate
permanently
P.falciparum
,astheremovalofthecompoundallowedtheinfectiontocontinue
Conclusions:
MAdisplaysanti-malarialactivityatmultipleintraerythrocyticstagesoftheparasiteand,depending
onthedoseandincubationtime,behavesasaplasmodialparasitostaticcompound.Thisnovelparasitostaticeffect
appearstobeunrelatedtopreviousmechanismsproposedforcurrentanti-malarialdrugs,andmayberelevantto
uncovernewprospectiveplasmodialtargetsandopensnovelpossibilitiesoftherapiesassociatedtohostimmune
response.
Background
anti-malarialdrugshaveaneffectonalimitedselection
Despitetheincreasingnumberofsyntheticandnaturalofplasmodialtargets:inhibitionoftheconversionof
compoundswhicharereportedtoinhibittheinfectivetoxichaemtohaemozoininthevacuole(chloroquine,
cycleofthemalaria-related
Plasmodium
species[1-5]quinine,mefloquineandotheralkaloids),theinhibition
onlyafewofthemhavebeenfoundtobeusefulintheofsynthesisofparasitenucleicacidsbyhinderingofthe
treatmentandpreventionofthedisease[6].Currentdihydrofolatepathway(pyrimethamine,sulphadoxine,
proguanil),thetriggeringofoxidativestress(artemisinin
andderivatives,primaquine)orinhibitionofthemito-
1
*Correspondence:apuyet@vet.ucm.es
chondrialelectrontransportchain(atovaquone).Some
DepartamentodeBioquímicayBiologíaMolecularIV,Universidad
ComplutensedeMadrid,FacultaddeVeterinaria,E28040Madrid,Spain
drugsmayaffectmultipleprocesses,likechloroquine,
Fulllistofauthorinformationisavailableattheendofthearticle
©2011Monerizetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
Moneriz
etal
.
MalariaJournal
2011,
10
:82
http://www.malariajournal.com/content/10/1/82
whichmayalsocauseoxidativedamageattheerythro-
cyticstage[7].Thewidespreaduseofthemosteffective
andaffordabledrugs,suchaschloroquineandtheanti-
folatecombinationpyrimetamine-sulphadoxine,has
favouredtheappearanceofresistantPlasmodiumvar-
iantsmakingthecontrolofthediseasemoredifficultin
endemicareas[8].Resistancetoartemisininhasbeen
alsorecentlyreported[9,10].Alternativeexistingmedi-
cationsare,ontheotherhand,virtuallyunaffordablein
thecountriesmostseriouslyaffected,ortheirefficacy
maybecompromisedinthenearfuturebytheemer-
genceofnewresistantvariants.Hence,thesearchof
newsyntheticornaturalcompoundstargetingnovel
biochemicalpathwaysorcellfunctionsisstillan
imperativeneed.
Thesearchofnaturalproductsshowinganti-malarial
activityforitsdirectuseorasleadsfornewdrugsis
oneofthealternativeswhicharecurrentlyexplored
[11].Maslinicacid(2
a
,3
b
-dihydroxyolean-12-en-28-oic
acid)(MA)isnaturalolenane-typepentacyclictriterpene
foundintheolivefruitandcanbereadilyobtained
fromolivepomaceoils[12].Severalpharmacological
activitieshavebeenreportedforthiscompound,suchas
anti-tumor[13,14],anti-oxidant[15,16],HIVprotease
inhibitor[17],antimicrobial[18],vasorelaxation[19],
andanti-diabeticactionmediatedbyinhibitionofglyco-
genphosphorylase[20,21]andproteintyrosinephospha-
tase1B[22].Inaddition,ithasbeenshownthatMA
displayslowtoxicityonnon-tumoralcells[23,24]indi-
catingthatitshouldbesafeforuseinhumans.Several
linesofevidencepointtoapossibleanti-malarialactivity
ofMA.HIVproteaseinhibitorshavebeenrecently
showntohinderthepre-erythrocyticstagesofPlasmo-
dium[25].Anti-parasiticactivitiesofMAwerealso
identifiedagainstotherApicomplexans[26].Ithasbeen
alsoproposedthattheobservedgrowthinhibitionof
Toxoplasmagondii
culturestreatedwithmaslinicacid
mayberelatedtoinhibitionofproteaseactivity,leading
todefectsinglidingmotilityandultrastructuralaltera-
tionsoftheparasite[24].Suchapossibilitysuggestsa
potentialactivityofmaslinicacid,orstructurallyrelated
molecules,onparasiticprocessesdifferentfromthose
targetedbycurrentanti-malarialdrugs.Inaddition,
othertriterpenoidmoleculesstructurallyrelatedtoMA
displayverydifferentcytotoxicandantiparasiticactiv-
ities.Thus,cucurbitane-typetriterpenoidshavebeen
recentlyshowntodisplayanti-malarialactivity[27],
whileursane-typeurosolicacidwasreportedtoinhibit
growthof
Trypanosomacruzi
[28]showingincontrast
lowanti-plasmodialactivity[29].However,hybridmole-
culesconsistingonursolicacidboundtopharmaco-
phoresshowedenhancedinhibitoryactivityon
Plasmodium,presumablybyfacilitatingtheaccessofthe
boundpharmacophoretohaem[30].Maslinicacidhas
Page2of10
alsobeenusedascorecompoundtosynthesizemodified
derivativesdisplayingnewactivities:bindingofhetero-
cyclicringsatMAC-2andC-3positionsincreasesig-
nificantlytheinhibitoryactivityuponhumanprotein
tyrosinephosphatase1B[22],whileattachmentof
hydrophobicgroupsatC-28increaseinhibitionof
humanglycogenphosphorylase[31].Couplingofamino
acidsordipeptidesatC-28allowedtoretaintheanti
HIV-1activitywhileloweringalongsidethecytotoxicity
[32].ShouldMAshowsignificantanti-malarialactivity,
itwouldbefeasibletousethismoleculeasaleadcom-
poundforthedevelopmentofanewfamilyofhighly
activeandlowtoxicanti-plasmodialdrugs.
Toexplorethesuitabilityofmaslinicacidasanti-
malarialagent,theactivityofthistriterpeneoncultures
of
P.falciparum
3D7andthechloroquine-resistantDd2
strainshasbeentested.Asnoneofthecurrentlyused
anti-malarialdrugstargetsplasmodialproteases,MA
activitywascomparedtothoseofchloroquineandato-
vaquoneasexamplesofeffectivedrugswhichinterfere
theintra-erythrocyticstageoftheparasiticcycle.
Methods
Drugsandinhibitors
Maslinicacid(MA)wasobtainedfrompressedfruitsof
olive(
Oleaeuropaea
)byamethodpreviouslypublished
[12].Chloroquinediphosphatesaltwaspurchasedfrom
Sigma-Aldrich.Atovaquonewaskindlyprovidedby
Glaxo-SmithKline.Stocksolutionsofatovaquone
30mMandMA300mMwerepreparedin100%
dimethylsulfoxide(DMSO).Chloroquinewasdissolved
indistilledwaterat100mM.Allcompoundswerestored
at-20°Cuntiluse.Forthedrugassays,serialdilutions
weremadeinculturemediumandaddedto96-wellcul-
tureplates.Controlculturesweretreatedwithequivalent
amountsofDMSOdilutedinculturemedium.
Invitroculturesof
Plasmodiumfalciparum
Plasmodiumfalciparum
strainsDd2(cloneMRA-150)
and3D7(cloneMRA-102)obtainedfromtheMR4[33]
wereusedforthisstudy.Erythrocyteswereobtained
fromtypeA+humanhealthylocaldonorandcollected
intubeswithcitrate-phosphate-dextroseanticoagulant
(Vacuette
®
).Theculturemediaconsistedofstandard
RPMI1640(Sigma-Aldrich)supplementedwith0.5%
AlbumaxI(Gibco),100
μ
Mhypoxanthine(Sigma-
Aldrich),25mMHEPES(Sigma-Aldrich),12.5
μ
g/mL
gentamicine(Sigma-Aldrich)and25mMNaHCO
3
(Sigma-Aldrich).Eachculturewasstartedbymixing
uninfectedandinfectederythrocytestoachievea1%
haematocritandincubatedin5%CO
2
at37°Cintissue
cultureflasks(Iwaki).Theprogressofgrowthinthecul-
turewasdeterminedbymicroscopyinthinblood
smearsstainedwithWright
’
seosinmethyleneblue
Moneriz
etal
.
MalariaJournal
2011,
10
:82
http://www.malariajournal.com/content/10/1/82
solution(Merck),usingthefreelyavailablePlasmoscore
software[34]tomonitortheparasitaemia.Thedetailed
descriptionofthecultureandsynchronizationmethods