Rev. bras. farmacogn. vol.25 número4

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w w w. s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

The

catechol-O-methyltransferase

inhibitory

potential

of

Z-vallesiachotamine

by

in

silico

and

in

vitro

approaches

Carolina

dos

Santos

Passos

a,b,1

_,

_Luiz

_Carlos

_{Klein-Júnior}

a,1

_,

_Juliana

_Maria

_de

_Mello

_Andrade

a

_,

Cristiane

Matté

c

_,

_Amélia

_Teresinha

_Henriques

a,∗

a_Laboratório_de_{Farmacognosia,}_Faculdade_de_Farmácia,_Universidade_Federal_do_Rio_Grande_do_Sul,_Porto_Alegre,_RS,_Brazil b_Department_of_{Pharmacochemistry,}_School_of_{Pharmaceutical}_Sciences,_Université_de_Genève,_Genève,_Switzerland

c_Programa_de_{Pós-graduac¸}_ão_em_Ciências_Biológicas:_Bioquímica,_ICBS,_Universidade_Federal_do_Rio_Grande_do_Sul,_Porto_Alegre,_RS,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29May2015 Accepted3July2015 Availableonline26July2015

Keywords:

Monoterpeneindolealkaloids Vallesiachotamine

Catechol-O-methyltransferase Docking

Neurodegenerativediseases

a

b

s

t

r

a

c

t

Z-Vallesiachotamineisamonoterpeneindolealkaloidthathasa␤-N-acrylategroupinitsstructure. ThisclassofcompoundshasalreadybeendescribedindifferentPsychotriaspecies.Ourresearchgroup observedthatE/Z-vallesiachotamineexhibitsamultifunctionalfeature, beingabletoinhibit targets relatedtoneurodegeneration,suchasmonoamineoxidaseA,sirtuins1and2,andbutyrylcholinesterase enzymes.Aimingatbettercharacterizingthemultifunctionalprofileofthiscompound,itseffecton cathecol-O-methyltransferaseactivitywasinvestigated.Thecathecol-O-methyltransferaseactivitywas evaluatedinvitrobyafluorescence-basedmethod,usingS-(5′_-adenosyl)-_l_-methionine_as_methyl_donor

andaesculetinassubstrate.Theassayoptimizationwasperformedvaryingtheconcentrationsofmethyl donor(S-(5′_-adenosyl)-_l_-methionine)_and_enzyme._It_was_observed_that_the_highest_{concentrations}_of

bothfactors(2.25Uoftheenzymeand100␮MofS-(5′_-adenosyl)-_l_-methionine)_afforded_the_more

reproducibleresults.TheinvitroassaydemonstratedthatZ-vallesiachotaminewasabletoinhibitthe cathecol-O-methyltransferaseactivitywithanIC50closeto200␮M.Moleculardockingstudies indi-catedthatZ-vallesiachotaminecanbindthecatecholpocketofcatechol-O-methyltransferaseenzyme. Thepresentworkdemonstrated forthefirsttimetheinhibitoryproperties ofZ-vallesiachotamine oncathecol-O-methyltransferaseenzyme,affordingadditionalevidenceregardingitsmultifunctional effectsintargetsrelatedtoneurodegenerativediseases.

Introduction

Z-Vallesiachotamine(1)isamonoterpeneindolealkaloid(MIA) firstlydescribedforVallesiaglabra(Cav.)Link(=V.dichotomaRuiz etPav),Apocynaceae(Djerassietal.,1966).Ithasa␤-N-acrylate

group, which confers a characteristic UV profile, with a high

molar attenuation coefficient (ε) at 290nm region. Therefore, vallesiachotamine-likealkaloids can beeasily detectedin

alka-loidfractions byLC-DAD,based alsoonpreviousknowledge of

thespecies or genus.This MIAhad already beenisolated from

several different families, including Apocynaceae (Evans et al., 1968;Chengetal.,2014),Loganiaceae(Zhongetal.,2014), and Rubiaceae(Heitzmanetal.,2005).SpecificallyinRubiaceaefamily,

this alkaloid had already been described for severalspecies of

PalicoureeaeRobbr.&Manen(PsychotrieaeCham.&Schltdl.s.lat.)

∗ _{Corresponding}_author.

E-mail:[email protected](A.T.Henriques).

1_These_authors_contributed_equally_to_this_work.

tribe (Klein-Júnior et al., 2014), including Psychotria dichroa

(Standl.) C.M. Taylor(=Cephaelisdichroa (Standl.) Standl.) (Solis etal.,1993),PsychotriabahiensisDC(Pauletal.,2003),Psychotria cuspidata Bredem. ex Schult. (=Palicourea acuminata (Benth.) Borhidi)(Bergeretal.,2012),PalicourearigidaKunth(Soaresetal., 2012)and more recently, for Psychotria suterella Müll.Arg. and

PsychotrialaciniataVell.(Passosetal.,2013a).

H

OH O

OCH₃ N

1

N H

The pharmacological potential of vallesiachotamine was

also reported. Shang et al. (2010) evaluated the in vitro

http://dx.doi.org/10.1016/j.bjp.2015.07.002

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anti-inflammatoryactivityofseveralalkaloids,howeverno signif-icanteffectwasobservedforvallesiachotamine,whichdisplayed

only41.9% ofcyclooxygenase-2inhibitionat 100␮M.The

cyto-toxicity inhumanmelanoma cellswas analyzedby MTTassay,

giving anIC50 valueof 14.7␮M. By flow cytometryanalysis, it

wasobservedthatthisMIAinducedG0/G1arrest,increasingthe

sub-G1hypodiploidcellsnumber.Theauthorsconcludedthatthe

cytotoxicityobservedforthiscompoundismainlydueto apopto-sisandnecrosisprocesses(Soaresetal.,2012).Inadditiontothe reportedactivities,differentcentraleffectswerealsodescribedfor

Z-vallesiachotamine,includingbutyrylcholinesterase (BChE)and monoamineoxidase-A(MAO-A)inhibitoryactivity(Passosetal., 2013b)andsirtuins(SIRT1andSIRT2)modulation(Sacconnayetal., 2015).

Another important target related to neurodegenerative

dis-orders is the cathecol-O-methyltransferase (COMT) enzyme

(Mannisto and Kaakkola, 1999). COMT is a methyltransferase

S-adenosylmethionine-dependentresponsible for catalyzingthe

methylationofcatecholamines,suchasdopamine,leadingtotheir inactivation.Twoisoformsaredescribed:asoluble(S-COMT)and

a membrane bounded(MB-COMT). S-COMTand MB-COMT can

bedifferentiatedbytheiraffinityforsubstratesandtheirtissue

distribution.TheMB-COMThasahighaffinityforcatecholamine

neurotransmitters,beingfoundinneuronsand glial cellsof rat brains(Shirakawaetal.,2004).However,littleisknownregarding theregionaldistributionandcellularlocalizationofCOMTinthe humanbrain.Kastneretal.(2006)foundimunoreactivityforCOMT intoneuronsand glialcells inthestriatumin postmortem

stud-iesofhumanbrains.Additionally,thepresenceof COMTmRNA

wasdemonstratedinneuronsof striatumandprefrontalcortex

(Matsumotoetal.,2003).

COMTisthemajorenzymeresponsiblefordopamine

degra-dation in the prefrontal cortex. In the striatum, in addition to

COMT,MAO-Benzymeandthedopaminetransporter(DAT)are

involvedintheinactivationofthisneurotransmitter(Rybakowski etal.,2006).TheroleofCOMTinthedopaminergictransmission makesthisenzymeaninterestingtargetforthediscoveryofnew

substances forthetreatmentofdiseases suchasschizophrenia,

obsessive-compulsivedisorder,bipolardisorder,anxiety,andpanic disorder (Brisch et al., 2009).Furthermore, theCOMT inhibitor entacaponeisclinicallyusedinassociationwithlevodopainthe treatmentofParkinson’sdisease(PD)(Hamaueetal.,2010).

TakingintoaccountthealreadydescribedBChE,MAO-A,and

sirtuininhibitoryeffectsdescribedforvallesiachotaminealkaloids,

thepresentstudyaimedatdevelopingandimplementinga

proto-colfortheevaluationofCOMTactivitysuitableforthescreening ofplantextractsandisolatednaturalproducts.Further,weused theoptimizedprotocoltoevaluatethepotentialeffectsof vallesia-chotamineonCOMTactivity,andaninsilicoapproachtoelucidate

themolecularmechanismsinvolvedintheinteractionsbetween

vallesiachotamineandCOMT.

Materialsandmethods

Chemicals

Potassiumphosphatemonobasic,potassium phosphate

diba-sic, l-cysteine, magnesium chloride hexahydrate, esculetin,

scopoletin,DMSO,S-(5′_-adenosyl)-_l_-methionine_iodide_(SAM),

3,5-dinitrocatechol(DNC),and porcineCOMT werepurchasedfrom

SigmaChemicalCo.(St.Louis,MO,USA).Z-Vallesiachotaminewas isolatedfromPsychotrialaciniataVell.collectedfromtheAtlantic Forestof Cocaldo Sul(SantaCatarina,BrazilS26◦_80’;_W48◦_95’;

ICN182552, HerbariumoftheFederalUniversity ofRioGrande

doSul),accordingtopreviouslydescribed(Passosetal.,2013b).

Briefly,thedriedleaveswereextractedwithEtOHatroom

tem-perature.Thesolventwasremovedundervacuumandtheresidual

extractdissolvedin1NHClandexhaustivelyextractedwithCH2Cl2

inordertoremovenon-polarconstituents.Subsequently,theacid

extractswerealkalinizedwith25%NH4OH(pH9–10)and

parti-tionedwithCH2Cl2,resultingintheP.laciniataalkaloid-enriched

extract.Thisextractwasfractionatedbyreversed-phasemedium

pressurechromatography(RP-MPLC)withaH2O-CH3CNgradient

step. Fractioncontaining Z-vallesiachotamine wassubmitted to

preparativeHPLCwithisocraticelution(H2O:CH3CN,40:60,v/v),

rate5ml/min,anddetectionat280nm,affordingtheisolated com-pound(purity>95%).

COMTenzymaticassayoptimization

TheCOMTinhibitoryactivitywasassessedbasedonmethods

previouslydescribedintheliterature(Kurkelaetal.,2004;Yalcin andBayraktar,2010),usingaesculetinasCOMTsubstrateandSAM asthemethyldonor.TheO-methylationofaesculetincatalyzedby

COMTtoaffordscopoletinwasmonitoredbyfluorescence

mea-surements(ex=355nmandem=460nm).Thegeneralreaction

ispresentedbelow:

ES+SAMCOMT−→SAH+Scopoletin

whereESisaesculetin,SAMisthemethyldonorS-(5′

-adenosyl)-l-methionineiodide,COMTiscatechol-O-methyltransferase,and

SAHisS-adenosyl-homocystein.

Forsettinguptheexperimentalconditions,different

concen-trationsoftheporcineCOMTandofthemethyldonorsubstrate,

SAM,weretested.COMTwasevaluatedinconcentrationsof0.75,

1.50and2.25U/wellwhileSAMwastestedinconcentrationsof

20,60,and100␮M.Theconcentrationofthecatecholsubstrate aesculetinwaskeptconstantat0.6␮M.ThecontrolforCOMT inhi-bition,DNC,wastestedinconcentrationcorrespondingtoitsIC50

(35nM)(Kurkelaetal.,2004).Calibrationcurvesofaesculetinand scopoletin(0.1–2␮M)wereconstructedintriplicateinorderto cal-culatetheratesoftheO-methylationofaesculetinintoscopoletin.

EvaluationofvallesiachotamineinCOMTenzymaticassay

For the assay, 190␮l potassium buffer (pH 7.4) containing

20mMl-cysteine,5mMmagnesiumchloride,15␮lof100␮M aes-culetin(finalconcentration6␮M),15␮lof150U/mlporcineCOMT (finalconcentration2.25U/well),and5␮lofsample(final

concen-trationvaryingfrom15.62to500␮M)solubilizedin100%DMSO

wereused.Intheblankwells,theenzymewasreplacedbythe

corresponding amountof buffer. The plates werepreincubated

for5minat37◦_C, _followed_by_the_addition_of ₂₅_␮_l_of ₁₀₀_mM

SAM(finalconcentration100␮M).Forthecontrols,sampleswere

replaced by the corresponding amount of DNC solution (final

concentrationcorrespondingtotheIC50value,35nM)orDMSO.

Readings were performed at temperature of 37◦_C, _for ₆₀_min,

in4minintervals.Amicroplatereader(SpectraMax® _Molecular

Devices,CA,USA)wasusedforfluorescencemeasurements.The

excitation andemission wavelengthswereadjustedto355and

460nm,respectively.CalculationswereperformedusingExceland

werebasedonthefollowingequation:

%inhibition=100−test×100

_NC

wheretestisthedifferencebetweenthetestfluorescencereading

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Molecularmodeling

ThecrystallographicstructureofhumansolubleCOMT(human

S-COMT)(PDBID:3BWM)(Rutherfordetal.,2008)wasretrieved fromtheproteindatabank(http://www.wwpdb.org/).Proteinwas preparedforstructuralinspectionanddockingusingMOE2014.09.

Thecrystallized water moleculesand the co-crystallizedligand

DNCwereremoved,andthemissinghydrogenatomswereadded.

ThemoleculardockingprotocolwasdevelopedusingGOLD,version 5.2(CCDC).COMTpocketwascenteredinthespaceoccupiedbythe co-crystallizedligandDNCanddelimitedbyan8 ˚Aradius.Foreach

ligand,100docking solutionswere generatedby using100,000

GOLD Genetic Algorithm interactions (Preset option). Docking

poseswereevaluatedandrankedaccordingtotheChemPLP

sco-ringfunction.Dockingprotocolwasvalidatedbyre-dockingthe

co-crystallizedligandDNCintheCOMTactivesite.RMSDvalues

betweenthebestrankeddockingsolutionandtheco-crystallized ligandlowerthan2.0 ˚Awereacceptedassatisfactory.

Resultsanddiscussion

Oneof theaims of this study wastooptimize the

parame-tersfor theevaluationofalkaloid fractionsandMIAsonCOMT.

Thisenzymeisresponsibleforthemethylationofdopamineand

otherendogenousandxenobioticcatecholsubstrates.Therefore,

itsinhibitioncanincreasedopaminelevelsandleadtoan

improve-mentofthemotorsymptomsassociatedwithPD(Dickinsonand

Elvevág,2009).Infact,COMTinhibitorshavebeenusedclinically forsomeyearsincombinationwithlevodopa.Thefirstgeneration oftheseinhibitors,astropolone,demonstratedlowinvivoefficacy withhightoxiceffects.Thesecondgeneration,representedby tol-caponeandentacapone,replacedfirstgenerationCOMTinhibitors, howevertheystillhaveseveraldopaminergicandgastrointestinal side-effects.Nowadays,opicaponeisunderclinicaltrials,starting thethirdgenerationofthisclass,withhigherpotencyandlower toxicity(Jatanaetal.,2013;Bonifácioetal.,2014).

Differentmethodshavealreadybeenreportedfortheinvitro

evaluationofCOMTinhibition,includingspectrophotometric, fluo-rimetricandradioactivemethods.Mostofthesemethodsdescribe thequantificationoftheproductandoftheremainingsubstrate

byusingchromatographictechniques;however,theyarenotwell

applicableforscreeningassays.YalcinandBayraktar(2010)

devel-opedafluorescence-basedmethodfortheCOMTevaluation,using

aesculetinasthecatecholsubstratewhichismethylatedtoafford thefluorescentproductscopoletin.

AimingatoptimizingascreeningmethodbasedonYalcinand

Bayraktar(2010)study,thelinearityofbothesculetinand scopo-letinwerecheckedinfivedifferentconcentrations,bytriplicate

experiments. It was possible to observe that both compounds

demonstratedalinearbehaviorintheconcentrationsevaluated,

withcoefficientof determination higher than 0.99, which is in

accordance to the literature for enzymatic assays (Tiwari and

Tiwari,2010).

TheactivityofCOMTwasevaluatedusingtheenzymeobtained

fromporcineliver.Incubationtemperature(37◦_C),_incubation_time

(60min)andsubstrateconcentration(0.6␮M)werekeptconstant

inallexperiments.The parametersevaluatedwerethe

concen-trationofthemethyldonorsubstrate(SAM)and theamountof

enzymeintheincubationmedium.Thecombinedvariationofthese parametersallowsustoobservethathigherconcentrationsofthe

enzymeandofthedonorledtomorereproducibleresults.With

0.75Uoftheenzyme,and20␮Mofthesubstrate(lowestlevels), therelativestandarddeviation(RSD)was59.9%,whilewith2.25U oftheenzymeand100␮MofSAM(highestlevels),theRSDwasjust 6.6%,whichisinaccordancetobioanalyticalmethodsvalidation

(TiwariandTiwari,2010).Takingthisintoaccount,bothenzyme andmethyldonorhigherlevelswerechosentoevaluate vallesia-chotamineeffect.

YalcinandBayraktar(2010)evaluatedphenolicandalkaloids enrichedextractsobtainedfromCistusparviflorusLam.,Vitex agnus-castusL.and Peganumharmala L.Theauthorsobserved thatan

alkaloid fraction from P. harmala demonstrated better

inhibi-tion results than the phenolic extracts, with an IC50 value of

1.09␮g/ml.Takingintoaccountthatharmalineandharmalolwere

themaincompoundsdetectedfor P.harmala,theeffectofboth

␤-carboline(␤C)alkaloidswerealsoevaluated,exhibitingIC50

val-uesof0.98and3.59␮M,respectively.Kineticexperimentsrevealed

thatharmalineshowedamixed-typeCOMTinhibitionregarding

the methyl donor substrate SAM while harmalol displayed an

uncompetitivemodeofinhibition.

In the present study, the COMT inhibitory effect of Z

-vallesiachotaminewasinvestigated.Justafewisknownregarding alkaloidseffectsonCOMT;however,consideringtheCOMT inhibi-tiondisplayedbyplant-derived␤C,itwasexpectedthatPsychotria

MIA, as E/Z-vallesiachotamine, might also inhibit this enzyme.

Whenevaluatingthecompoundinarangefrom15to500␮M,it

waspossibletodetermineanIC50valueof196␮M(92%maximum

inhibition).Comparedtoplanar␤-carbolines,theloweractivity

observedmaybeduetothedifferentiatedgeometrygivenbythe

monoterpenoidunitthatisnotpresentinP.harmalaalkaloids.In addition,thesaturationoftheN-containingringaffordsflexibility totheligand,andthisfeaturecandecreasethepotencyofTH␤C alkaloidsasvallesiachotamineforCOMTinhibition.

Passos et al. (2013b) evaluated E and Z-vallesiachotamine

regarding its monoamine oxidases (MAOs) and cholinesterases

(ChEs) inhibitory activities. The alkaloids were able to inhibit butyrylcholinesterase(BChE)activitywithIC50of7.08and9.77␮M,

respectively.Bydockingstudies,itwasobservedthattheindole ringofE/Z-vallesiachotaminecanestablishhydrogenbondswith Ser-198andHis-438residuesofBChE,whiletheresiduesTrp-82, Trp-231,Leu-286,Phe-329,andIle-442canstabilizethecompound throughvanderWaalscontactswiththecarbonatomsofthering

system.Inaddition,bothcompoundswereabletoinhibit

MAO-Aactivity,withIC50 of2.14and0.85␮M.Moleculardocking on

MAO-ArevealedthatthebindingofE/Z-vallesiachotaminein MAO-Aisstabilizedbyhydrophobicinteractionsbetweentheligandand

thenon-polarside chainsofamino acidsresidues inthe

MAO-Apocket.Polar contactsbetweentheligandandtwo conserved

water molecules,andGln-215side chainalsocontribute tothe

complexstabilization.Finally,thecarboxymethylmoietiesofthe compoundsareclosetoFADcofactor,allowinganucleophilicattack

bytheN5of FAD,creatinga covalentbond andreinforcingthe

irreversiblebinding.

Recently, the potential of E/Z-vallesiachotamine on sirtuins

(SIRT1andSIRT2)modulationwasalsoevaluated.SIRTareclass

IIIhistonedeacetylases,responsibleforthedeacetylationofthe

N-acetyl-lysine tails of histones and of non-histone substrates, suchas␣-tubulin.Sirtuininhibition, especially SIRT2,hasbeen

proposed as a new target to treat neurodegenerative diseases

(DillinandKelly,2007;Maxwelletal.,2011;DonmezandOuteiro, 2013).ThenonselectiveinhibitionofSIRT1andSIRT2enzymesby

nicotinamidehasshowntopromotetherestorationofcognitive

deficitsinamicemodelofAlzheimer’sdisease(Greenetal.,2008).

By docking studies, Sacconnay et al. (2015) demonstrated that

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SAM SAM

B

A

Asp-141 Asp-141

Asp-169 Asp-169

Asn-170 Asn-170

Pro-174 Pro-174

Leu-198 Leu-198

Trp-38 Trp-38

Arg-201 _Arg-201

Cys-173 Cys-173

Met-40

Glu-199

Met-40 Glu-199

Mg2+ Mg2+

Fig.1. MoleculardockingresultsofZ-vallesiachotamine(A)andE-vallesiachotamine(B)inthecathecol-O-methyltransferase(COMT)catecholbindingpocket(PDBID:3BWM; thecarbonatomsoftheproteinareshowningray,thecarbonatomsofSAMareshowninorange,andMg2+_ion_is_shown_as_a_purple_sphere)._(A)_The_best_ranked_solution

obtainedforZ-vallesiachotamine(green)dockinginCOMT.(B)ThebestrankedsolutionobtainedforE-vallesiachotamine(magenta)dockinginCOMT.(Forinterpretationof thereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthisarticle).

effectof vallesiachotaminewasconfirmedin a dose-dependent

way.

Aiming at betterunderstandingthe interactions betweenZ

-vallesiachotamineandCOMT,moleculardockingcalculationswere

performed.AsdemonstratedinFig.1A,Z-vallesiachotaminewas

abletobindhumanCOMTinthesame pocketoccupiedbythe

co-crystallized DNC. Nonpolar contacts, namely ␲–␲ stacking

interactions with Trp-38, and van der Waals interactions with

Met-40, Pro-174, and Leu-198, seem to stabilize the

protein-ligandcomplex.On thecontrarytowhat isobserved forCOMT

inhibitorscontainingcathecolgroups, nocoordination withthe

Mg2+_ion_was_verified_for_Z_{-vallesiachotamine,}_which_can_explain

theIC50 valuearound 200␮M.In order to getadditional clues

regardingtothebindingmodeofvallesiachotamine-likealkaloids

onCOMTenzyme,thesamedockingprotocolwasperformedto

evaluatethepotentialinteractionsbetweenE-vallesiachotamine

andCOMT.E-vallesiachotaminewasabletobindtheDNCpocket

ofhumanCOMT,withtheTH␤Cnucleusinthesameorientation

observedforitsZisomer.Inadditiontothenonpolarcontactswith Trp-38,Met-40,Pro-174,andLeu-198,thealdehydeoxygenofE -vallesiachotaminecancoordinate withMg2+_._This_feature _could

suggest that these two structure-related MIAmight be ableto

inhibitCOMTshowingdifferentpotenciesandmodesofinhibition. Takingintoaccounttheobtainedresults,differentMIAmaybe evaluatedusingtheproposedscreeningassayinordertoconfirm ornottheirpotentialasCOMTinhibitors,andtodeterminetheir structure–activityrelationship.Thesesecondarymetabolites rep-resentasourceofchemicaldiversityand,basedonthis,theymight

beusedasscaffoldsforthedevelopmentofnewchemical

enti-tiesactingontargetsrelatedtoneurodegeneration,andpossessing favorablepharmacokineticandtoxicologicalfeatures.

Authorcontributions

CSP(PostdoctoralResearcher)performedtheinsilico

experi-mentsand partof theinvitro methodoptimization.LCKJ(PhD

Student)performedtheinvitroexperimentsandwrotethepaper.

JMMA(PhDStudent)performedpartoftheinvitroexperiments.

BothCSPandJMMAcontributedtowritethemanuscript.CMand

ATH supervised the experimentalwork and contributed tothe

manuscript. Alltheauthors haveread thefinalmanuscript and

approvedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisworkwassupportedbyCNPq(grant#478496/2011-7)and

byFAPERGS(grant#2281-2551/14-2SIAFEM).LCKJandATHthank

CNPqforthefellowships.JMMAthankstoCAPESforthePhD schol-arship.

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