Tetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungal activity

21 Pages·2017·5.42 MB·English

RESEARCHARTICLE
Tetrahydrocarbazoles are a novel class of
potent P-type ATPase inhibitors with
antifungal activity
MaikeBublitz1,LasseKjellerup2,3,KarenO’HanlonCohrt2,SandraGordon2,Anne
LouiseMortensen2,JohannesD.Clausen2,ThomasDavidPallin4,JohnBondoHansen2,
AnjaThoeFuglsang3,WilliamDalby-Brown2,Anne-MarieL.Winther2*
1 DepartmentofBiochemistry,UniversityofOxford,Oxford,UnitedKingdom,2 Pcovery,CopenhagenN,
Denmark,3 DepartmentofPlantandEnvironmentalSciences,UniversityofCopenhagen,Frederiksberg,
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Denmark,4 CharlesRiverLaboratories,Harlow,UnitedKingdom
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*[email protected]
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Abstract
WehaveidentifiedaseriesoftetrahydrocarbazolesasnovelP-typeATPaseinhibitors.
Usingasetofrationallydesignedanalogues,wehaveanalyzedtheirstructure-activityrela-
OPENACCESS
tionshipusingfunctionalassays,crystallographicdataandcomputationalmodeling.We
Citation:BublitzM,KjellerupL,CohrtKO,Gordon
foundthattetrahydrocarbazolesinhibitadenosinetriphosphate(ATP)hydrolysisofthefun-
S,MortensenAL,ClausenJD,etal.(2018)
TetrahydrocarbazolesareanovelclassofpotentP- galH+-ATPase,depolarizethefungalplasmamembraneandexhibitbroad-spectrumanti-
typeATPaseinhibitorswithantifungalactivity. fungalactivity.Comparativeinhibitionstudiesindicatethatmanytetrahydrocarbazolesalso
PLoSONE13(1):e0188620.https://doi.org/
inhibitthemammalianCa2+-ATPase(SERCA)andNa+,K+-ATPasewithanevenhigher
10.1371/journal.pone.0188620
potencythanPma1.Wehavelocatedthebindingsiteforthiscompoundclassbycrystallo-
Editor:Agust´ınGuerrero-Hernandez,Cinvestav-
graphicstructuredeterminationofaSERCA-tetrahydrocarbazolecomplexto3.0Åresolu-
IPN,MEXICO
tion,findingthatthecompoundbindstoaregionabovetheioninletchanneloftheATPase.
Received:June30,2017
AhomologymodeloftheCandidaalbicansH+-ATPasebasedonthiscrystalstructure,indi-
Accepted:November10,2017 catesthatthecompoundscouldbindtothesamepocketandidentifiespocketextensions
Published:January2,2018 thatcouldbeexploitedforselectivityenhancement.Theresultsofthisstudywillaidfurther
optimizationtowardsselectiveH+-ATPaseinhibitorsasanewclassofantifungalagents.
Copyright:©2018Bublitzetal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
Introduction
DataAvailabilityStatement:Allcrystallographic
structuredatafilesareavailablefromtheProtein Invasivefungalinfections(IFIs)areasignificantthreattohumanhealth,especiallyamong
DataBankdatabase(https://www.wwpdb.org/, immunocompromised,elderlyorhospitalizedindividuals.Despitetheavailabilityofanumber
accessionnumber5NCQ).
oftreatments,IFIsresultinapproximately1.5milliondeathsworldwideannually[1].IFIsare
Funding:T.D.P.isanemployeeatCharlesRiver generallyassociatedwithhighmortalityrates,oftenabove50%,andcanapproach90%for
Laboratoriesandreceivedfundingintheformof someinfections.ThemajorIFIsarecausedbyCandida,AspergillusandCryptococcusspecies
salary,butCharlesRiverLaboratoriesdidnothave
[1].Manyofthecurrentlyavailabletherapiesexhibitpoortoxicologyprofiles(amphotericin
anyadditionalroleinthestudydesign,data
B)[2],extensivedrug-druginteractions(azoles),andarebeginningtosufferfromacquired
collectionandanalysis,decisiontopublish,or
resistanceamongpathogenicspecies(azolesandechinocandins)[3,4].Inaddition,many
preparationofthemanuscript.L.K.,K.O.C.,S.G.,A.
L.M.,J.D.C.,J.B.H.,W.D.B.,andA.L.Wareorwere antifungalshavealimitedspectrumofactivityandappropriatetreatmentisoftendelayedby
PLOSONE|https://doi.org/10.1371/journal.pone.0188620 January2,2018 1/21
TetrahydrocarbazolesinhibitthefungalH+-ATPase
employeesofPcovery.PcoveryApShasreceived challengesindiagnosis[5].Consequently,safer,broad-spectrumantifungaldrugswithnovel
fundingfromWellcomeTrustResearchCouncil, mechanismsofactionareurgentlyrequired[6].
UK(100480/Z/12)https://wellcome.ac.uk;Novo ThefungalH+-ATPasePma1belongstoafamilyofmembrane-embeddedATPasesthat
Seeds,DKhttp://www.novoholdings.dk/seedsand
pumpionsacrosscellularmembranes,aprocessenergizedthroughtransientphosphoryla-
BoehringerIngelheimVentureFund,D.http://www.
tionbyATP.Pma1pumpsH+outofthecell,generatingalargemembranepotential,which
boehringer-ingelheim-venture.com.Thefunder
providedsupportintheformofsalariesforauthors drivessecondarytransporterstoimportionsandmetabolites,suchasglucoseandamino
L.K.,K.O.C.,S.G.,A.L.M.,J.D.C.,J.B.H.,W.D.B., acids[7].Pma1hasbeenshowntobeanessentialmembraneproteinthroughpma1genedis-
andA.L.W,butdidnothaveanyadditionalrolein ruption,RNAinterferencestudies[8]andloss-of-functionmutationsofPma1inyeast[9].
thestudydesign,datacollectionandanalysis,
Pma1ispresentinallfungiwithahighdegreeofsequencesimilarityamongdiversefungal
decisiontopublish,orpreparationofthe
pma1genes(50–96%)butisnotpresentinmammaliancells.AselectivePma1inhibitoris
manuscript.A.L.W.wasinpartsupportedby
IndustrialpostdoctoralfellowshipsfromInnovation thereforeverylikelytohavebroad-spectrumantifungalactivityandnotargetassociatedtox-
FundDenmark(012-2011-5).M.B.wassupported icity.SeveralclinicallyimportanttherapeuticstargetothermembersoftheP-typeATPase
bytheDanishCouncilforStrategicResearch.L.K. family.Forexample,cardiacglycosidestargettheNa+,K+-ATPaseandprotonpumpinhibi-
wassupportedbyInnovationFundDenmark,DK tors(PPIs),suchasomeprazole,targetthegastricH+,K+-ATPase[10].Theestablishment
(401900019B)https://innovationsfonden.dk.These
oftheP-TypeATPasesasadruggableclassoftargetssuggeststhatitshouldbepossibleto
fundershadnoroleinstudydesign,datacollection
developPma1inhibitorsaspotentantifungalagents.Notably,Pma1inhibitorscouldact
andanalysis,decisiontopublish,orpreparationof
themanuscript.Thespecificrolesoftheauthors fromtheextracellularside,similartoPPIs,andcircumventthechallengesassociatedwith
arearticulatedinthe‘authorcontributions’section. crossingthefungalplasmamembrane.
HerewereportacompoundlibraryscreeningcampaignthatidentifiedaseriesofPma1
Competinginterests:L.K.,K.O.C.,S.G.,A.L.M.,J.
D.C.,J.B.H.,W.D.B.,andA.L.Wareorwere inhibitorsthatexhibitbroad-spectrumantifungalactivity.Computermodeling,supportedby
employeesofPcovery.T.D.P.isanemployeeat structuralbiologyindicatesthatthecompoundsbindtoagrooveattheintracellularmem-
CharlesRiver.Thisdoesnotalterouradherenceto braneinterface,similartootherP-typeATPaseinhibitorsthatblocktheionentrychannel.
PLOSONEpoliciesonsharingdataandmaterials.
Pcoveryhasfiledapatentrelatingtothe
compoundsdescribedinthispublication,but
neitherPcoverynorCharlesRiverhaveanyproduct
Results
indevelopmentormarketedproductrelatedtothis
publication.Therearenoothercompetinginterests
TetrahydrocarbozolecompoundsinhibitATPhydrolysisbyP-type
todeclare.
ATPases
20,240compoundswerescreenedforinhibitionofPma1ATPhydrolysisatasingleconcentra-
tion(20μM)toidentifyinitialhitswithpotencyinthelowmicromolarrange.Onehundred
compoundsexhibited>30%Pma1inhibition,andwereselectedforPma1IC determination,
50
andthenevaluatedforantifungalactivityagainstSaccharomycescerevisiae(baker’syeast)and
Candidaalbicans(ATCC90028).Two-thirdsofthePma1inhibitorsalsoinhibitedfungal
growthofS.cerevisiaeandC.albicansatconcentrationsbelow<100μM,whiletheremaining
compoundsdidnotinhibitfungalgrowth.Aseriesoftetrahydrocarbozolesstoodoutasinhibi-
torsofbothPma1activityandfungalgrowthinthelowmicromolarrange(<20μM).These
compoundswererepurchasedandconfirmedasPma1inhibitors(compound1–3,Fig1,
Table1).
Ninetetrahydrocarbazolecompounds(4–12,Fig1)weresynthesized(seeS1Filefordetails)
andtheracemiccompoundsweretestedforactivityagainstPma1andtherelatedmammalian
Ca2+-ATPase(SERCA,rabbit)andNa+,K+-ATPase(pig)(Table2).Compounds4and5were
weakinhibitorsofPma1,andcompounds11and12exhibitednoactivityagainstPma1.Com-
pounds,6–10inhibitedPma1inthelowmicromolarrange(IC valuesof2–9μM).Com-
50
pounds6,7,and8weremorepotentininhibitingSERCAandtheNa+,K+-ATPasethan
Pma1,andcompound12wasweaklyselectiveforNa+,K+-ATPaseonly(IC =11.8μM).
50
Compounds9and10werepotentPma1inhibitors(IC =5–7μM)andexhibitedantifungal
50
activityinthesameconcentrationrangeagainstSaccharomycescerevisiae,Candidaalbicans
andCandidakrusei(Table3,andseebelow).
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TetrahydrocarbazolesinhibitthefungalH+-ATPase
Fig1.Chemicalstructureofthetetrahydrocarbazolescaffold(top),threeinitialhitsfromthelibraryscreening
(1–3)andninerationallydesignedtetrahydrocarbazoleanaloguesusedinthisstudy(4–12).Thechiralcenteris
indicatedbyanasterisk.
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Table1. ATPhydrolysisandgrowthinhibitionbylibraryhitcompounds. MICisdefinedas50%growthinhibition,n=3.
ATPhydrolysisIC50[μM] GrowthinhibitionMIC[μM]
ID SaccharomycescerevisiaePma1 Saccharomycescerevisiae Candidaalbicans
1 18.2±4.7 10 20
2 12.9±3.2 12 10
3 14.8±3.5 12 5
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TetrahydrocarbazolesinhibitthefungalH+-ATPase
Table2. IC50valuesofcompounds4–12againstATPhydrolysisactivityoffungalandmammalianP-typeATPases. n=3.
ID ATPhydrolysisIC50[μM]
SaccharomycescerevisiaePma1 CandidaalbicansPma1 MammalianSERCA MammalianNa+,K+-ATPase
4 75.0±3.2 46.0±14.5 1.55±0.06 37.2±13.6
5 85.9±10.3 129.7±32.2 61.7±18.8 131.9±35.2
6 2.79±0.36 3.89±1.71 0.09±0.03 0.72±0.30
7 2.43±0.39 2.47±0.26 0.11±0.06 0.38±0.12
8 3.57±1.81 3.77±1.04 0.26±0.06 1.07±0.38
9 4.73±2.23 5.56±1.55 3.27±1.89 9.76±4.47
10 6.69±2.06 9.70±2.15 5.54±3.16 5.50±2.74
11a >20 >20 >20 >20
12a >20 >20 >20 11.8±6.8
aThecompoundsweretestedinaconcentrationupto166μM,butcompoundprecipitationwasobservedatconcentrationsabove20μM.
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Tetrahydrocarbazolesexhibitbroadspectrumfungistaticactivity
Compounds6–10exhibitedthebroadestspectrumofantifungalactivity,inhibitingthegrowth
oftheyeastsSaccharomycescerevisiae,Candidaalbicans,CandidakruseiandCandidaglabrata.
Thecompoundswerelesspotentthanvoriconazole(VRC)andamphotericinB(AMB)against
allfungalisolatestested,butmorepotentthanfluconazole(FLC)onS.cerevisiae,C.kruseiand
C.glabrata(Table3).Theywerealsomorepotentthanother,structurallydifferentcompound
seriesidentifiedinthisandpreviouslibraryscreens,thecarbazoles[11]andthepyrido-thieno-
pyrimidines[12].ThelesspotentPma1inhibitors4and5displayedweakantifungalactivity.
Compounds11and12,despitetheirveryclosestructuralsimilarityto10,exhibitedneither
substantialPma1inhibitionnorantifungalactivity(IC andMIC>20μM).Finally,oneof
50
thesynthesizedtetrahydrocarbazolecompounds,9,exhibitedantifungalactivityagainsttheC.
glabratastrainCg003,whichoverexpressestheeffluxpumpsCdr1pandCdr2passociatedwith
multidrugresistance[13].
Table3. Fungalgrowthinhibitionbycompounds4–12. MICisdefinedas50%growthinhibition,n=3–6.FLC:fluconazole;VRC:voriconazole;AMB:amphotericinB.
GrowthinhibitionMIC[μM] EC50[μM]
ID SaccharomycescerevisiaeATCC Candidaalbicans CandidakruseiATCC CandidaglabrataATCC Candidaglabrata HepG2(72
9763 SC5314 6258 90030 Cg003 h)
4 7.5±0.0 100±43 66±16 23.7±0.0 75±0.0 3.50±1.87
5 11.6±8.1 150±0.0 150±0.0 46.5±27 >75 4.70±3.40
6 5.0±2.9 7.5±0.0 7.5±0.0 12.1±7.9 >75 16.69±3.54
7 1.5±0.0 15±0.0 4.7±0.0 15±0.0 >75 14.87±0.26
8 6.2±2.6 7.5±0.0 12.9±9.4 5.3±2.7 >75 13.60±1.33
9 7.5±0.0 7.5±0.0 7.5±4.8 7.5±0.0 23.7±0.0 12.45±3.68
10 7.5±0.0 6.8±4.6 6.8±4.6 21.0±6.6 >75 18.64±1.12
11a >20 >20 >20 >20 >20 >20
12a >20 >20 >20 >20 >20 >20
FLC 20.68±8.68 0.75±0.48 67.17±26.98 26.12±0.0 >208 >50
VRC 0.27±0.19 0.03±0.02 1.02±0.70 0.61±0.28 6.11±2.64 >50
AMB 0.02±0.01 0.10±0.04 0.44±0.34 0.10±0.04 0.18±0.08 0.69±0.01
aThecompoundsweretestedinconcentrationsupto75μMinthefungalgrowthassayandupto50μMagainstHepG2cells,butcompoundprecipitationwasobserved
atconcentrationsabove20μM.
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TetrahydrocarbazolesinhibitthefungalH+-ATPase
Compoundswereevaluatedformammaliancytotoxicityinastandardhumanhepatocyte
cellproliferationassay(HepG2).AllninetestedtetrahydrocarbazolesinhibitedHepG2prolifer-
ation(EC :3–20μM)morestronglythanVRC,butlessthanAMB(EC :<1μM)(Table3).
50 50
Compounds4and5weremorepotentatinhibitingHepG2proliferationthanfungalgrowth.
Althoughcompounds6,8,9and10inhibitedfungalgrowthmorestronglythanHepG2prolif-
eration,theissueofmammaliancytotoxicitywillneedtobeaddressedduringfurtherdevelop-
mentofthecompounds.However,thebroadrangeofEC valuesobtainedintheproliferation
50
assay,fromverycytotoxictomoderateornotoxicity,indicatethatthecytotoxicityprofileof
thesecompoundscanbeoptimizedtoincreasetheselectivitytowardsfungalcells.
Compounds6and8wereevaluatedintime-killexperimentsagainstCandidaalbicans.
Here,exposureto6or8(20μM)resultedinarapidreductionincolony-formingunits(CFU),
comparedtodimethylsulfoxide(DMSO)treatedcontrolsatT=0h(Fig2A).After3hexpo-
sure,>99%reductioninCFUwasobservedforcellstreatedwith6or8,butafter6hexposure,
theseculturesyieldedCFUcountssimilartoDMSO-controls(Fig2A).ThelowCFUcountsat
T=3hweresimilartoobservationsforAMB,whichisfungicidalafter3hexposureinC.albi-
cans,anddistinctfromVRCwhichexhibitsfungistaticactivityafterboth3and24hofincuba-
tion.Thus,thefungalgrowthdataobservedforCandidacellsexposedto6and8suggeststhat
thetetrahydrocarbazolesbothdelaygrowthanddisplayedfungistaticactivityafter24hof
incubation.
Compounds6and8elicitalongpost-antifungaleffect(PAFE)afterbriefexposureinC.
albicanscells(Fig2B).PAFEisassessedbytheabilityoffungalcellstogrowanddividefollow-
ingcompoundexposure,andisdeterminedbyfollowingthenumberofCFU/mLincom-
pound-freeculture.Fig2BshowsthePAFEafter1hexposureofC.albicanstoAMB,VRCor
compounds6or8.ExposuretoAMB(0.54μM)significantlydelayedthegrowthofC.albicans
cells,withCFUcountsonlybeginningtorise7haftercompoundremoval(PAFE=7h).Cells
exposedtoVRC(2.8μM)exhibitednosignificantdelayingrowthfollowingthesameexposure
periodascomparedtountreatedcells.Exposuretocompounds6or8(20μM)ledtoa5h
delayingrowthascomparedtoDMSO-treatedcontrols(PAFE=5h),asimilareffectto
thatobservedwithAMB.Oncecellsthathadbeenexposedtotetrahydrocarbazolesresumed
growththeydividedrapidly,asevidencedbyanexponentialincreaseinCFUcountsbetween3
and7hfollowingcompoundremoval(Fig2B).
Tetrahydrocarbazolesinducemembranedepolarizationwithoutaffecting
membraneintegrity
WeassessedthemembranepotentialandintegrityofS.cervevisiaecellsexposedto10μMtet-
rahydrocarbazolesusingthefluorescentprobesbis-(1,3-dibutylbarbituricacid)trimethineoxo-
nol(DiBAC (3))andpropidiumiodide(PI).DiBAC (3)canenterdepolarizedcellswhereit
4 4
bindstointracellularproteins,leadingtoanenhancementofthefluorescentsignal[14].PI
exhibitsincreasedfluorescenceuponbindingtoDNA,andiscommonlyusedasamarkerfor
lossofmembraneintegrityandthepresenceofdeadcells[15].Nosignificantdifferencesin
thefluorescencesignalforDiBAC (3)wereobservedforS.cerevisiaecellstreatedwith4or5
4
ascomparedtoDMSOafter30minutesexposure(Fig3).ExposureofS.cerevisiaeto10(S.cer-
evisiaePma1IC =7μM)ledtoaslightincreaseintheDiBAC (3)fluorescenceintensity,
50 4
whileexposuretotheslightlymorepotentPma1inhibitors6–9(S.cerevisiaePma1IC =
50
2–5μM)ledtoasignificantincreaseinthefluorescencesignalindicativeofmembranedepo-
larizationinducedbythesecompounds(Fig3).Noneofthecompoundsinducedanincrease
inPIfluorescence,indicatingthatmembraneintegritywasnotaffected.Anoverviewofthe
resultsfromthemembranepotentialassaysispresentedinFig3andFigureAinS1File.
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TetrahydrocarbazolesinhibitthefungalH+-ATPase
Fig2.AntifungalactivityoftetrahydrocarbazolesagainstC.albicans.A)Time-killanalysisandB)Post-antifungal
effectoftetrahydrocarbazoles:●=DMSO-treatedcontrolcells,(cid:14)=cellstreatedwithAMB(0.54μM),(cid:3)=cellstreated
withVRC(2.8μM),□=cellstreatedwith6(20μM)and^=cellstreatedwith8(20μM).ValuesinA)andB)arethe
mean(±SEM)forthreeindependentexperiments.SampleswithdrawnattheindicatedtimesinA)andB)were
evaluatedforCFU.
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Theobservedeffectsindicateaconcurrenceofantifungalactivitywithalossinmembrane
potential,whichwouldbeconsistentwithamechanisminvolvingPma1inhibitionratherthan
unspecificmembranedamage.
CrystalstructureofaninhibitoryP-typeATPase•tetrahydrocarbazole
complex
Tocomplementthefunctionaldataofthetetrahydrocarbazolecompoundswithstructural
information,wecollectedX-raydiffractiondataofaninhibitorycomplexofcompound7
boundtoSERCA(rabbitisoform1a).ThiscomplexwasformedinaCa2+-freeso-calledE2
stateandusedanATPanalogue,TNPATP,tostabilizetheflexiblenucleotide-bindingregion
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TetrahydrocarbazolesinhibitthefungalH+-ATPase
Fig3.Membranedepolarizationeffectoftetrahydrocarbazoles.PercentageoffungalcellpopulationmeasuredasPIand
DiBAC4(3)positive(+)andnegative(-)after30minutesexposuretotetrahydrocarbazoles(10μMfinalconcentration).
ErrorbarsindicateSEM.Compounds6–10leadtomembranedepolarization(increaseinDiBAC(3)signal)withminimal
4
effectonmembraneintegrity(noincreaseinthePIsignal).n=3forlysisbufferandn=6forDMSOcontrolandthe
compounds.
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ofSERCA,whichhasbeenwellcharacterizedbothfunctionallyandstructurally[16–26].The
3.0Åcrystalstructureoftheinhibitorycomplexshowscompound7boundatthecytosolic
membraneinterface,inagroovebetweentransmembranehelicesM1,M2,M3andM4(Fig
4Aand4B).
Despitethemodestresolutionof3.0Å,differenceelectrondensitymapsclearlyallowthe
placementofboundTNPATPattheATPaseactivesite(FigureBinS1File),twolipidmole-
culesattheluminalmembraneinterface(modeledasDOPC,FigureBinS1File),andofcom-
pound7whichiscradleddeepinsidethecavitymarkingthecytoplasmicendoftheCa2+inlet
channelofSERCA(Figs4A,5Aand5B).Itspositionsuperimposeswithpreviouslyco-crystal-
lizedSERCAinhibitors2,5-di-t-butyl-1,4-benzohydroquinone(BHQ)[22]andcyclopiazonic
acid(CPA)[25](Fig4BandFigureCinS1File).
Thetetrahydrocarbazolecoreof7isclearlyvisibleinanmF -DF electrondensityomit
o c
mapcontouredat3σ,butthebromo-phenylmoietyispoorlydefined,probablyduetoan
inherentflexibilityinthelinkerconnectingittothetetrahydrocarbazolecore.However,an
anomalousdifferencemapvisualizingtheisolatedsignalfromtheheavybromineatomin7
revealstwodistinctpeaks(5.7and7.3σ)inclosevicinitytothetetrahydrocarbazolecore(Fig
5A).Amodelwithtwoalternativeconformationsofthebromo-phenylmoiety,withanoccu-
pancyof0.5each,allowedastraightforwardplacementofthebromineatomsintobothanom-
alouspeaks.Oneofthetwoorientationsallowsforastaggeredstackinginteractionbetween
thebromo-phenylgroupandthetetrahydrocarbazolecore.Thetetrahydrocarbazolecore
(modeledwithanoccupancyof1)isheldinplacebythreepolarcontactstoSERCAresidues
D59onM1,N101onM2andD254onM3,aswellasseveralhydrophobicinteractionsinvolv-
ingV62,L65,L98,L253,I307,P312,I315,andtheC andC -atomsinthesidechainofE309
β γ
(Fig5Aand5B).ThepocketextendsfurthertowardstheinnercoreoftheSERCAprotein,
boundedbyF256andP308,andalsoupwardstowardstheP-domain(Fig5C,FigureDinS1
File).
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TetrahydrocarbazolesinhibitthefungalH+-ATPase
Fig4.CrystalstructureoftheSERCA•7•TNPATPcomplex.A)CartoonrepresentationoftheSERCA•7•TNPATP
complex.Domainsandligand-bindingsitesareindicated.Compound7(carboninyellow,oxygeninred,brominein
darkred,fluorineinpalecyan,nitrogeninblue)andTNPATP(carboninmarine,oxygeninred,phosphorousin
orange,nitrogeninblue)areshowninstickrepresentation.B)Slicedsurfacerepresentationrevealingthepositionof
theligand-bindingpocketatthecytosolicmembraneinterface(TNPATPomittedforclarity,colorsasinA).
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Fig5.Thebindingpocketofcompound7.A)Compound7withpolarandhydrophobicinteractionsandmFo-DFcdifferenceelectrondensity
mapbeforetheligandwasmodeled(green,contouredat3σ)andanomalousdifferencemap(orange,contouredat4.5σ,calculatedusingphasesof
thefinalrefinedmodelandanomalousdifferencedatafrom56–6Åresolution).B)Slightlyrotatedviewoftheligandbindingsite,revealingthe
positionofthefunctionallyimportantresidueE309inSERCA.C)Close-upviewofthemostlyunchargedsurfaceoftheligand-bindingsiteandits
extensiontowardstheP-domain(asterisk).CarbonatomsarecoloredblueinSERCAandyellowin7,oxygenred,nitrogenblue,brominedarkred,
andfluorineinpalecyan.
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TetrahydrocarbazolesinhibitthefungalH+-ATPase
Interestingly,theSERCA•7•TNPATPcomplexproteinhascrystallizedinapreviously
unreportedcrystalform,reflectinganoveloverallconformationoftheATPase.Structural
alignmentrevealsthemostsimilarknownSERCAstructuresare–asexpected–alsointhe
Ca2+-freeE2state,stabilizedbytheinhibitorthapsigargin(Tg)andTNPAMP,TNPADPand
TNPATPasnucleotideanalogs(PDBID3AR5,3AR6,and3AR7,all-atomRMSD=1.90Å,
1.94Åand1.99Å,respectively).Whereasthetransmembraneregionssuperposealmost
identically(all-atomRMSDwith3AR7=0.736Å),thethreecytosolicdomains(N-,P-,and
A-domains)intheSERCA•7•TNPATPstructurehaveundergoneaconsiderablepositional
shiftrelativetothetransmembranehelices(FigureEinS1File).Thethreecytosolicdomains
adoptpositionsroughlyhalfwayin-betweentheCa2+-freeE2andtheCa2+-boundE1state.
Inparticular,theA-domainhasundergoneasignificantmovementandtilt(FigureEinS1
File),withthedeviationstartingatthecytosolicendsofM1andM2,inthevicinityofthe
bindingsiteofcompound7(FigureEinS1File).Itisthereforetemptingtospeculatethat
compound7inducesthisnewconformation,especiallysinceneitherTg-norTNPATP-
boundSERCAstructuresdisplayanysuchsubstantialconformationalchangesascompared
totheirligand-freecounterparts.
Pma1homologymodelanddockingoftetrahydrocarbazolecompounds
TheoveralltopologicalorganizationofallstructurallycharacterizedP-typeATPasefamily
members,suchasSERCA,theNa+,K+-ATPase,theplantH+-ATPase,theCopACu2+-ATPase
andtheZn2+-ATPase[16–18,20,24,27–32],isverysimilaranditisthereforeunlikelythatthe
Pma1structuredeviatessignificantlyfromitsotherfamilymembers.Wegeneratedhomology
modelsofC.albicansPma1andpigNa+,K+-ATPasebasedontheSERCA•7•TNPATPcom-
plexstructure,toassessthepotentialforprotein-ligandinteractionsintherespectivebinding
pockets.InthePma1model,thebindingpocketisconsiderablylargerthaninSERCA,extend-
ingdeeperintothecoreoftheproteintowardstransmembranehelixM5,andformingacon-
tinuouschanneltowardsthegroovebetweenM3,M5,andM7,whichisaknownbindingsite
oftheinhibitorTginSERCA(FigureDinS1File).
Inaninitialcontrol,weattemptedtodockbothR-andS-isomersofcompound7backinto
theSERCA•7•TNPATPcrystalstructurewithanemptycompoundbindingsite.Thedocking
posewiththesecondhighestaffinityscoregivenbytheVinaprogram(-9.2kcal/mol)forthe
S-isomerplacedthecompoundalmostidenticaltothatmodeledinthecrystalstructure(Fig
6A),whereastheR-isomerdidnotdockinasimilarpose.Thehighestscoringsolutionforthe
S-isomerplacedtheligandintothethapsigarginbindinggroovebetweenM3,M5andM7,a
relativelywideanddeephydrophobiccleftapparentlypronetofalse-positivesamplingaswe
foundthissitesampledbyseveralofthealternativedockingposesthroughout.Whendocked
intothePma1homologymodel,however,thehighestscoringdockingposesofbothenantio-
mersofcompound7(-9.1and-9.7kcal/molfortheR-andS-isomer,respectively)adopta
verysimilardockingposetothebindingmodeobservedintheSERCAcrystalstructure,with
keypolarinteractionstoQ101,N267,andN130(Fig6B).Thehighestscoringposesforthe
othercompoundsalloccupythesamepocket,withrelativelysimilaraffinityscoresbetween
-8.3and-9.8kcal/mol.Generally,compoundswithasubstituentnotlargerthanaphenylmoi-
etyinpositionR (4–8)favoradockingposeinwhichthetetrahydrocarbazolecoreoccupiesa
1
similarpositiontothatobservedintheSERCA•7•TNPATPcrystalstructure.Intheenzyme
andfunctionalassaysthecompoundshaveonlybeentestedasracemicmixtures,butthedock-
ingposesof4–8areverysimilarfortheSandR-isomers.TheR phenylgroupssamplethe
1
spacebetweenthetwoconformationsobservedintheSERCA•7•TNPATPcomplex(Fig6C).
Compounds9–12,withtheirlargerbiphenylmoietiesintheR position,displayalarger
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TetrahydrocarbazolesinhibitthefungalH+-ATPase
Fig6.Dockingposesofcompounds4–12.Oxygen,nitrogen,bromine,chlorineandfluorineatomsinred,blue,dark
red,green,andlightblue,respectively.A)Docking(green)andexperimental(yellow)bindingmodeof7toSERCA.
SERCAisshownascartoonwithcarboninblue,andaminoacidsD59,D254andN101areshownassticks.
Compound7isshownasstickswithcarboninyellow.B-F)Dockingof4–12intotheC.albicansPma1homology
model.ThePma1modelisshownascartoonwithcarboningray,andaminoacidsQ101,N130andN267areshownas
sticks.B)Dockingof7(S)(green)and7(R)(orange)C)Dockingof4(magenta),5(yellow),6(blue),7(green),8
(pink)(allS).D)Dockingof9(S)(magenta),and10(S)(yellow).E)Dockingof11(S)(yellow),12(S)(blue),and12
(R)(orange).F)Dockingof9(brown),10(cyan),and11(yellow)(allR).
https://doi.org/10.1371/journal.pone.0188620.g006
varietyofdockingposes.Hence,theS-isomersofcompounds9–12dockwithslightlyshifted
and/ortiltedtetrahydrocarbazolecoresrelativetotheconfigurationof7,andwiththebiphenyl
moietiespointingroughlyinthesamedirectionsaseitheroneofthetwoconformations
observedin7(Fig6Dand6E).Withtheexceptionofcompound12,wherebothRandS-iso-
merdockwiththeirtetrahydrocarbazolecoreinsimilarpositions,theR-isomersofcom-
pounds9–11adoptcompletelydisplacedpositionswithinthebindingsite(Fig6F).
Fromourstructuralanddockingdata,thepoorbindingaffinityofcompounds11and12
forbothPma1andSERCAcanberationalizedbystericclashesinthebindingsites.Com-
pound11posessesamethylgroupatpositionR forwhichthereisnospaceinthetetrahydro-
2
carbazole-bindingpocket,resultinginastericclashwithD59inSERCA,andQ101inthe
Pma1model,andthemethylgrouponthenitrogeninR ofcompound12causesastericclash
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PLOSONE|https://doi.org/10.1371/journal.pone.0188620 January2,2018 10/21