High-Resolution Genetics Identifies the Lipid Transfer Protein Sec14p as Target for Antifungal

19 Pages·2016·2.31 MB·English

RESEARCHARTICLE
High-Resolution Genetics Identifies the Lipid
Transfer Protein Sec14p as Target for
Antifungal Ergolines
IreosFilipuzzi1*,SimonaCotesta1,FrancescaPerruccio1,BrittaKnapp1,YueFu2,
ChristianStuder1,VerenaPries1,RalphRiedl1,StephenB.Helliwell1,Katarina
T.Petrovic1,N.RaoMovva1,DominiqueSanglard3,JianshiTao2,DominicHoepfner1
1 NovartisInstitutesforBioMedicalResearch,NovartisCampus,Basel,Switzerland,2 GenomicsInstitute
oftheNovartisResearchFoundation,SanDiego,California,UnitedStatesofAmerica,3 Instituteof
a11111 Microbiology,UniversityofLausanneandUniversityHospitalCenter,Lausanne,Switzerland
*[email protected]
Abstract
Invasiveinfectionsbyfungalpathogenscausemoredeathsthanmalariaworldwide.We
OPENACCESS
foundtheergolinecompoundNGx04inanantifungalscreen,withselectivityovermam-
Citation:FilipuzziI,CotestaS,PerruccioF,Knapp
B,FuY,StuderC,etal.(2016)High-Resolution maliancells.High-resolutionchemogenomicsidentifiedthelipidtransferproteinSec14p
GeneticsIdentifiestheLipidTransferProtein asthetargetofNGx04andcompound-resistantmutationsinSec14pdefinecompound-
Sec14pasTargetforAntifungalErgolines.PLoS
targetinteractionsinthesubstratebindingpocketoftheprotein.Beyonditsessentiallipid
Genet12(11):e1006374.doi:10.1371/journal.
transferfunctioninavarietyofpathogenicfungi,Sec14pisalsoinvolvedinsecretionof
pgen.1006374
virulencedeterminantsessentialforthepathogenicityoffungisuchasCryptococcus
Editor:J.AndrewAlspaugh,DukeUniversity
neoformans,makingSec14panattractiveantifungaltarget.Consistentwiththisdual
SchoolofMedicine,UNITEDSTATES
function,wedemonstratethatNGx04inhibitsthegrowthoftwoclinicalisolatesofC.neo-
Received:May25,2016
formansandthatNGx04-relatedcompoundshaveequalandevenhigherpotencyagainst
Accepted:September20,2016
C.neoformans.FurthermoreNGx04analoguesshowedfungicidalactivityagainstaflu-
Published:November17,2016 conazoleresistantC.neoformansstrain.Insummary,wepresentgeneticevidencethat
Copyright:©2016Filipuzzietal.Thisisanopen NGx04inhibitsfungalSec14pandinitialdatasupportingNGx04asanovelantifungal
accessarticledistributedunderthetermsofthe startingpoint.
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
AuthorSummary
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
Emergingresistancetoantibioticsledtoaningloriousrevivalofinfectiousdiseases.Fur-
files.
thermore,inthepast30years,onlyonenovelanti-fungaltargethasbeendiscovered
Funding:Theauthorsreceivednospecificfunding whichwasusedtodeveloptherapiesagainst.Thereforepathogen-selectivetargetsand
forthiswork.
knowledgeaboutpossibleresistancedeterminantsareofutmostimportancetosuccess-
CompetingInterests:Ihavereadthejournal’s fullydevelopnewmedicines.Herewedescribetheidentificationofanti-fungalergolines,
policyandtheauthorsofthismanuscripthavethe targetingthelipidtransferproteinSec14p,andinhibitingthegrowthoftwoclinicaliso-
followingcompetinginterests:IF,SC,FP,BK,YF,
latesofthepathogenicfungusCryptococcusneoformans.Both,compoundandtargetrep-
CS,VP,RR,SBH,KTP,NRM,JTandDHare
resentattractivepointsforfurtherinvestigations:Sec14pasitdifferssignificantlyfromthe
employeesofNovartisandmayownstockinthe
humanhomologandasithasbeenimplicatedinfungalviabilityandpathogenicity,and,
company.DSisanemployeeoftheUniversityof
PLOSGenetics|DOI:10.1371/journal.pgen.1006374 November17,2016 1/19
AntifungalErgolinesTargetSec14p
Lausanneanddeclaresnocompetingorfinancial
interests. ergolinesastheyareusedintheclinicagainstavarietyofdiseasesdemonstratingbotheffi-
cacyandsafety.
Introduction
Severeinvasivefungalinfections(IFIs)arelife-threateningandassociatedwithhighmortality
[1].Candidaalbicans(mortality:20–50%)[2],Cryptococcusneoformans(mortality:20–70%)
[3]andAspergillusfumigatus(mortality:50–90%)[4]representthemajorhumanfungalpath-
ogensthathavedevastatingeffectsonmostly,butnotexclusively,immunecompromisedindi-
vidualssuchasHIVpatients,prematureinfants,diabetics,chemotherapypatients,organ
transplantrecipientsandagingpeople[1,5].Currentlymarketeddrugssufferfromlimitedeffi-
cacyandbioavailability,narrowspeciesspectrumandhightoxicity[6,7]andthedevelopment
ofnoveldrugsagainstfungal-selectiveproteintargetsischallengingduetothehighevolution-
aryconservationbetweenfungiandman.Thisresultsinalimitednumberofdruggablenodes
andthusrestrictedchemicalmatter[8].Onlyonenewantifungaldrugclasstotreatsystemic
infectionshasbeenapprovedintheUSinthelastthreedecades,theβ-glucansynthaseinhibi-
torsechinocandins(reviewedin[9]).Efficacyofechinocandinshoweverislimited,astheysuf-
ferfrompoorbioavailability[10].Newfungal-selectivetargetsarethusneededtoscreenfor
novelchemotypestofighttheseinfections[6,8].
ThevirulenceofC.neoformansislargelydependentonitssecretionmachinery,asmanyof
themoleculesrequiredtoexertitsvirulenceareextracellular[11,12].Secretedfactorsinclude
themajorpolysaccharidebuildingblockofthecapsule,enzymesinvolvedintheassembly,
maintenanceandintegrityofthecellwallaswellasotherproteins[13–17].SofarthreeSec
proteins,Sec4p,Sec6pandSec14p,havebeenimplicatedinsecretionoffactorsessentialtovir-
ulenceofC.neoformans[12].AmongthesethreeSecproteins,thephosphatidylinositoland
phosphatidylcholinetransferproteinSec14pismostlikelydruggable[18],whereasSec4p,a
Rabfamilymember,orSec6p,ananchoringprotein,representbyfarmorechallengingtarget
classeslikeGTPasesandprotein-proteininteractions,respectively.Sec14phasbeenshownto
beessentialnotonlyforvirulence,butalsoforgrowth[11],adualrolewhichmakesthis
enzymeanattractivedrugtarget.HereweshowthecharacterizationofafungalSec14pinhibi-
torbyusingasuiteofgeneticassaystode-convolutethemodeofactionofthescreeninghit,
NGx04,prioritizedduetoanestablishedtrackrecordofergolinesinmedicine[19–25].Che-
mogenomicprofilingidentifiedhypersensitivityandresistanceofdeletionstrainsrelatedtothe
actionofSec14p.SaturatingmutagenesisoftheSEC14geneidentifiedfourkeyresiduesthat
reducedNGx04potency.Allidentifiedresidueslocalizedintothelipid-bindingcavityofthe
Sec14proteinsuggestingthatNGx04isanactive-siteinhibitorandcompetitionwiththe
Sec14plipidsubstratessupportedthishypothesis.Resistance-residueguidedinsilicodocking
ofNGx04intothelipid-bindingcavityoutlinedcriticalsitesforcompound-proteininteraction
mostofwhichareconservedintheproteinsequenceofthepathogenC.neoformansbutnotin
thehumanhomolog.TestingNGx04againsttwoclinicalisolatesofC.neoformansconfirmed
antifungalactionandlackofpronouncedcytotoxicityinhumancells.Interestingly,themost
potentNGx04derivativewasfoundtoexertfungicidalactivityagainstastrainresistanttoflu-
conazole,acommonlyuseddrugagainstcryptococcosis.
Insummary,wepresentevidencethatNGx04inhibitsfungalSec14pandinitialdatasup-
portingNGx04asanovelantifungalstartingpoint.
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Results
Haploinsufficiencyprofiling(HIP)andhomozygousprofiling(HOP)of
NGx04
Ahigh-throughputscreenoftheNovartiscompoundarchiveagainstS.cerevisiaewasper-
formedtoidentifynovelantifungalcompounds[26,27].Theergot-relatedcompoundNGx04
(Fig1A)wasamongthevalidatedhitsscoringanIC <20μM.Duetothelonghistoryof
50
ergolinesinmedicine[19–25]thisscaffoldwasselectedfortargetidentificationbychemoge-
nomicprofiling[26].Haploinsufficiencyprofiling(HIP)andhomozygousprofiling(HOP)are
gene-dosage-dependentmethodsthatassesstheeffect(s)ofcompoundsagainstpotentialtar-
getsencodedbytheS.cerevisiaegenome[28].HIPindicatespathwaysdirectlyaffectedbythe
compound,whereasHOP, withbothgenecopiesdeleted,indicatessyntheticlethalityandiden-
tifiescompensatingpathwaystothosedirectlyaffectedbythecompound.TestingNGx04at
sub-lethaldoses(8μM)intwoindependentbiologicalreplicatesandcorrelatingtheprofilesas
previouslypublished[26]identifiedtheheterozygousdeletionofSEC14toresultinsignifi-
cantlyreducedfitnesscomparedtotherestofthegenome-wideheterozygousdeletioncollec-
tion(HIP)(Fig1B).Incontrast,heterozygousdeletionofKES1,whichencodesaproteinthat
negativelyregulatesSec14p-dependentsecretion[29,30],resultedinincreasedfitness(Fig1B).
HypersensitivityoftheheterozygousSEC14strainwasfoundtobeuniquewhenanalyzingthe
scoresofthesetwostrainsinourdatabasecapturingprofilesof>4000diversecompounds
(Fig1D).Chemical-syntheticinteractionsofNGx04(HOP)wereidentifiedwithhomozygous
deletionsofthegenesencodingtheregulatorysubunit(SRF1)andthecatalyticsubunit
(SPO14)ofPhospholipaseD,apreviouslyidentifiedsubstrateofSec14p[31,32],andITR1(Fig
1C),themajormyo-inositoltransporterinS.cerevisiae[33].HypersensitivityofSEC14/sec14,
srf1/srf1,spo14/spo14oritr1/itr1andhyposensitivityofKES1/kes1againstNGx04wascon-
firmedbytitratingNGx04acrossthedeletionstrainsandcomparinginhibitiontothecontrol
stainho/ho(Fig1EHIPandFig1FHOP).Takentogether,thisdatasuggeststhatNGx04inter-
fereswithcellularviabilityatthelevelofthephosphatidylinositolandphosphatidylcholine
transferproteinSec14p.
FunctionalvariomicsscreenforNGx04-resistantmutants
To verifyourfindingsfromthechemogenomicprofilingstudies,asaturationmutagenesis
screeningapproach[34]wasusedandsupportedSec14pasthetargetofNGx04.Apoolcon-
tainingapproximately105plasmid-based,mutagenizedSec14p-variants[34]wasplatedonto
solidgrowthmediasupplementedwith150μMNGx04,aconcentrationthatpreventedcolony
formationofwild-typecells.Eighty-eightsurvivingcolonieswereisolated,plasmidDNA
encodingtheSec14proteinsisolatedandsequenced.Thisapproachyieldedmutationsinfour
distinctaminoacidsofSec14p:Y111H(identified14times),Y151H/S(identified27times),
V154I(identified15times)andS173A/P/L(identified32times)(Fig2A).To confirmthatthe
mutationsweretheresistance-definingfactors,theplasmidscontainingtheSEC14mutants
wereretransformedintowildtypeyeast.AplasmidcontainingthewildtypecopyofSEC14
wasusedascontroltoexcluderesistanceduetoSEC14genecopy-numbereffects.Spottingfor
singlecolonyformationonlethaldosesofNGx04(Fig2B)aswellasgrowthinhibitiontitra-
tions(Fig2C)revealedthateachofthefouridentifiedmutationswassufficienttoconferresis-
tancetoNGx04.AsthecellscontainedalsotheendogenouswildtypeSEC14allelethisdata
revealedthatthedrugresistancewasdominantandprovidedfurthersupportthatSec14pisthe
growthinhibitorytargetofNGx04.
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Fig1.Chemogenomicprofiling.Structure,haploinsufficiencyprofiling(HIP)andhomozygousprofiling(HOP)ofNGx04.A)Thestructureof
NGx04isshown.B)HIPandC)HOPwereperformedaroundIC (8μM)induplicatesandtheaverageoftheprofilesisshown.TheMADLscoreis
30
givenonthey-axisandindicateshowsensitiveaparticulardeletionstrainistoagivencompound,comparedtoitsno-drugcontrol.TheZ-scoreis
givenonthex-axisandisastatisticalmeasureofhowfrequentlyaparticulardeletionstrainisaffectedwithinthesofarprofiledcompounds[26].
Circlesdepictnon-essentialdeletions,whereassquaresessentialdeletions.Hyper-sensitivedeletionsarefoundbottomleftandhyper-resistant
deletionstoprightofthepanels.D)HypersensitivityofaheterozygousSEC14/sec14strainacross4011diversecompounds(x-axis)isdisplayedas
Z-score(y-axis).NGx04(arrow)wasfoundtoexertthestrongesteffect.ValidationofthehitcandidatesfromHIPE),HOPF)wasperformedbysingle
straingrowthinhibition.Theindicatedstrainsweregrownfor24hinthepresenceofincreasingconcentrationsofNGx04andgrowthmeasuredby
turbidity(OD ).Duplicatevaluesweredeterminedandusedforlogisticregression.ThecalculatedIC valuesaredisplayed.
600 50
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Fig2.Resistancemapping.FunctionalvariomicsscreenforNGx04-resistantmutants.A)TheSEC14ORFs
of88NGx04-resistantcolonieswereamplifiedbyPCRandsequenced.Fourmutationsmappingtothelipid-
bindingpartofSec14pwerefound.Theidentifiedaminoacidchangeswiththeirrespectivefrequenciesaregiven.
B)TheplasmidscodingforvariousSEC14proteinswerere-transformedintoafreshwildtypeyeaststrainand
serialdilutionsoftheculturesspottedoneither150μMofNGx04orDMSOcontrol,incubatedfor3daysat30˚C
andscoredforgrowth.C)Thestrainsweregrownfor24hinthepresenceofincreasingconcentrationsofNGx04
andgrowthmeasuredbyturbidity(OD ).Duplicatevaluesweredeterminedandthe18htimepointwasusedfor
600
logisticregression.ThecalculatedIC valuesdisplayed.
50
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AntifungalErgolinesTargetSec14p
Phosphatidylinositol(PI)andphosphatidylcholine(PC)competition
CrystalstructuresofSec14pandSec14p-likeproteinsfromvariousspeciesincludingS.cerevisi-
sae(PDBID:1AUA) andhuman(PDBID:1OLM)havebeensolved.Althoughthelipid-bind-
ingpocketoftheenzymeiswellcharacterized[35–37]Sec14phasnotbeencrystalizedwhen
boundtophosphatidylethanolamine(PtdEtn)substrate.ThecrystalstructureoftheS.cerevisi-
saeSfh1protein(PDBID:3B7Q),sharing62.3%identitywithSec14p,showsthatphosphatidyl-
coline(PC)hydrophobictailformsvanderWaals interactionswithaclusterofhydrophobic
residues,whereasthephosphatemoietyisinteractingwiththeside-chainhydroxylgroupsof
residuesS175andT177,andthenitrogencontactsthephenolicoxygenofY113(Fig3A).Since
theNGx04resistance-conferringresidueswerelocatedintheequivalentcatalyticsiteofSec14p
(Fig3A)(i.e.S173,correspondingtoS175,andT175toT177inSfh1,tworesiduesinvolvedin
H-bondswithPC)wereasonedthatNGx04wouldbindinthelipid-bindingpocketofthe
protein.
To testthishypothesis,theS.cerevisisaex-raystructureofSec14pwasusedtodockthe
NGx04ligandintothelipid-bindingcavity(Fig3B).Intheresultingdockingmodel,theester
moietyoftheligandformedanH-bondwithY111whiletheergolineamineformedanH-
bondwithT175.TheergolinemoietyalsomadevanderWaals interactionswiththeresidues
Y122,Y151,S173,M177andF212.Thecalculateddockingmodelwasinagreementwiththe
experimentaldatafromthemutagenesisstudy,asthreeofthefouridentifiedresistanceconfer-
ringmutationsweredirectlyinvolvedinligandinteractions(Y111,Y151andS173)andthe
identifiedV154residue,althoughnotdirectlyinvolvedinligandinteractions,wasonly4.5Å
awayfromNGx04.ThemodeloutlinesthatalargerresidueatthispositionlikeIsoleucine
wouldbedeleteriousforligandbinding.Asourdockingexperimentwasbiasedtowardsthe
lipid-bindingcavityweaimedatfurthersupportingevidenceforNGx04bindinginthisprotein
pocket.To dosowetestedifNGx04wouldcompeteforphospholipid-bindingintheSec14p
cavity.WildtypeS.cerevisiaecellsweregrowninpresenceofincreasingdosesofNGx04in
mediumcontaininganexcessofeitherphosphatidylinositol(PI)orphosphatidylcholine
(PC)(200μM).Bothlipids,PIandPC,werefoundtoalleviateNGx04-mediatedgrowth
inhibition,supportingacompetitivemodeofinhibitionatthelipid-bindingcavityofSec14
(Fig3C).
MammaliancellproliferationandcytotoxicityofNGx04
Onekeyrequirementofantifungalmedicinesisselectiveinhibitionofthepathogenoverthe
mammalianhost.To explorepotentialundesiredactivityofNGx04againstmammaliancells
weusedtheCancerCellLineEncyclopediaCCLE,acollectionof512humancancercelllines
(broadinstitute.org/ccle).Dose-responsecurvesforNGx04weregeneratedinaproliferation
assayandIC valuesdetermined.Althoughalargenumberofanti-proliferativecompounds
50
werepublishedtobeactiveacrosstheCCLEcollection[38,39]onlyonefourthofthecelllines
wasfoundtobesensitivetoNGx04asdefinedbyAmax<50%andIC <30μM.Furthermore
50
thelargemajorityofcells(76%)werenotnotablyaffectedbyNGx04(Fig4A).SinceHepG2
cellsareknowntobeasuitabletooltoprofiletoxicityofcompounds[40],weusedthisliver-
derivedlinetoassesscytotoxicityofNGx04.Adose-responsecurvewasgeneratedandanIC
50
valueof157.6μMdeterminedbylogisticregression(Fig4B),againindicatingpotentialselec-
tivityofNGx04forthefungalprotein.SuperimpositionoftheS.cerevisisaeSec14pcrystal
structure(PDBID:1AUA, blue)andthecrystalstructureofthehumanorthologueSEC14L2
(PDBID:1OLM,green)(Fig4C)revealedthatalthoughhavingasimilaroverallstructurefold,
theactivesiteofthetwoenzymesdifferconsiderably.16of21aminoacidresidueswithina5Å
radiusfromanyatomofthedockedNgx04ligandwerenotconservedbetweenthetwo
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Fig3.Activesiteantagonism.Phosphatidylinositol(PI)andphosphatidylcholine(PC)competition.
A)ThecrystalstructureofPhosphatidylcholine(PC)boundtotheyeastSfh1protein(PDBID:3B7Q),(where
RisoleoylandR’ispalmitol).PChydrophobictailformsvanderWaalsinteractionswithaclusterof
hydrophobicresidue,whereasthephosphatemoietyisinteractingwiththeside-chainhydroxylgroupsof
residuesS175andT177,andtheethanolaminenitrogencontactsthephenolicoxygenofY113.B)The
proposeddockingposeofNGx04intheputativebindingsiteoftheyeastproteinstructureisshown(PDB
ID:1AUA).Inthesuggestedbindingmode,theestermoietyoftheligandformsahydrogenbondwithY111
whiletheergolineamineformsH-bondswiththeT175.TheergolinemoietyisalsoformingvanderWaals
interactionswiththefollowingresidues:Y122,Y151,S173,M177andF212.C)Wildtypeyeastwasgrownin
presenceofincreasingconcentrationsofNGx04,supplementedwitheither200μMPI,200μMPCorno
phospholipid,incubatedat30˚Candgrowthfollowedovertimebymeasuringturbidity(OD ).Duplicate
600
valuesweredeterminedandthetimepointsat18hwereusedforlogisticregression.
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proteins.CriticaldifferencesintheactivesiteofthehumanproteinaretheresiduesF178,
C128,I151andL186(correspondingtoS201,Y151,T175andM209inyeast)whichforma
smallerandmorehydrophobicpocketcomparedtotheyeastprotein(Fig4C).Visualizingthe
Fig4.Fungalselectivity.MammaliancellgrowthinhibitionandcytotoxicityofNGx04.A)Dose-responsecurves
forNGx04weregeneratedingrowthinhibitionassayandIC valuesdeterminedinduplicates[39].IC valuesand
50 50
growthinhibition(Amax)aredisplayed.76%ofthetestedcelllineswerenotsignificantlyinhibitedbyNGx04.B)Adose-
responsecurvesforNGx04wasgeneratedinaMTScytotoxicityassayusingHepG2cellsintriplicatesandatoxicity
curvegeneratedbylogisticregression.C)OverlayofthecrystalstructureoftheyeastSec14p(PDBID:1AUA,protein
structurehighlightedinblue)anditshumanorthologueSEC14L2(PDBID:1OLM,proteinstructurehighlightedingreen)
revealsthattheaminoacidresiduesbelongingtotheputativebindingsitearenotconservedbetweentheyeastandthe
humanprotein.D)OverlayofthedockedNGx04intheyeastSec14p(PDBID:1AUA)anditshumanorthologue
SEC14L2(PDBID:1OLM).Theaminoacidresiduesoftheyeastproteinstructurehavebeenhiddenwhereasthe
surfaceoftheputativebindingsiteofthehumanproteinstructurehasbeenaddedtohighlightthedifferenceinthe
overallshapebetweentheactivesiteofthetwospecies.
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AntifungalErgolinesTargetSec14p
solventaccessiblesurfaceofthehumanSec14pwithNGx04asapotentialligandrevealedthat
NGx04istoobigtooccupythissmallerbindingpocket(seeclearligandbumpstowardsthe
solventaccessiblesurface)(Fig4D).Inaddition,allfourNGx04-interactingaminoacidsidenti-
fiedbyfunctionalvariomicsaremissinginthehumanenzyme(S1Fig).Insummary,itisplau-
siblethattheobservedlackofpotentactivityofNGx04againsthumancelllinescanbe
explainedbydifferencesinthecatalyticpocketandtheabsenceofproposedNGx04interacting
residuesinthehumanprotein.
C.neoformansgrowthinhibitionbyNGx04scaffolds
Sec14pisreportedtobeessentialforvirulenceaswellasforgrowthinC.neoformans[11,12].
TheS.cerevisiaeandC.neoformansproteinsshow53%similaritybypair-wisealignmentwith
higherconservationoftheresiduesinthelipid-bindingpocket(S2Fig).Furthermore,threeof
thefoursitesidentifiedtobecriticalforNGx04sensitivity,areconserved(S2Fig).Incombina-
tionwiththecurrenthighunmetmedicalneedrelatedtoCryptococcusinfectionsweprioritized
testingofNGx04and18relatedergolinescaffoldanalogsagainstthispathogen.Twoclinical
isolatesofC.neoformans(ATCC36556andATCC90113)wereplatedintriplicatesonincreas-
ingdrugconcentrationsandMIC-2(50%inhibition)andMIC-0(novisiblefungalgrowth)
concentrationswerescoredafter72hrs.(Fig5).NGx04andanaloguesincludingNGx03,
NGx16,NGx07,NGx09andNGx19werefoundtoinhibitC.neoformansgrowthinthetested
doserange.NGx03appearedtobethemostpotentcompoundagainstATCC36556witha
MIC-2of3.13μg/mlandaMIC-0of6.25μg/mlwhereasNGx19showedabroaderspectrum
withaMIC-2of12.5μg/mlagainstthefirstisolateandaMIC-2of6.25μg/mlagainstthesecond
strain.
Fluconazole(FLU)iscommonlyusedtotreatcryptococcosis[41].Repeatedexposureor
prolongedtreatmentwithazoles,however,canleadtoresistance[42–44].We thustestedthe
abilityofthemostpotentcompoundstoinhibitgrowthoftheFLU-resistantC.neoformans
strainBPY22[45]andfoundactivityforNGx03(MIC-2of16μg/mlandMIC-0of32μg/ml)
andweakeractivityforNGx16andNGx19(S3Fig),indicatingthepotentialofergolinesto
fightFLU-resistantC.neoformans.We nextassessedthemechanismofinhibition,sincefungi-
cidalagents,comparedtofungistaticagents[46],areassociatedwithdecreasedmortalityand
foundNGx03andNGx16toexhibitafungicidaleffectonFLU-sensitiveandFLU-resistantC.
neoformansstrains(S6Fig).NGx03wasamongthemostactivecompoundwhentestedinsus-
ceptibilityassaysandthuscorroborateswithitsfungicidaleffectinthetestedstrains.
Analysisofthestructure-activityrelationship(SAR)ofthetestedergolines(Fig5)revealed
thatanalogueswherethearomaticaminogroupwassubstitutedbya2,2,2-trichloroethyl-car-
boxylatemoietyscoredanti-fungalactivity(NGx03andNGx07),whiletheanalogueswithan
unsubstituted,acetylatedorformulatedaromaticaminogrouplackednotableactivity.Nitro
substitutionofthearomaticringappearedtobetoleratedinallpositionsastheanaloguewith-
outanysubstituentwasfoundtobeequipotent.Insummary,antifungalactivityagainstC.neo-
formansaspredictedbytheanalysisofidentifiedNGx04interactingresiduescould
experimentallybeconfirmed.OurlimitedSARexperimentidentifiedthatbuildingoffthearo-
maticringwastoleratedenablingfuturemedicinalchemistryapproacheswiththeaimtoassess
andoptimizepotency,spectrumandotherpharmacokineticparametersofthisscaffold.
Discussion
SevereIFIsareoftenlife-threateningconditions.Theclinicalanti-fungalarsenalhoweveris
verylimitedandtheβ-glucansynthaseinhibitorechinocandinistheonlydrugdevelopedover
thelastthreedecadesthatmodulatesanoveltarget.Inaddition,theemergenceofresistance
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