Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids

15 Pages·2015·2.55 MB·English

RESEARCHARTICLE crossmark
Identification of a New Class of Antifungals Targeting the Synthesis of
Fungal Sphingolipids
VisesatoMor,aAntonellaRella,aAmirM.Farnoud,aAshutoshSingh,aMansaMunshi,aArielleBryan,aShamoonNaseem,a
JamesB.Konopka,aIwaoOjima,bErikaBullesbach,cAlanAshbaugh,dMichaelJ.Linke,d,eMelanieCushion,d,eMargaretCollins,e
HariKrishnaAnanthula,fLarrySallans,qPankajB.Desai,fNathanP.Wiederhold,gAnnetteW.Fothergill,gWilliamR.Kirkpatrick,h
ThomasPatterson,hLaiHongWong,iSunitaSinha,iGuriGiaever,iCoreyNislow,iPatrickFlaherty,jXuewenPan,k
GabrieleVargasCesar,lPatriciadeMeloTavares,lSusanaFrases,mKildareMiranda,l,nMarcioL.Rodrigues,l,oChiaraLuberto,p
LeonardoNimrichter,lMaurizioDelPoetaa
DepartmentofMolecularGeneticsandMicrobiology,StonyBrookUniversity,StonyBrook,NewYork,USAa;DepartmentofChemistryandInstituteofChemicalBiology DDDD
andDrugDiscovery,StonyBrookUniversity,StonyBrook,NewYork,USAb;DepartmentofBiochemistryandMolecularBiology,MedicalUniversityofSouthCarolina, oooo
Charleston,SouthCarolina,USAc;DepartmentofVeteransAffairsMedicalCenter,Cincinnati,Ohio,USAd;UniversityofCincinnatiCollegeofMedicine,Cincinnati,Ohio, wwww
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USAe;DepartmentofPharmaceuticalSciences,UniversityofCincinnati,Cincinnati,Ohio,USAf;DepartmentofPathology,FungusTestingLaboratory,UniversityofTexas lolololo
HealthScienceCenteratSanAntonio,SanAntonio,Texas,USAg;DivisionofInfectiousDiseases,UniversityofTexasHealthScienceCenteratSanAntonio,SanAntonio, aaaa
Texas,USAh;DepartmentofPharmaceuticalSciences,UniversityofBritishColumbia,Vancouver,BritishColombia,Canadai;DepartmentofBiomedicalEngineering, dddd
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WorcesterPolytechnicInstitute,Worcester,Massachusetts,USAj;DepartmentofBiochemistryandMolecularBiology,BaylorCollegeofMedicine,Houston,Texas,USAk; dddd
InstitutodeMicrobiologiaProfessorPaulodeGóes,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,Brazili;LaboratóriodeUltraestruturaCelularHerthaMeyer, f f f f
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InstitutodeBiofísicaCarlosChagasFilho,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,Brazilm;DiretoriadeMetrologiaAplicadaaCiênciasdaVida,Instituto oooo
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NacionaldeMetrologia,QualidadeeTecnologia(INMETRO),Xerém,RiodeJaneiro,Braziln;FundaçãoOswaldoCruz(Fiocruz),CentrodeDesenvolvimentoTecnológico

emSaúde(CDTS),RiodeJaneiro,Brazilo;DepartmentofPhysiologyandBiophysics,StonyBrookUniversity,StonyBrook,NewYork,USAp;DepartmentofChemistry, hhhh
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UniversityofCincinnati,Cincinnati,Ohio,USAq tptptptp
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ABSTRACT Recentestimatessuggestthat>300millionpeopleareafflictedbyseriousfungalinfectionsworldwide.Currentanti- mmmm
fungaldrugsarestaticandtoxicand/orhaveanarrowspectrumofactivity.Thus,thereisanurgentneedforthedevelopmentof bbbb
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newantifungaldrugs.Thefungalsphingolipidglucosylceramide(GlcCer)iscriticalinpromotingvirulenceofavarietyof ....
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human-pathogenicfungi.Inthisstudy,wescreenedasyntheticdruglibraryforcompoundsthattargetthesynthesisoffungal, ssss
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butnotmammalian,GlcCerandfoundtwocompounds[N=-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide
....
(BHBM)anditsderivative,3-bromo-N=-(3-bromo-4-hydroxybenzylidene)benzohydrazide(D0)]thatwerehighlyeffectivein oooo
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vitroandinvivoagainstseveralpathogenicfungi.BHBMandD0werewelltoleratedinanimalsandarehighlysynergisticor ////

additivetocurrentantifungals.BHBMandD0significantlyaffectedfungalcellmorphologyandresultedintheaccumulationof oooo
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intracellularvesicles.Deep-sequencinganalysisofdrug-resistantmutantsrevealedthatfourproteinproducts,encodedbygenes N N N N
APL5,COS111,MKK1,andSTE2,whichareinvolvedinvesiculartransportandcellcycleprogression,aretargetedbyBHBM. oooo
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IMPORTANCE Fungalinfectionsareasignificantcauseofmorbidityandmortalityworldwide.Currentantifungaldrugssuffer eeee
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fromvariousdrawbacks,includingtoxicity,drugresistance,andnarrowspectrumofactivity.Inthisstudy,wehavedemon-
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stratedthatpharmaceuticalinhibitionoffungalglucosylceramidepresentsanewopportunitytotreatcryptococcosisandvari- eeee
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ousotherfungalinfections.Inadditiontobeingeffectiveagainstpathogenicfungi,thecompoundsdiscoveredinthisstudywere 2 2 2 2
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welltoleratedbyanimalsandadditivetocurrentantifungals.Thesefindingssuggestthatthesedrugsmightpavethewayforthe ,,,,

developmentofanewclassofantifungals. 2222
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Received20April2015 Accepted19May2015 Published23June2015 bbbb
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CitationMorV,RellaA,FarnoudAM,SinghA,MunshiM,BryanA,NaseemS,KonopkaJB,OjimaI,BullesbachE,AshbaughA,LinkeMJ,CushionM,CollinsM,AnanthulaHK,
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SallansL,DesaiPB,WiederholdNP,FothergillAW,KirkpatrickWR,PattersonT,WongLH,SinhaS,GiaeverG,NislowC,FlahertyP,PanX,CesarGV,deMeloTavaresP,FrasesS, uuuu
MirandaK,RodriguesML,LubertoC,NimrichterL,DelPoetaM.2015.Identificationofanewclassofantifungalstargetingthesynthesisoffungalsphingolipids.mBio6(3): eeee
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e00647-15.doi:10.1128/mBio.00647-15. tttt
EditorJulianE.Davies,UniversityofBritishColumbia
Copyright©2015Moretal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttribution-Noncommercial-ShareAlike3.0Unportedlicense,
whichpermitsunrestrictednoncommercialuse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
AddresscorrespondencetoMaurizioDelPoeta,[email protected].
Recent estimates suggest that (cid:2)300 million people are af- misingmedicalconditionssuchasAIDSorthoseusingimmu-
flicted by serious fungal infections worldwide, with the nosuppressive medical treatment, such as some cancer thera-
most common and life-threatening invasive mycoses being pies (3). Three classes of antifungal drugs are currently
cryptococcosis,candidiasis,aspergillosis,andpneumocystosis available:azoles(e.g.,fluconazole),polyenes(e.g.,amphoteri-
(1,2).Thenumberoffungalinfectionsisrisinginpartdueto cinB),andechinocandins(e.g.,caspofungin).However,these
the increasing number of individuals with immunocompro- drugsarestaticandtoxic,haveanarrowspectrumofactivity,
May/June2015 Volume6 Issue3 e00647-15 ® mbio.asm.org 1
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FIG1 BHBMandD0inhibitthesynthesisoffungalbutnotmammalianglucosylceramide.(A)Thin-layerchromatographyanalysisofthesynthesisof f
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glucosylceramide(GlcCer)uponinvivolabelingofC.neoformans(Cn)orJ774cellswith[3H]palmitateandtreatedwithBHBMorD0attheindicated o
concentrations.(B)StructureofN=-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide(BHBM)and3-bromo-N=-(3-bromo-4-hydroxybenzylidene) m
benzohydrazide(D0).TheMICandminimumfungicidalconcentration(MFC)areshown.Representativedataofthreeindependentexperimentsareshown. h
t
t
p
andinteractwithotherdrugssuchaschemotherapyagentsand phenotypewasutilizedforscreeningofcompoundsthatcanin- :/
/
m
immunosuppressants(4). hibit fungal GlcCer. A ChemBridge DIVERSet-CL library con-
b
Duetothelimitednumberofantifungalsavailableonthemar- taining49,120compoundswasscreenedtoidentifymoleculesthat io
ket,theirincreasingusestimulatesthedevelopmentofdrugresis- inhibitthegrowthofC.neoformansatneutralandalkalinepH.A .
a
tance,andthus,newclassesofantifungalsaredesperatelyneeded. totalof220compoundswithaMICof(cid:2)1(cid:3)g/mlwereselected s
m
Thefungalsphingolipidglucosylceramide(GlcCer)iscriticalin andscreenedinacidic(4.0)pH.The18compoundsthatwere
.
promoting virulence of several plant- and human-pathogenic inactive (MIC (cid:2) 32 (cid:3)g/ml) were subjected to an in vivo or
g
fungi,includingCryptococcusneoformans,Candidaalbicans,and labeling assay for the inhibition of GlcCer synthesis. Two /

Aspergillusfumigatus(5–10). compounds,identifiedasN=-(3-bromo-4-hydroxybenzylidene)- o
n
GlcCer regulates fungal cell replication in environments of 2-methylbenzohydrazide(BHBM)and3-bromo-N=-(3-bromo- N
neutraloralkalinepH(5,11).WhenfungalcellslackingGlcCer 4-hydroxybenzylidene) benzohydrazide (D0), a derivative of o
areexposedtoneutral/alkalinepH,theycannotprogressthrough BHBM,significantlyinhibitedthesynthesisofGlcCerinC.neo- ve
thecellcycle,andcytokinesisdoesnotoccur(5,9,12).GlcCeris formansbutnotmammalian(J774.16)cells(Fig.1).Bothcom- m
mostlikelyapanfungalvirulencefactorrequiredduringinfection poundswerefungicidalinvitrowithminimumfungicidalconcen- be
topromotefungalgrowthatneutral/alkalineenvironmentsinthe trations (MFC) of 4 (cid:3)g/ml for BHBM and 1.2 (cid:3)g/ml for D0. r
host(e.g.,alveolarspaces,cerebrospinalfluid,andbloodstream); 2
Becauseoftheirstructure,wedesignatedthisclassofcompounds 3
assuch,itisapromisingnoveldrugtarget. ,
“hydrazycins.” 2
AntibodiestofungalGlcCerexertantifungaleffectsinvitroand BHBMandD0exertantifungalactivity.Theantifungalactiv- 0
in vivo (13); however, inhibitors that block fungal, but not 1
ityofBHBMwasexaminedagainstavarietyofclinicallyrelevant 8
mammalian,GlcCersynthesisarenotavailable.Inthisstudy,
fungi(Table1).BHBMshowedgoodinvitroactivityagainstvar- b
wescreenedasyntheticdruglibraryforcompoundsthatspe- y
ious Cryptococcus species with the MIC ranging between
cifically target the synthesis of fungal GlcCer. Two such 0.25 (cid:3)g/ml and 8 (cid:3)g/ml. Importantly, a fluconazole-resistant gu
compounds were found [N=-(3-bromo-4-hydroxybenzylidene)- C. neoformans strain (MIC (cid:2) 64 (cid:3)g/ml) and various virulent es
2-methylbenzohydrazide (BHBM) and 3-bromo-N=-(3-bromo- t
Cryptococcus gattii strains were highly susceptible to BHBM
4-hydroxybenzylidene)benzohydrazide(D0)]andwerehighlyef-
(MICsof1to2(cid:3)g/mland0.5to2(cid:3)g/ml,respectively).Other
fective in vitro and in vivo against a series of pathogenic fungi.
fungiagainstwhichBHBMwashighlyactiveincludedRhizopus
Thesecompoundsaresafeandwelltoleratedinanimals,demon-
oryzae,Blastomycesdermatitis,Histoplasmacapsulatum,Pneumo-
strategoodpharmacokineticproperties,andarehighlysynergistic
cystismurina,andPneumocystisjirovecii(Table2).BHBMshowed
or additive to current antifungals. The compounds target the
moderateactivity(MICrangeof2to32(cid:3)g/ml)againstCandida
transportoffungalvesicles,whichishowfungalceramideistrans-
krusei,Candidaglabrata(dependingonthestrain),Candidaguil-
portedforthesynthesisofGlcCer.Thus,wehaveidentifiedanew
liermondii, Candida parapsilosis, A. fumigatus, and Coccidioides
classofantifungalsthatcouldpotentiallybeusedaloneorincom-
spp. and poor activity (MIC (cid:2) 32 (cid:3)g/ml) against C. albicans,
binationwithexistingantifungaldrugs.
Candidaparapsilosis,andPaecilomycesvariotii(Table1).Inaddi-
RESULTS tion,bothBHBMandD0werehighlysynergisticwhencombined
BHBMandD0inhibitthesynthesisofGlcCer.GlcCerregulates with fluconazole and amphotericin B and additive when com-
thegrowthofC.neoformansinneutralandalkalinepH(14).This binedwithcaspofungin(seeTableS1inthesupplementalmate-
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NewAntifungalsagainstFungalSphingolipids
TABLE1 InvitroantifungalactivitiesofN=-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide(BHBM)determinedbytheMICsagainst
severalfungalclinicalisolatesandreferencestrains
Species(straina)(n) MICrange((cid:3)g/ml)
Cryptococcusneoformans(FTLstrains)(13) 0.25(cid:5)8
Cryptococcusneoformans(MDPLstrainsandSisolates)(8) 0.5(cid:5)2
Cryptococcusneoformans(MDPLstrainsandRisolates)(4) 1(cid:5)2
Cryptococcusgattii(FTLstrains)(10) 0.5(cid:5)2
Cryptococcusgattii(MDPLstrains)(3) 0.5(cid:5)1
Candidaalbicans(FTLstrains)(3) (cid:2)32
Candidaalbicans(MDPLstrains)(5) (cid:2)32
Candidakrusei(MDPLstrain)(1) 32
CandidakruseiATCC6258(MDPLstrain) 16
CandidakruseiQCFTLstrains 8
Candidaglabrata(FTLstrains)(10) 0.125(cid:5)2
D
Candidaglabrata(MDPLstrains)(3) 4(cid:5)(cid:2)32
o
Candidaparapsilosis(MDPLstrains)(3) (cid:2)32 w
CandidaparapsilosisQCFTLstrains (cid:2)16 n
Candidaguilliermondii(FTLstrains)(3) 2(cid:5)(cid:2)16 lo
a
Aspergillusfumigatus(MDPLstrain)(1) 8
d
Aspergillusfumigatus(FTLstrains)(3) (cid:2)16 e
Rhizopusoryzae(FTLstrains)(3) 2 d

Blastomycesdermatitis(FTLstrains)(10) 0.5(cid:5)1 fr
Histoplasmacapsulatum(FTLstrains)(10) 0.125(cid:5)1 om
Coccidioidesspp.(FTLstrains)(10) 8–16

PaecilomycesvariotiiQCFTLstrains (cid:2)16 h
t
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aFTLstrains,strainsfromtheFungusTestingLaboratoryattheUniversityofTexasHealthScienceCenteratSanAntonio;MDPLstrains,strainsfromMaurizioDelPoeta’s p
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laboratory.Sisolates,clinicalisolatessensitivetofluconazole;Risolates,clinicalisolatesresistanttofluconazole;QC,qualitycontrol. //
m
b
io
rial).BHBMwassynergisticandD0wasadditivewhencombined BHBM significantly decreased the intracellular replication of .a
withtunicamycin.Incontrasttofluconazole,C.neoformanscells C. neoformans inside macrophages. This effect was dose depen- s
m
didnotdevelopresistancetoeitherBHBMorD0after15daysof dent,particularlyafter12and24hofincubation(Fig.2C).Drugs .
o
in vitro passages (120 generations) with a population size of showedlittlecytotoxicactivityagainsttheJ774cellswitha50% r
g
106cells/ml(datanotshown). effective concentration (EC ) of 50 (cid:3)g/ml for both drugs (see /
BHBMandD0arefungicidalagainstintracellularC.neofor- Fig.S1inthesupplementalm50aterial),leadingtoafavorableEC / o
50 n
mans. Since both BHBM and D0 exhibited fungicidal activity MIC80selectivityindex(50forBHBMand(cid:2)100forD0).Inad- N
againstC.neoformans,weexaminedtheirkillingactivityusinga dition, toxicity was also tested in A549 and L2 mammalian cell o
time-killassay.BHBMshowedconcentration-dependentkilling lines,andEC sof112(cid:3)g/mland72(cid:3)g/mlwerefoundforthetwo ve
(Fig.2A),whereasD0showedtime-dependentkilling(Fig.2B). celllines,resp50ectively.TheseEC concentrationsdidnotinhibit m
BHBMislessinhibitorytowardGlcCersynthesiscomparedtoD0, mammalian GlcCer synthesis (d5a0ta not shown), suggesting that be
anditisonlyathigherconcentrationsthatitsinhibitoryaction drugtoxicityattheseconcentrationsisnotduetotheinhibitionof r
2
becomes comparable to that of D0 (Fig. 1). Thus, the dose re- mammalianGlcCer. 3
sponseassociatedwithBHBMmightbedirectlyrelatedtoitsabil- BHBMandD0havepotentantifungalactivityagainstcryp- , 2
ity to inhibit GlcCer synthesis, resulting in increased fungicidal tococcosis. The efficacy of BHBM and D0 was tested in mouse 0
1
activitywithincreasedGlcCerinhibition.C.neoformansisafac- modelsofcryptococcosis.Treatmentwasinitiatedthesameday 8
ultative intracellular pathogen and is able to replicate within thatmicewereinfectedintranasallywith5(cid:3)105C.neoformans b
phagocyticcells,suchasmacrophages.Thus,theabilityofBHBM y
cellswithadrugconcentrationof1.2mg/kgofbodyweight/day.
g
tokillphagocytosedC.neoformanscellswasalsoexamined.The Most (90%) BHBM-treated mice and 70% of D0-treated mice u
J774.16macrophagecelllinewasinfectedwithC.neoformansin e
survivedC.neoformansinfectionforupto60days,whereas100% s
thepresenceofopsonins.Theopsoninsandextracellularfungal ofuntreatedmicediedwithin33days(P(cid:4)0.001forbothBHBM- t
cellswerethenwashedoff,andBHBMwasaddedtotestitsactivity
andD0-treatedmiceversusuntreatedmice)(Fig.3A).Thefungal
against intracellular fungal cells. Analysis of CFU revealed that
burdeninthelungsandbrainwerestudiedduringthecourseof
infection.Drugtreatmentsignificantlyreducedthefungalburden
inbothorgans,andnocellsweredetectedinthebraintissueatthe
TABLE2 InvitroantifungalactivitiesofBHBMdeterminedbythe
endofthestudy(seeFig.S2inthesupplementalmaterial).Itis
percentageinhibitionofATP(IC )againstPneumocystismurinaand
50 possiblethatthedrugswereabletoactinthelungsortheblood-
Pneumocystisjiroveciia
streamandpreventfungaldisseminationtothebrain.However,
BMBMIC ((cid:3)g/ml)afterexposurefor:
50 thetotalabsenceoffungalcellsinthebrainatlatertimepoints
Pneumocystisisolates 24h 48h 72h 96h (Fig.S2B)suggeststhatperhapsthedrugsmayactdirectlyinthe
P.murinaHAisolates 1.02 (cid:4)0.01 (cid:4)0.01 2.02 braintissue.Drugsdidnotshowsignificantinvivotoxicity.Blood
P.jiroveciiVMisolates 0.912 0.159 0.074 0.072 workofBHBM-andD0-treatedmiceafter60daysoftreatment
aIC ,50%inhibitoryconcentration. showedaslightbutinsignificantincreaseofliveraspartateamino-
50
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FIG2 KillingactivityofBHBMandD0.KillingactivitywasdeterminedusinganinvitrokillingassayinwhichthecompoundswereaddedtoC.neoformanscells, p
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whichwerethenincubatedat37°C,5%CO2,andpH7.4.ThenumberofCFUwerecountedduringthe96-hincubation.(AandB)BothBHBM(A)andD0(B) //m
showedfungicidalactivity.BHBMshowedconcentration-dependentkilling,whereasD0showedtime-dependentkilling.D0ismoreeffectiveinkillingC.neo-
formans(100%deadcellswithin24h),andthekillingactivitydoesnotoccurearlierthan24hwithhigherdoses.BHBMkillsmoreslowly,requiringatleast72h b
ofincubationtokillabout50%ofthecells.Valuesthataresignificantlydifferentareindicatedasfollows:*,P(cid:4)0.05comparingtreatedcellsversusuntreatedcells io
(nodrug);#,P(cid:4)0.001comparingtreatedcellsversusuntreatedcells(nodrug).(C)IntracellularactivityofBHBMwasassessedbyincubatingmacrophageswith .a
s
internalizedC.neoformanscellswithdifferentconcentrationsofBHBMintheabsenceofopsonins.Valuesthataresignificantlydifferentareindicatedasfollows: m
*,P(cid:4)0.05comparingextracellularorintracellulartreated(0.25,1,or4(cid:3)g/ml)versusextracellularorintracellularuntreated(0(cid:3)g/ml),respectively.Statistical
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analysiswasperformedusinganalysisofvariance(ANOVA).Datawerecompiledfromthreeindependentexperiments. o
r
g
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transferase(AST)(125(cid:6)75U/liter)inBHBM-treatedmicecom- mice) and 10.5 (cid:6) 4.2 days (P (cid:4) 0.05 versus untreated mice) n

paredtountreatedmice(50(cid:6)9U/liter).Allotherbloodparam- postinfection,respectively.Thiswassimilartothesurvivalpattern N
o
eters for liver and kidney function were normal, as were the observedwithfluconazole,withanaveragesurvivalof10(cid:6)2days v
numberoferythrocytes,thrombocytes,andleukocytes. postinfection(P(cid:4)0.05versusuntreatedmice).Micetreatedwith em
PharmacokineticstudiesfollowingBHBMtreatmentwereper- amphotericin B succumbed at an average of 23.5 (cid:6) 6.5 days b
formedinimmunocompetentcontrolhealthymiceandimmuno- postinfection(P(cid:4)0.005versusuntreatedmice). e
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suppressed/infected (IS/IN) mice. In immunocompetent mice, BHBMandD0haveantifungalactivityagainstpneumocys- 2
3
thehalf-lifeofhigh-doseintravenous(i.v.)BHBMwas1.03h.The tosis.BHBMshowedmarkedinvitroactivityagainstP.murina ,

half-livesofBHBMafterlow-doseandhigh-doseadministration andP.jirovecii.Therefore,itsinvivoactivityinmurinemodelsof 2
0
were1.70and1.43h,respectively(seeTableS2inthesupplemen- pneumocystosis was tested. Since establishment of lethal pneu- 1
8
talmaterial).Ininfectedmice,thehalf-livesofBHBMafterlow- mocystosisinmurinemodelsrequiresimmunosuppression,mice

b
doseandhigh-doseadministrationwere0.79hand0.84h,respec- weretreatedwithcorticosteroidspriortoinfection.Fourexperi- y
tively. The changes in the half-lives of the drugs between mental groups of mice were included: (i) infected mice treated g
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immunocompetentandimmunosuppressed/infectedanimalsare with vehicle (negative control), (ii) infected mice treated with e
duetointeractionsbetweenthedrugsandcorticosteroidsusedfor trimethoprim-sulfamethoxazole(T/S),(iii)infectedmicetreated s
t
immunosuppression;wehavefoundthatthesedrugsdonotin- dailywithBHBM(3.2mg/kg),and(iv)infectedmicetreatedwith
teract with another immunosuppressant, cyclophosphamide D0 (1.25 mg/kg/day). Mice treated with 3.2 mg/kg BHBM sur-
(datanotshown). vived significantly longer than untreated mice did (P (cid:4) 0.05)
Asecondmousesurvivalstudywasperformedtotesttheeffi- (Fig.3C).D0appearedtodisplaysometoxicityinmiceimmuno-
cacyofBHBMandD0againstinvasivecryptococcalinfectionof suppressedwithcorticosteroids.Thus,CD4(cid:7)celldepletionwas
thecentralnervoussystem(CNS).Fluconazoleandamphotericin usedtoinduceimmunosuppressioninstudiesofD0againstpneu-
Bwerealsoincludedinthesestudiestocomparetheefficaciesof mocystosis.Inthismodel,1.25mg/kg/dayofD0significantlyim-
thesecompoundstothoseofcommonlyuseddrugs.Allmicein proved survival (P (cid:4) 0.05) (Fig. 3C) with no apparent signs of
the vehicle-treated control group succumbed 7.0 (cid:6) 1.1 days toxicity.Nosignificantdifferenceswereseeninthenumberofasci
postinfection. All drugs prolonged survival significantly com- ornucleiinlunghomogenatesofthevehicle-treatednegativecon-
pared with the control, although none of the drugs completely trol versus the BHBM treatment groups or the D0 treatment
protected the mice from infection (Fig. 3B). BHBM- and D0- groups(seeFig.S2CtoFinthesupplementalmaterial).Therewas
treatedmicesuccumbed11.1(cid:6)3.7(P(cid:4)0.05versusuntreated a significant reduction in both asci and nuclei count following
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FIG3 EffectsofBHBMandD0onsurvivalofmiceuponinfectionwithC.neoformans,P.murina,orC.albicans.(A)Cryptococcosis.Micewereinfected m
intranasallywithC.neoformans(Cn)andreceived1.2mg/kg/dayofeitherBHBMorD0intraperitoneally(10micepergroup).Valuesthataresignificantly b
differentareindicatedasfollows:*,P(cid:4)0.001comparingBHBM-orD0-treatedmiceversusuntreatedmice(nodrug).(B)Cryptococcosis.Survivalofmice io
infectedintravenouslywithC.neoformans(8micepergroup)andtreatedintraperitoneallyeitherwithBHBM,D0,fluconazole(FLC),oramphotericinB(AMB). .a
Valuesthataresignificantlydifferentareindicatedasfollows:^,P(cid:4)0.05comparingBHBM-orD0-treatedmiceversusuntreatedmi8ce(solvent);$,P(cid:4)0.005 s
m
comparing FLU-treated mice versus untreated mice and AMB-treated mice versus untreated mice. (C) Pneumocystosis. Survival of corticosteroid-
.
immunosuppressed(Cort)orCD4-depleted(CD4d)miceinfectedintranasallywithP.murinaafter13daysofintraperitonealtreatmentwithBHBM(3.2mg/ o
kg/day),D0(1.25mg/kg/day),ortrimethoprim-sulfamethoxazole(T/S)(12micepergroup).Valuesthataresignificantlydifferentareindicatedasfollows:&, rg
P(cid:4)0.05comparingBHBM-orD0-treatedmiceversusrespectiveuntreatedcontrols.(D)Candidiasis.SurvivalofmiceinfectedwithC.albicansSC5314(Ca) /
o
intravenouslyreceivednodrugor1.2mg/kg/dayofeitherBHBMorD0intraperitoneally(8micepergroup).Valuesthataresignificantlydifferentareindicated
n
asfollows:§,P(cid:4)0.01comparingBHBM-orD0-treatedmiceversusuntreatedmice.StatisticalanalysisforsurvivalstudieswasperformedusingKruskal-Wallis
N
testandbyStudent-Newman-KeulsttestformultiplecomparisonsusingINSTAT.
o
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m
13daysofT/Streatmentversusthenegativecontrolandversus GlcCerlevelsandincreaseddihydroceramide,sphingosine,and b
BHBMgroups(Fig.S2Cand S2D). sphingosine-1-phosphate (Fig. 4a). These changes were con- e
r
BHBMandD0haveantifungalactivityagainstinvasivecan- firmedbymassspectrometry(Fig.4btoe).Interestingly,thelevels 2
3
didiasis.MostCandidaspp.examinedwereresistantinvitroto ofC18hydroxyceramide,theceramidespeciesthatisadirectpre- ,

bothBHBMandD0.However,previousstudieshaveshownthat cursorofGlcCer,anditscorrespondingdesaturatedandmethyl- 2
0
GlcCerisrequiredforvirulencethroughamechanismotherthan atedformsdecreaseduponBHBM(Fig.4FtoH)orD0(datanot 1
8
facilitatinggrowthatneutral/alkalinepH(8),whichisthepHused shown) treatment, confirming a dysregulation of the ceramide

b
inourlibraryscreening.InvitrotreatmentofC.albicanscellswith synthesisupontreatmentwithBHBM.Nosignificantchangesin y
BHBMinhibitedGlcCersynthesisinadose-dependentmanner GlcCer levels were observed in mammalian cells after BHBM g
u
(datanotshown).Thus,theefficacyofBHBMorD0againstin- treatmentatvarioustimepoints(seeFig.S3inthesupplemental e
vasivecandidiasiswasevaluated.Invasivecandidiasiswasestab- material). s
t
lishedinCBA/Jmicebyintravenousinjectionwithalethaldoseof BHBMaffectsfungalcellmorphologyandvesicularstruc-
105C.albicanscells.Treatmentstartedthesamedaywitheither ture.TherelationshipbetweenlipidcompositionandGolgiarchi-
BHBMorD0usingthesamedoseregimensusedtotreatcrypto- tectureiswellestablished(15,16).Thus,wesetouttoevaluatethe
coccosis.After21daysofinfection,75%ofmicetreatedwithD0 effect of BHBM treatment on the Golgi accumulation of NBD
and62.5%ofmicetreatedwithBHBMwerestillalive,whereasall C6-ceramide {N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-
untreatedmicehadsuccumbedbyday13(Fig.3D). aminocaproyl-D-erythro-sphingosine}, indicating altered Golgi
BHBMandD0targetmetabolismand/ortransportofcertain function.TheperipheraldistributionreportedforC.neoformans
ceramidespeciesusedforsynthesisofGlcCer.Sincetheinitial wasobservedforNBDC6-ceramidestainingincontrolcells;how-
screeninghaddemonstratedthatBHBMandD0affectthesynthe- ever,asubstantialaccumulationofthiscompoundwasdetectedin
sisofGlcCer,biochemicalstudieswereperformedbythin-layer BHBM-treatedyeastsofC.neoformansasobservedbymicroscopy
chromatography(TLC)andmassspectrometrytounderstand andconfirmedbyfluorescence-activatedcellsorting(FACS)anal-
theeffectsofdrugsonthesphingolipidpathway.Lipidprofil- ysis (Fig. 5A and B). The majority of BHBM-treated yeasts dis-
ingbyTLCrevealedthatBHBMtreatmentledtoadecreasein playedaringaccumulationofNBDC6-ceramide.Similarresults
May/June2015 Volume6 Issue3 e00647-15 ® mbio.asm.org 5
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FIG4 MeasurementsofsphingolipidsupontreatmentwithBHBM.(a)Thin-layerchromatographyanalysisofsphingolipids(seediagram)isolatedfrom //
untreatedorBHBM-treatedC.neoformanscellsafterinvivolabelingwith[3H]palmitate(3H-Palm).(btoh)LipidanalysisbyLC-MS.(b)C18hydroxy((cid:8)8)9 m
b
mdihetyhdyrlo-gspluhcionsgyolcsienrea-m1-idpeho(GsplchCateer)(;S(PcH)C-11-8Pd);ih(fy)dCro18cehryamdriodxey(cCer1a8mdihdCee(Cr)1;8(dO)Hsp-hCinerg)o;s(ign)eCa1n8dhdyidhryodxryos(cid:8)p8h-icnegroasminidee(S(CPH18s)O;(He)-(cid:8)sp8h-Cinegro);sianned-1(-hp)hCo1s8phhaytderaonxdy io
.
(cid:8)8,9methyl-ceramide(C18OH-(cid:8)8,9Me-Cer).Valuesthataresignificantlydifferentareindicatedasfollows:*,P(cid:4)0.05comparingtreatedtountreated(no a
drug).Statisticalanalysiswasperformedusinganalysisofvariance(ANOVA)test.StatisticalsignificanceisacceptedataPvalueof(cid:4)0.05.Datawerecompiled sm
fromthreeindependentexperiments. .
o
r
g
/
wereobservedinC.albicans,confirminganaltereddistributionof romyces cerevisiae HIP-HOP (haploinsufficiency profiling- o
NBD C6-ceramide after BHBM in hyphal forms. In addition, homozygous deletion profiling) assay comprising of essential n

N
BHBMtreatmentsignificantlyreducedtheyeast-to-hyphadiffer- genesasheterozygousdiploidsandnonessentialgenesashomozy-
o
entiationinC.albicans(Fig.5A).Thisdefectinchangeofmor- gous diploids (18) was screened to find the potential pathways v
e
phology might explain the loss of virulence in candidiasis affectedbythedrug.Ofthisgenome-widecollectionof5,900mu- m
(Fig.3D),asCandidaspeciesthatarelockedintheyeastformare tants,23were(cid:2)2-foldmoresensitivetoBHBMthanthevehicle- b
knowntolosevirulence(17).Additionalfluorescentimagesare treated control (see Table S3 in the supplemental material). Of e
r
showninFig.S4inthesupplementalmaterial. these23genes,8werenotconsideredfurtherbecausetheycorre- 2
3
Electronmicroscopyexaminationofdrug-treatedcellsshowed spondedtodubiousopenreadingframesorlackedanyhomology ,

anaccumulationoflargevesiclesinsidethecells(Fig.6A),while to C. neoformans. Among the remaining 15 significant sensitive 2
0
controlcellsmaintainedaregularorganizationandstructureof genes, 9 genes (RET2, UBP3, SEC26, PEP7, SEC31, YML018C, 1
8
intracellular vesicles (Fig. 6B). Vesicle accumulation in drug- SNF8,GOS1,andRET3)areinvolvedintheregulationofvesicle

b
treatedcellswasobservedassinglevesicles(Fig.6AandD,arrows) sorting or transport between Golgi apparatus and endoplasmic y
and fused vesicles (Fig. 6A and D, arrowheads). Morphometric reticulum(ER).Thiswasinagreementwiththeobservationsof g
analysisrevealedthattherewasasignificantincrease(P(cid:4)0.05)in fungalcellmorphologyandvesicularstructureafterdrugtreat- ue
the volume occupied by the intracellular vesicles in BHBM- ment(Fig.5and6).Thesefindingswerealsoconsistentwiththe s
t
treatedcells(Fig.6C).Thenumbersofvesiclespersquaremicro- decreaseofGlcCerlevelsuponBHBMtreatment,asvesicletrans-
meterofcellsurfaceareawere0.84(cid:6)0.58inBHBM-treatedcells port is the main mechanism by which ceramide is transported
and0.93(cid:6)0.54inD0-treatedcellscomparedto0.33(cid:6)0.36in betweentheERandGolgiapparatusforthesynthesisofGlcCer
untreatedcells.Theincreaseinbothvolumeandnumberofintra- andothercomplexsphingolipids(19–21).Thesegenesarecon-
cellularvesiclessuggestsdefectsinsecretionprocessesinducedby served in C. neoformans, but they have not been characterized
thedrug(Fig.6C).Inaddition,plasmamembrane-limitedstruc- experimentally. The other six genes detected in the HIP-HOP
tures, which seemed to accumulate portions of the cytoplasm, screen (MTW1, REG1, SPT4, SAP4, GLC7, and ACT1) regulate
were frequently seen accumulated in the periplasmic space chromatin organization, cell cycle progression, or cell division,
(Fig. 6E). Transmission electron microscopy (TEM) images of functionsthatareknowntoberegulatedbyGlcCer(14,22).
D0-treatedcellsandadditionalimagesofBHBM-treatedcellsare TopinpointthecellulartargetsofBHBM,asecondapproach,
presentedinFig.S5inthesupplementalmaterial. generationofBHBM-resistantmutants,wasfollowed.Thedrug-
CellulartargetsofBHBM.Twoapproacheswereusedtofind sensitiveS.cerevisiaeRYO0622strainwasusedforthegeneration
thepotentialtarget(s)ofBHBM.Inthefirstapproach,aSaccha- ofmutants(23).Aprescreeningstudyexposingthisstraintovar-
6 ® mbio.asm.org May/June2015 Volume6 Issue3 e00647-15
NewAntifungalsagainstFungalSphingolipids
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m
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a
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FIG5 EffectofBHBMonGolgimorphologyinC.neoformansandyeast-to-hyphaldifferentiationinC.albicans.(A)ControlorBHBM-treatedcellswere m
stained with NBD C6-ceramide (Golgi and Golgi-derived compartments, green fluorescence). DIC, differential interference contrast; NBD-Cer, NBD .o
6-ceramide.Bars,5(cid:3)m.(B)FACSanalysisshowsNBDC6-ceramideaccumulationinC.neoformans.ThegrayhistogramcorrespondstoC.neoformansyeastin r
g
theabsenceofNBDC6-ceramide.CT,control(noBHBMtreatment).(C)PercentageofyeastandhyphalformsundercontrolconditionsandafterBHBM /
treatment.Statisticalanalysiswasperformedusinganalysisofvariance(ANOVA)test.StatisticalsignificanceisacceptedataPvalueof(cid:4)0.05.Theimagesin o
panelsAandBarerepresentativeofthreeseparateexperiments.ThedatainpanelCwerecompiledfromthreeindependentexperiments. n

N
o
v
e
iousconcentrationsofBHBMrevealedthatadrugconcentration sistanceofthesemutantstoBHBMtreatmentconfirmsthatthe m
of133(cid:3)g/mlcompletelyinhibitedyeastgrowth(100%inhibitory above-mentionedgenesareindeedthetargetsofBHBM,andthe b
concentration [IC ]). Incubating 106 cells of S. cerevisiae absenceofthesetargetsimpairsthekillingactivityofthedrug.Itis e
100 r
RYO0622strainwithBHBMattheIC resultedinsevenresis- worthnotingthatdeep-sequencinganalysisalsorevealedthepres- 2
100 3
tant colonies, the genomes of which were sequenced and com- enceofmutationsinanothergene,SLA2,thatisalsoinvolvedin ,
paredwiththeNCBIsacCer3referencegenomeusingtheGenome vesiculartransport(29).Althoughthe(cid:8)sla2mutantwasnotre- 2
0
AnalysisToolkit(GATK).Thisanalysisledtotheidentificationof sistanttoBHBM,itremainstobedeterminedwhetherpointmu- 1
8
mutationsinfourgenes,APL5,COS111,MKK1,andSTE2,that tationswithintheSLA2openreadingframedoinfactconferre-

b
were present in all resistant mutants. The proteins encoded by sistance. y
theselociareknowntobeinvolvedinvesicletrafficking,budding, g
andcellcycleprogression(24–27),againinagreementwiththe DISCUSSION ue
observations of cell morphology and known phenotypes in the Inthisstudy,wehaveidentifiedandcharacterizedanewclassof s
t
absenceofGlcCer(14,22).Thesefindingsalsocloselymatchthe antifungalcompounds,thehydrazycins(specificallyBHBMand
pathwayproposedbytheHIP-HOPanalysis.Interestingly,these D0),whichdecreasethesynthesisoffungal,butnotmammalian,
fourgeneseachinteractwithUBI4whichencodesubiquitinand GlcCer.Thesecompoundswereeffectiveagainstcryptococcosis,
which is conjugated to proteins to target them for degradation. candidiasis,andpneumocystosisinanimalmodelsandtargetfour
UBI4isamemberoftheendomembranerecyclingpathwaysas genesinvolvedinvesiculartraffickingandcellbudding.Thecom-
definedbyFinleyetal.(28). poundswerewelltoleratedbyanimalsandcanpotentiallypave
Toconfirmdrugresistance,individualmutantsalongwiththe thewayforthedevelopmentofanewclassofantifungals.
parent strain were grown in the presence of various concentra- BothBHBMandD0areacylhydrazonecompoundswiththe
tions of BHBM. Various concentrations of fluconazole and structureR R C(cid:9)NNH (Fig.1)(30–32).Hydrazonemolecules
1 2 2
methylmethanesulfonate(MMS)wereusedascontrols(Fig.7). havebeenshowntoexertdiversebiologicalfunctions,including
AllindividualmutantsshowedincreasedresistancetoBHBMin antibacterial, antifungal, antiviral, antiparasitic, anticancer, and
the range of 11 to 92 (cid:3)g/ml (Fig. 7), while all mutants showed antidepressant effects (33–36), but these activities are linked to
similarsusceptibilitytofluconazoleandMMS.Theincreasedre- specific structures of hydrazones, which we can readily modify
May/June2015 Volume6 Issue3 e00647-15 ® mbio.asm.org 7
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FIG6 Transmissionelectronmicroscopyexaminationofhigh-pressurefrozenandfreezesubstitutedC.neoformansyeastcells.(A,D,andE)Cellstreatedwith m
4(cid:3)g/mlofBHBMfor6h,showingaccumulationofintracellularvesicles(arrows)andregionscontaininga“reticulum”presumablyformedbythefusionof b
intracellularvesicles(arrowheads).M,mitochondrion.(B)Controlcellsshowingaregularaspect.(C)Morphometricanalysisresultsshowingthevolumetric io
densityoftheintracellularvesiclesincontrolandtreatedcells.(E)Accumulationofcytoplasm-richvesiclesintheperiplasmicspace.Imagesanddataare .a
representativeoftheresultsofthreeseparateexperiments. s
m
.
o
r
g
(datanotshown),potentiallyincreasingtheirantifungalactivity. phage cell line (see Fig. S1 in the supplemental material) and a /

Although previous literature exists on antimicrobial activity of goodselectivityindex(EC /MICratio(cid:2)50),whichispromising o
50 n
hydrazonecompounds,thisisthefirsttimethattheabilityofthese fortheiruseasantifungals. N
compoundstoblockfungalGlcCerhasbeendiscovered. BHBMandD0wereeffectiveagainstavarietyoffungalinfec- o
v
BHBMandD0exhibitedantifungalactivityagainstavarietyof tionsinvivo.Inmousemodelsofcryptococcosis,thedrugssignif- e
clinically relevant fungi (Table 1) and acted synergistically with icantly increased the survival of mice that were infected with a m
b
currentlyavailableantifungaldrugs(seeTableS1inthesupple- lethal dose of C. neoformans via the intranasal route (Fig. 3A). e
mentalmaterial).Theabilityofthesecompoundstoexertfungi- Analysisoffungalburdendemonstratedthatdrugtreatmentre- r
2
cidalactivityonphagocytosedC.neoformanscellsisparticularly duced the number of fungal cells in the brain throughout the 3
important(Fig.2).C.neoformansisabletosurviveandreplicate courseofthetreatment,leadingtotheabsenceoffungalcellsinthe ,
2
inside macrophages (37), and replication inside nonactivated brainafter60days(seeFig.S2inthesupplementalmaterial).This 0
1
macrophagesisanimportantfactorinitspathogenicity(38).Fur- observationindicatesthatdrugswereabletoreachthebrainand 8
thermore,BHBMandD0exhibitedlowcytotoxicityinamacro- cleartheinfection.Thishypothesisissupportedbytheobserva- b
y

g
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s
t
FIG7 EffectsofBHBM,fluconazole,andmethylmethanesulfonate(MMS)onwild-typeBY4741and(cid:8)apl5,(cid:8)cos111,(cid:8)mkk1,and(cid:8)ste2deletionstrains.
RelativegrowthinhibitionwascalculatedbytheaveragerateafternormalizingtheOD valuesindrug-treatedwellsagainsttheDMSOcontrolwellsoneach
600
assayplate.ThemutantstrainsshowincreasedresistancetoBHBMbutnottofluconazoleorMMS.Resultsarefromtwoindependentgrowthassays.
8 ® mbio.asm.org May/June2015 Volume6 Issue3 e00647-15
NewAntifungalsagainstFungalSphingolipids
tionthatBHBMisreadilyfoundinbraintissueuponadministra- difference in resistance to the unrelated drugs fluconazole or
tion(datanotshown),andthistissuedistributionmayexplainthe MMS(Fig.7),confirmingthatthesegenesencodeproteinsthat
lowserumhalf-lifeandtheeffectoncryptococcalmeningitis.In haveacriticalroleinthecellularresponsetoBHBM.Ofthese4
anaggressivemodelofcryptococcosis,inwhichtheinfectionwas genes,APL5playsanimportantroleinvesiculartransportandhas
establishedviatailveininjection,thedrugsperformedsimilarlyto beenshowntobeinvolvedincargoinclusionintovesiclesduring
fluconazoleinprolongingsurvivalinourmurinemodel(Fig.3B). thesecretoryandendocyticpathways(44,45).Furthermore,the
Theconcentrationoffluconazolewasalmosteighttimeshigher APL5proteindirectlyinteractswiththeVPS3andGOS1-encoded
thanthatofBHBMandD0,suggestingthatthesedrugscouldbe proteins,whichwereidentifiedbytheHIP-HOPassay(46,47).
more effective than fluconazole. While amphotericin B was the Similarly,MKK1directlyinteractswithRET2andRET3identified
mosteffectivedruginthismodeofinfection,thisdrugisassoci- bytheHIP-HOPassay(48).COS111andSTE2mayalsointeract
atedwithdrawbackssuchasadversereactionsandnephrotoxicity withgenesidentifiedbytheHIP-HOPassayinaprocessmediated
(39,40).BHBMandD0arewelltoleratedbyanimals;thus,itis byubiquitin(49–52).Infact,oneofthegenesidentifiedbythe
possibletoaugmenttheirdoseregimenforimprovedantifungal HIP-HOPassaytobehighlyupregulatedisUBP3,whichencodes
D
effect. aubiquitin-specificproteaseworkingtogetherwithUBI4toreg- o
Inadditiontocryptococcosis,bothdrugswereeffectiveinin ulate protein degradation and endocytosis. In fact, UBI4 physi- w
n
vivoinfectionmodelsofpneumocystosisandcandidiasis(Fig.3C callyinteractswithallthegenesidentifiedbytheHIP-HOPassay lo
andD).Inthecaseofpneumocytosis,BHBMwasabletoincrease exceptSPT4,SAP4,SNF8,andVPS3.UBI4alsointeractswithall a
d
survivalincorticosteroid-treatedmice,whileD0waseffectivein proteinsidentifiedbythedeep-sequencinganalysis.UBP3isalso e
improvingthesurvivalofCD4-depletedmice.AlthoughT/Swas essentialfortheregulationoftheCOPIsystem(encodedbyRET2, d

themosteffectivedrug,thisdrughassignificantsideeffects,and RET3, SEC26, and SEC31). Thus, UBI4 may be involved in the fr
o
relapseandrecurrenceofinfectionsareoftenobservedwhenus- regulationofendocytosisandvesicletransportbyinteractingwith m
ingsecondarytherapies(41).Thus,BHBMandD0canpotentially RET2,RET3,UBP3,REG1,SEC26,SEC31,andPEP7,allofwhich h
provide new options for pneumocystosis treatment. Previous aregenesidentifiedbytheHIP-HOPanalysis.UBImaybeahub tt
p
studieshaveshownantifungalactivityoffluconazoleagainstC.al- betweentheproteinsidentifiedbythedeep-sequencinganalysis :
/
/
bicansinvivodespiteinvitroresistance(42).Asimilarphenome- andthoseidentifiedbytheHIP-HOPassay.Vesiculartransportis m
nonwasobservedwithourdrugcompounds.Ithasbeenshown themainmechanismbywhichceramideistransportedfromthe b
that GlcCer-deficient mutants in C. albicans lack the ability to ERtotheGolgiapparatusforthesynthesisofGlcCer,whichin io
.
a
causevirulenceinthemousemodeldespiteshowingnogrowth turnregulatescellcycleprogression(5,9,12).Theresultsofdeep- s
defectinvitro(8).Thus,inhibitionofGlcCerbyBHBMandD0is sequencing analysis suggest that the drug compounds inhibit a m
.
thelikelymechanismforpartiallyprotectingthemiceagainstin- proteinproductintheendocytic/vesicletransportpathwaythat o
r
vasivecandidiasis. leadstotheformationofGlcCer;thiswillstripthedrugofitsmain g
/
BothBHBMandD0inhibitthesynthesisofGlcCerandsignif- target,leadingtoincreasedresistance. o
icantlyaffectthelevelsofvarioussphingolipids(Fig.4).However, Insummary,wehaveidentifiedhydrazonemolecules(BHBM n

N
GlcCerisnottheonlytargetofthesedrugs.LossofGlcCerhasa andD0)thatexhibitpotentantifungalactivitythroughaspecific
o
static effect on fungal growth (6), whereas BHBM and D0 are inhibitionoffungalGlcCer.Thesemoleculesexertedantifungal v
e
fungicidal.Interestingly,sphingosinespeciesaccumulateinfungal activity in the mouse models of cryptococcosis, pneumocystis, m
cellsuponBHBMorD0treatment(Fig.4),aphenomenonthatis andcandidiasisandwerewelltoleratedbyanimals.BHBMandD0 b
notobservedwhentheglucosylceramidesynthasegene(GCS1)is also affected fungal cell morphology and vesicular trafficking. e
r
deleted(5),andthesesphingolipidsarehighlytoxic.Thesesphin- Thesemoleculestargetfourgenesthatareinvolvedinvesicular 2
3
golipids do not significantly accumulate in mammalian cells secretionandcellcycleprogression.Tothebestofourknowledge, ,

treatedwithBHBMorD0(seeFig.S3inthesupplementalmate- thisstudymarksthefirstdrugsthatactbasedontheinhibitionof 2
0
rial),andthismayalsoexplainthelackoftoxicityinthissystem. fungalGlcCer.Giventheantifungalpotencyofthesedrugs,fur- 1
8
TheresultsofHIP-HOPanalysisprovidedanindicationthat therstudiesarewarrantedfortheirpreclinicalandeventuallyclin-

b
thedrugsaffectthegenesinvolvedinvesiculartransport,cellbud- icaldevelopment. y
ding,andcellcycleprogression.Ofthe23genesidentifiedinthis g
u
analysis, 9 were involved in the regulation of vesicle sorting or MATERIALSANDMETHODS e
transportbetweenGolgiapparatusandER,andendocytosisand s
Strains,media,andreagents.Aseriesoffungalclinicalisolatesandref- t
another6geneswereinvolvedincellbuddingandcellcyclepro- erencestrainswereusedinthisstudy,includingCryptococcusneoformans
gression(seeTableS3inthesupplementalmaterial).Theseobser- (strainH99),Cryptococcusgattii,Candidaalbicans,Candidakrusei,Can-
vationssuggestedthatvesiculartransport,celldivision,andcell didaglabrata,Candidaparapsilosis,Candidaguilliermondii,Aspergillusfu-
cycleprogressionaretargetedbythedrugs.Thisprovidedapo- migatus,Rhizopusoryzae,Blastomycesdermatitis,Histoplasmacapsulatum,
tentialtargetpathway,whichwassupportedbytheelectronmi- Coccidioidesspp.,Paecilomycesvariotii,Pneumocystismurina,Pneumocys-
croscopyimagesshowinganaccumulationofintracellularvesicles tis jirovecii, and Saccharomyces cerevisiae. These strains were obtained
fromexistingcollectionsattheFungalTestingLaboratory(Universityof
(Fig.6).
TexasHealthScienceCenteratSanAntonio,SanAntonio),DelPoeta’s
Further understanding of the drug targets was provided by
laboratory(StonyBrookUniversity),Cushion’slaboratory(Universityof
generation of mutants resistant to BHBM. Mutations in four
Cincinnati),andNislow’slaboratory(UniversityofBritishColumbia).
genes,APL5,COS111,MKK1,andSTE2,werefoundinallseven
Yeastpeptonedextrose(YPD),yeastnitrogenbase(YNB),Luria-Bertani
coloniesthatshowedresistancetoBHBM.Thecorrespondingsin- (LB), Roswell Park Memorial Institute (RPMI 1640), and Dulbecco’s
gledeletionmutantsofeachofthesegenes(fromtheyeastdeletion modifiedEagle’smedium(DMEM)werepurchasedfromInvitrogenLife
collections [43]) showed increased resistance to BHBM, but no Technologies(GrandIsland,NY).Fluconazole,amphotericinB,dexa-
May/June2015 Volume6 Issue3 e00647-15 ® mbio.asm.org 9
Moretal.
methasone, cyclophosphamide, and tunicamycin were purchased anddissolvedin2mlmethanoland1mlchloroform.Lipidswereex-
fromSigma-Aldrich(St.Louis,MO).Caspofunginandposaconazole tractedbythemethodofBlighandDyer(54)followedbybasehydrolysis.
wereobtainedfromMerck(Rahway,NJ).Voriconazolewasobtained LipidsamplesweredriedinaSpeedVacvacuumconcentrator,suspended
fromPfizer(ReyBrook,NY).N=-(3-bromo-4-hydroxybenzylidene)-2- in30(cid:3)lof1:1methanol-chloroform,andloadedonaTLCplatewith
methylbenzohydrazide (BHBM) and 3-bromo-N=-(3-bromo-4- GlcCerasthestandard.
hydroxybenzylidene)benzohydrazide(D0)wereobtainedfromChem- Invitrosusceptibilitytesting.MICsweredeterminedfollowingthe
Bridge (San Diego, CA). Cryptococcal capsular monoclonal antibody methodsoftheClinicalandLaboratoryStandardsInstitutes(CLSI)with
MAb18B7wasagiftfromArturoCasadevall’slaboratory(JohnsHopkins modifications.RPMI1640orYNBmedium(pH7.0,0.2%glucose)buff-
University). eredwithHEPESwasusedforMICstudies.HEPESwasusedinsteadof
Library screening. The DIVERSet-CL library was obtained from morpholinepropanesulfonicacid(MOPS),becauseMOPSwasfoundto
ChemBridge(SanDiego,CA)ina96-wellplateformatandcontained inhibittheactivityofBHBMandD0.BHBMandD0wereseriallydiluted
10mMcompoundperwellin100%dimethylsulfoxide(DMSO).Ineach from32to0.03(cid:3)g/mlor19to0.02(cid:3)g/ml,respectively,ina96-wellplate
well,10compoundsweremixedtogether.Thecompoundswerefirstdi- withtherespectivemedium.Theinoculumwaspreparedasdescribedin
lutedto1mMeach(1:10dilutionwith10%DMSO)withYNBmedium theCLSIprotocolM27-A3guidelines(55).Theplateswereincubatedat
D
bufferedwithHEPESatpH7.4containing2%glucoseandsubsequently 37°Cwith5%CO2for24to96h.TheMICsweredeterminedasthelowest o
dilutedto300(cid:3)M(1:3.3dilution)withthesamemedium(3%DMSO).A concentrationofthedrugthatinhibited50%ofgrowthcomparedtothe w
100-(cid:3)laliquotofthissolutionwasplacedintoeachwellofa96-wellplate control.Theminimumfungicidalconcentration(MFC)wasalsodeter- n
andstoredat(cid:5)20°Cuntiluse.Then,4(cid:3)104C.neoformansH99cellsin minedbytaking100(cid:3)lofserialdilutionsfromeachwell,spreadingiton lo
a
100(cid:3)lofYNBmediumbufferedatpH7.4withHEPESwereaddedtoeach YPDagarplates,andcountingtheCFU.MFCwasdefinedasthelowest d
well. The final concentration of the tested drugs was 150 (cid:3)M in YNB drugconcentrationthatyieldedthreeorfewercolonies(i.e.,99%ofthe e
d
mediumcontaining1.5%DMSO.Theplateswereincubatedat37°Cin inoculumwaskilled).
f
r
thepresenceof5%CO for48h.Theopticaldensityat495nm(OD ) Invitrokillingassay.C.neoformanscellsfromaculturegrownover- o
2 495 m
wasrecordedusingtheFilterMax5multimodemicroplatereader(Mo- nightwerewashedinPBSandresuspendedinYNBbufferedwithHEPES
lecularDevices,Sunnyvale,CA).CompoundcocktailsshowinganODof atpH7.4.Thecellswerecounted,and2(cid:3)104cellswereincubatedwith1, h
(cid:4)80%comparedtotheODinthecontrolwell(1.5%DMSObutnodrug) 2,or4(cid:3)g/mlofBHBMorD0inafinalvolumeof10mlwithafinal tt
p
wereselectedforfurtherstudies. concentrationof0.4%DMSO.Thetubeswerethenincubatedat37°C :
/
/
Labeling of fungal cells with tritiated palmitate ([3H]palmitate). with5%CO onarotaryshakerat200rpm.Aliquotsweretakenattime m
2
C.neoformanscellsweregrowninYNB(pH7.4)at37°Cinthepresenceof pointsanddiluted,and100-(cid:3)lportionswereplatedontoYPDplates. b
5%CO for16h.Thecellswerecentrifugedfor10minat3,000rpmat YPDplateswereincubatedina30°Cincubatorandafter72h,thenum- io
2 .
roomtemperature.Supernatantwasremoved,andthecellpelletwassus- bersofCFUwerecountedandrecorded. a
pendedandcounted.Next,900(cid:3)lcontaining5(cid:3)108C.neoformanscells InvitrotestingagainstP.murinaandP.jirovecii.Acharacterized sm
wasplacedintoa15-mlround-bottomCorningcentrifugetube.Then, P.cariniistrainisolatedfromratlungtissuewasdistributedintotriplicate .
o
100-(cid:3)lvolumesofdifferentconcentrationsofBHBMorD0dilutedin wellsof48-wellplateswithafinalvolumeof500(cid:3)landafinalconcentra- r
g
YNBcontaining0.1%DMSOwereadded,resultinginfinalconcentra- tionof5(cid:3)107nuclei/ml.Thecellswereincubatedwitheither0.1,1,10, /

tionsof0.25,1,and4(cid:3)g/mlor0.075,0.3,and1.2(cid:3)g/ml,respectively.The or100(cid:3)g/mlofBHBMorD0.Thenegativecontrolsweremediaalone o
n
tubeswereincubatedinashakerincubatorat225rpmat37°Cinthe andampicillin10(cid:3)g/ml,andthepositivecontrolwaspentamidineis-
N
presenceof5%CO2for4h.[3H]palmitate(30(cid:3)Ci/ml)(PerkinElmer, ethionateat1(cid:3)g/ml.At24,48,and72h,10%ofthewellvolumewas o
Waltham,MA)wasaddedtothecultureandincubatedforanadditional removed,andtheATPcontentwasmeasuredusingPerkinElmerATP- v
e
2h.Cellswithoutthedrugwereincludedasanegativecontrol.Thecells liteMluciferin-luciferaseassay.TheluminescencegeneratedbytheATP m
werethenpelleted,washedoncewithdistilledsterilewater,andresus- contentofthesampleswasmeasuredbyusingaspectrophotometer(Po- b
pendedin1.5mlofMandalalipidextractionbuffer.Lipidswereextracted larStar Optima BMG, Ortenburg, Germany). Reduction in ATP con- e
r
bythemethodsofMandalaetal.(53)andBlighandDyer(54),followed sumptionbyPneumocystiscellswasconsideredameasureofdrugefficacy. 2
bymethanolicbasehydrolysisaspreviouslydescribed.Extractedlipids Foracontrolfortoxicity,ATPconsumptionwasalsomeasuredinA549 3
,
weredriedinanSPD210SpeedVacsystem(ThermoFisherScientific). andL2mammaliancells.Asampleofeachgroupwasexaminedmicro- 2
Driedlipidswereresuspendedin30(cid:3)lof1:1methanol-chloroformand scopicallyonthefinaldayoftheassaytoruleoutthepresenceofbacterial 0
1
loadedonthin-layerchromatography(TLC)silicagel60plates(EMD contamination. 8
Millipore,Billerica,MA).Glucosylceramide(GlcCer)standardfromsoy- IntracellulareffectofBHBM.ToassesswhetherBHBMwouldbe b
bean(AvantiPolarLipids,Alabaster,AL)wasaddedinaseparatelaneasa effective against intracellular C. neoformans, J774.16 macrophage cells y

control. The sample was resolved in a tank containing chloroform- wereincubatedwithC.neoformanscellsata1:1ratiointhepresenceof g
u
methanol-water(65:25:4)asthemobilephase.TheTLCplateswerethen opsonins(complementandantibodyMAb18B7againstthecryptococcal e
s
driedandexposedtoiodinevaporfortheidentificationoftheGlcCer capsularantigen).After2hofincubation,about60to80%ofmacro- t
standardband,whichwasmarked.TheTLCplatewasthenenhancedby phageshadatleastoneC.neoformanscellinternalized.Atthistime,wells
spraying with Enhancer (PerkinElmer) and exposed to X-ray film at werewashedtoremoveextracellularfungalcellsandfreshDMEMwith
(cid:5)80°Cfor72h,afterwhichthefilmwasdeveloped. differentconcentrationsofBHBMwasaddedtoeachwell.Theplateswere
Labelingmammaliancellswithtritiatedpalmitate([3H]palmitate). incubatedat37°Cwith5%CO .At0,6,12,and24h,extracellularcells
2
The murine macrophage cell line J774.16 (ATCC) was maintained in werecollectedbywashingandplatedontoYPDagarplatesforcounting
DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin- theCFUofextracellularcells.Macrophageswerealsolysedandplated
streptomycin (pen-strep) and were used until passage eight. Cells at a ontoYPDagarplatesforcountingtheCFUofintracellularfungalcells.
densityof5(cid:3)106cells/mlwereculturedinasix-wellcultureplatefor14h Synergismassay.Synergisticactivitywasassayedbycalculatingthe
toachieveadherence.BHBMorD0atthesameconcentrationsusedfor fractionalinhibitoryindex(FIC).Briefly,ina96-wellplate,drugA(either
fungalcells(seeabove)wasaddedtotheplatefor4h.Then,30(cid:3)Ci/mlof BHBMorD0)wasseriallydilutedfrom16to0.015(cid:3)g/ml(11dilutions),
[3H]palmiticacidwasadded,andtheplatewasfurtherincubatedfor2h. whereasdrugB(eitherfluconazole,amphotericinB,caspofungin,ortu-
Labeled,untreatedJ774.16cellswereincludedasacontrol.Thecellswere nicamycin)wasseriallydilutedfrom12to0.19(cid:3)g/ml,5to0.078(cid:3)g/ml,
harvestedbyadding0.05%trypsin-EDTAandscrapingwithacellscraper, 70to1.09(cid:3)g/ml,and6to0.09(cid:3)g/ml(sevendilutions),respectively.The
andthenthecellswerewashedoncewithphosphate-bufferedsaline(PBS) FICwasdefinedas(MICcombined/MICdrugAalone)(cid:7)(MICcom-
10 ® mbio.asm.org May/June2015 Volume6 Issue3 e00647-15