Match-making for posaconazole through systems thinking

Match-making

for

posaconazole

through

systems

thinking

Matthias

A.

Fu¨gi

,

Marcel

Kaiser

,

Marcel

Tanner

,

Roger

Schneiter

,

Pascal

Ma¨

ser

,

and

Xue

Li

Guan

1_{SwissTropicalandPublicHealthInstitute,CH-4051Basel,Switzerland} 2_{UniversityofBasel,CH-4000Basel,Switzerland}

3_{UniversityofFribourg,CH-1700Fribourg,Switzerland}

CurrentlyavailabledrugsforChagas’diseasearelimited bytoxicity andlowefficacyinthechronicstage. Posa-conazole, the most advanced new anti-chagasic drug candidate,didnotfullyconfirmitsinitialpotentialina PhaseIIclinicaltrialforchronicChagas’disease. Given thatposaconazoleishighlyactiveagainstTrypanosoma cruziinvitro,andwasverywelltoleratedinclinicaltrials, it should not be abandoned. Rather, a combination therapymay provide a highlypromising outlook. Sys-tems-scaleapproachesfacilitatethehuntfora combina-tionpartner for posaconazole, which acts byblocking sterol biosynthesis. Mounting evidence suggests the functional interactions between sterols and sphingoli-pids in vivo. Here, we propose combining sterol and sphingolipid biosynthesis inhibitors to advance drug developmentinChagas’disease.

Chagas’disease:aglobalburdenandtheunmetneed fornewdrugs

Worldwide, an estimated 7 million–8 million people are infectedwith the protozoan parasite Trypanosoma cruzi [1]. Chagas’ disease is endemic in 21 South American countriesbut,asaresultofpopulationmobility,alsooccurs outsidethe continent [2]. Chagas’disease poses aglobal challengeduetothelackofsafeandeffective treatment. Effortstowardstheurgentlyneedednewdrugshave cul-minatedintheclinicaldevelopmentoftriazolicantifungals forChagas’disease. The mostadvanced drug candidates wereposaconazoleandE1224,aprodrugofravuconazole. Unfortunately, although very well tolerated, both com-pounds failed to meet the high expectations in recent clinicalPhaseIItrials:theydidnotcurechronicChagas’ diseaseas indicated by the high relapse rates observed during follow-up. Considering that posaconazole and E1224arehighlyactiveagainstT.cruziandwelltolerated inclinicaltrials,weproposenottoabandonthetriazoles but to find a suitable partner for combination therapy. Combinationtherapyisanattractiveapproachbecauseit may improve treatment efficacy while decreasing the

likelihoodofresistancedevelopment[3].Systemsbiology aimsat revealinginterconnectionsofbiologicalnetworks and these works serve as useful resources for rational identificationofpotentialinteractingpartnersfor chemo-therapy.Basedongenetic,physical,andfunctional inter-actions betweensterols andsphingolipids[4] anddueto thesyntheticlethalityofSaccharomycescerevisiaedouble mutants of sterol and sphingolipid anabolism [5], our opinion is that inhibitors of sphingolipid biosynthesis are promising combination partners for posaconazole or ravuconazole.

CurrentdrugsforthetreatmentofChagas’disease Chagas’diseaseremainedwithoutaneffectivetreatment for severaldecades after its originaldescription in1909 [6].Nifurtimoxandbenznidazole,discoveredover40years ago and still the only available drugs for the specific treatmentofChagas’disease,are limitedby toxicityand lowefficacyintheestablishedchronicformofthedisease [7].Thesemajordrawbacks,alongwithupcomingreports ofresistant T. cruzi [8]and thespread ofthe diseaseto nonendemiccountries[2],spurredreneweddrugresearch anddevelopment(R&D)forChagas’disease.Thetriazoles posaconazoleandE1224weretheonlycandidatestopass the preclinical phase and enter clinical proof-of-concept trials.However,theresultsinPhaseIIclinicaltrialswere disappointing. While the parasitemia dropped below de-tection limit after treatment, 10 months later, most patientsagaintestedpositiveforT.cruzi[9].Either can-didate was less efficacious than benznidazole. This out-comeisarguablyattributabletolimitedsystemicexposure resultingfromtheliquidsuspensionofthedrugand sub-optimal treatment duration [10]. Even so, these results haveaggravatedthesituationinthealreadyslimChagas’ portfolio, where the most advanced alternatives to the triazoleshavenotyetreachedclinicalPhaseI.

Quovadis posaconazole?

In 1995, V.M. Girijavallabhan described posaconazole (SCH 56592) as a novel, orally active, broad-spectrum antifungal agent [11]. Posaconazole (Nofaxil)was devel-opedbySchering-PloughandwasapprovedbytheUSFood andDrugAdministration(FDA)forthetreatmentof inva-sive fungalinfection in humans in 2006 [12].Similar to other triazoles, posaconazole isa potent inhibitor of the Correspondingauthor:Guan,X.L. ([email protected]).

Keywords:Chagas’disease;combinationtherapy;systemsbiology;sterols; sphingolipids.

Published in 7UHQGVLQ3DUDVLWRORJ\± which should be cited to refer to this work.

Cyp450-dependentlanosterol14a-demethylase(Cyp51)in yeasts and molds [13]. Inhibition of Cyp51 blocks the synthesis ofergosterol, whichis an essential component inthecellmembraneoffungalpathogens.Accumulationof methylated sterol precursorsanddisruption ofthe close packing ofacyl chains ofphospholipids in ergosterol-de-pletedcellmembranes ultimatelyleadstogrowth inhibi-tionofthefungi[14].Similartofungi,T.cruzisynthesizes ergosterolandissensitivetosterolbiosynthesisinhibitors [15].Posaconazoleexhibitedexcellentinvitroandinvivo efficacyagainstbothdrug-sensitiveand-resistantisolates [16,17]. Suitable combination partners for posaconazole might be found in the sterol biosynthesis pathway to enhancetheblockadeofthishighlyinterconnected meta-bolicnetwork.Thelinksbetweendistinctstepsofthesterol biosynthesispathwaycanbeexemplifiedinS.cerevisiae:in thepresenceoferg6deletion,theerg2geneproductworks inefficiently[4],resultinginanerg6singledeletionmutant exhibitingapartialphenotypeofanerg2erg6double mu-tant.Furthermore,cellshaveevolvedcompensatory mech-anismswithinmetabolicpathwayssuchthataccumulating substratesresultingfrominhibitionofaspecificenzymatic stepcanbealternativelymetabolizedasasalvage mecha-nism.Clearly,sterolbiosynthesisisofprovendruggability and targeting multiple steps in the same pathway can potentiateantiparasiticactivity.Lovastatin,ablockbuster usedforhypercholesterolemia,enhancedthe antiprolifera-tiveeffectsofketoconazoleandterbinafineagainstT.cruzi invitroandinvivo[18].Evidencefromyeastpointsinthe samedirectionbecauseatleastadozenproteins interact-ing with Erg11 (Cyp51 ortholog in S. cerevisiae) can be found in the sterol metabolic pathway [19]. Druggable pathwaysthatinteractwithsterolmetabolismalso repre-sentcomplementarytargets.Glycerophospholipid biosyn-thesisinhibitors,suchasajoeneoralkyl-lysophospholipids (ALP,e.g.,miltefosine),havebeenshowntohave antipro-liferativeeffectsonT.cruziepimastigotesandamastigotes [20–22]. Growth inhibitioncorrelatedwith a decreasein thephosphatidylcholinetophosphatidylethanolamine ra-tio(PC:PE) and, inthe caseofALP,alsowith amarked effectonsterolcompositionduetoinhibitionofsterol 22-desaturase (Erg5), a finding that probably explains the antiproliferativesynergismofthesedrugswiththeCyp51 inhibitor ketoconazole against both proliferative stages (epimastigotesandintracellularamastigotes)ofthe para-site[21,22].Here,weproposetocombinetheanti-chagasic triazoleswithinhibitorsofsphingolipidsynthesis,as sug-gestedbysystemsapproaches.

Systems-basedmatchmaking

Systemsapproacheswerepioneeredinmodelorganismsto understandhowbiologicalsystemsactasawhole. Emer-genceofcomplexbehaviorisobservedwhenthebiological systems are treated as networks. These can be protein interactionnetworks,metabolicnetworks,or genetic net-works.Allareamenabletolarge-scaleinteractionstudies, particularlyinS.cerevisiae,whereglobalapproachessuch aschemicalgeneticsscreens,mutantlibraryscreens, pro-tein–protein interactions, and other -omics technologies can be automated. These have led to the availability of databasescontainingawealthofinformationthatcanbe

minedtogeneratenewhypothesesofcellularandsystem functions. It can also guide drug discovery in modern medicinebyprovidingarationalbasistopinpoint interre-latedpathways.TheinteractingpartnersforCYP51inS. cerevisiae,forinstance,arefoundontheBioGRIDdatabase [23],containing103physicaland184geneticinteractions. Candidate pathwayscan be furthernarrowed by pheno-typesandfunctionality.Specifically,interactorsofCyp51 thatcausesyntheticlethalitywillbeappealing.

Capitalizingonsterol–sphingolipidinteractionsasa combinatorialtreatment

Posaconazole blocks sterol biosynthesis; thus, druggable pathways interacting with sterol metabolism and func-tions represent highly complementarymatches for posa-conazole.Sterolshavebeenshowntomodulatemembrane thickness in artificial membranes andthis property has beenproposedtohavearoleinmembraneprotein localisa-tioninvivo[24].Itisincreasinglyknownthatproteinsand lipidsdonotfreelydiffuseovertheentiresurfaceofthecell and it has beenproposed that eukaryotic plasma mem-branes contain micro- and/or nanodomains (reviewed in [25–27])thatactasplatformscreatingmembrane hetero-geneities with many proposed functions. There is clear biophysical evidence that sterols and sphingolipids can segregatefromotherlipidsinsimpleartificialmembrane systemstoformliquidordereddomains[28].

Sterol–sphingolipidinteractionshavealsobeen demon-stratedin vivo.Evidence inthebudding yeast,S. cerevi-siae, suggests a genetic interaction between mutants in sterolandsphingolipidbiosynthesis[4,5,29,30].For exam-ple,mutantsthataffectthehydroxylationpatternof sphin-golipidsdisplaysyntheticgrowthdefectswithmutationsin late-acting ergosterolbiosyntheticgenes[4].Bycontrast, mutations that affect the synthesis of the sphingolipid-specificvery-longchainC26fattyaciddisplaystrong syn-theticlethalitywithmutationsinERG6,a methyltransfer-asethatcatalyzestheadditionofafungal-specificmethyl groupatpositionC24inthealiphaticsidechainor ergos-terol [5].Figure 1summarizesexperimental evidenceon genetic interactionsbetween the sterol and sphingolipid syntheticgenes.While thebulkofexperimentalevidence comes from high throughput screens and needs to be treated with caution, there issolidsupport forsynthetic lethality betweenELO3 andERG6[5],andforsynthetic growthdefectsofsterolsyntheticgeneswithISC1,SUR2, and SCS7 [4]. Strikingly, synthetic lethality has been demonstratedbetweenCYP51andSCS7[31].

Inyeastandhighereukaryotes,ithasfurtherbeenshown that sterols and sphingolipids are important for proper traffickingoftransporters(aminoacidsandprotonpumps) tothecellsurface andtheirstabilityattheplasma mem-brane[32–35].Adaptationtochangesinsterolcomposition byadjustingsphingolipidlevelsandvariantsisnotunique totheunicellulareukaryotesbutisalsopresentinMetazoa, exemplifiedbythefruitfly,Drosophilamelanogaster.Asa sterolauxotroph,D.melanogastercannotsynthesizesterols butthislipidisrequiredforlarvalgrowthanddevelopment. Adropinsterollevelscauseddevelopmentalarrestbutcells remainviable,possiblyduetoacompensatoryincreasein sphingolipidlevelsandcomposition[36].

Figure 2 shows the structures of mannosyl diinositol phosphoryl ceramide [M(IP)2C] and ergosterol, which is themostabundantsphingolipidandsterolspeciesinyeast. Togetherwith glycerophospholipids,sterolsand sphingo-lipidscomprisethemajorclassesofeukaryoticmembrane

lipids.Manymembranecharacteristics,suchas composi-tionandintegrity,turnoveror trafficking,andsignaling, fulfilltherequirementsforbonafidedrugtargetsin para-sites:theymust be(i)essentialforparasite survival; (ii) druggable; and (iii) sufficiently different from the host. Indeed, ‘membrane-lipid therapy’ was coined by Pablo Escriba´ andisdefinedasthetherapeuticapproachbased ontheregulation ofthemembrane-lipidcompositionand structure to modulate cell functions [37]. In a broader sense,wethinkofmembranetherapyasinterferingwith membranesdirectlyorviacurtailinglipidbiosynthesis.

WhiletherecurrentlyisnoevidenceinT.cruzionthe interactionsofsterolsandsphingolipids,itisintuitivethat thesimultaneousinhibitionofbothsterolandsphingolipid metabolismwillhaveamajorimpactonmembrane homeo-stasis.Moreover, thereare several linesofevidence that theselipidshavecriticalrolesintrypanosomatids. Endoge-noussterolsandsphingolipidsarerequiredforproliferation oftrypanosomes[38–40].Interestingly,reduced inositolpho-sphoceramide(IPC)levelsduetoinhibitionofserine palmi-toyltransferase(Spt2)inT. bruceihavebeenshowntobe compensatedforbyincreasedlevelsofphosphatidylcholine andcholesterol,demonstratingatightinteractionofsterol andsphingolipidhomeostasis[41].Asinyeast,IPCrather than glycerophospholipidsisutilizedas lipid anchor con-stituentofglycoproteinsandfree glycosylinositolphospho-lipids(GIPLs)inT. cruzi[42].Furthermore, inhibitionof IPCsynthesisimpairedT.cruzidifferentiation[43]. Erg7 Erg11 Erg25 Erg26 Erg6 Erg2 Erg3 Erg5 Erg4

Lcb1 Lcb2 Tsc10 Sur2 Elo3 Lag1 Aur1 Scs7 Sur1 Csg2 Ipt1 Isc1

Sterol biosynthec genes

Sphingolipid biosynthec genes

TRENDS in Parasitology Figure1.Geneticinteractionsbetweensterolandsphingolipidbiosyntheticgenes inyeast.Onlygenesforwhichaninteractionhasbeenexperimentallydocumented are shown. Data are from BioGRID v. 3.2.117 [23]. Green, positive genetic interaction;red,negativegeneticinteractionorsyntheticgrowthdefect;black, syntheticlethality;gray,equivocalresultsfromdifferentscreens.Forgenesymbol definitions,pleaseseeFigure2.

SUR2 AUR1 ISC1 ELO3 ERG26 ERG3 ERG6 ERG5 ERG4 ERG11 ERG2 ERG25 SCS7 CSG2 SUR1 IPT1 TSC10, LAG1 ERG7 LCB1, LCB2 OH OH OH OH OH OH NH OH OH OH OH OH OH HO HO HO HO H H O O O O O O O O– O– O O P P TRENDS in Parasitology

Figure2.Structuresofergosterolandthesphingolipid,mannosyldiinositolphosphorylceramide[M(IP)2C].Genesshownencodeenzymesthatcatalyzemajorstepsin

sterolandsphingolipidmetabolism,respectively.Thelistofgenesisnotexhaustivebutbasedonsterol/sphingolipidinteractions(seealsoFigure1).Forergosterol:ERG7, 2,3-oxidosqualenecyclase;ERG11,C14demethylation;ERG25,C4amethyloxidation;ERG26,C3decarboxylation;ERG6,C24methylation;ERG2,C8isomerization;ERG3,C5

desaturation; ERG5, C22 desaturation; ERG4, C24 reduction. For M(IP)2C: LCB1, serine C-palmitoyltransferase; LCB2, serine C-palmitoyltransferase; TSC10,

3-dehydrosphinganine reductase; SUR2, long chain base hydroxylase; ELO3, fatty acyl elongase; LAG1, sphingosine N-acyltransferase; AUR1, phosphatidylinositol:ceramidephosphoinositoltransferase;SCS7,fattyacylhydroxylase;SUR1,mannosylinositolphosphorylceramide(MIPC)synthase;CSG2,MIPC synthaseregulatorysubunit;IPT1,inositolphosphotransferase;ISC1,inositolphosphosphingolipidphospholipaseC.

Therelationbetweensterolsandsphingolipids,evident inyeastandthefruitfly,couldindicateapotential evolu-tionarily conserved adaption mechanism for membrane homeostasis. Thus, concomitant perturbation of these two classes of lipids may promote synergistic lethality. Therefore, it will be interesting to test the interactions between posaconazole or ravuconazole and sphingolipid inhibitorsonT.cruzifocusingon100%cidalityratherthan potentialsynergism.

Concludingremarksandoutstandingquestions

Sterilecidalityalsoagainstnonproliferatingtrypanosomes is imperative to Chagas’ disease chemotherapy. To this aim,threedifferentstrategieshavebeenproposedtoselect a suitable combination partner for azoles. The partner couldbeadrugsuchasbenznidazole,whichis100%cidal itselfandadditiveinactionwithposaconazole[44–46].It couldalsobeadrugthatisnot100%cidalitselfbutshows synergistic interaction with posaconazole, such as amio-darone, amlodipine, or clemastine [46,47]. In addition,

aimingtocompletelyblocksterolsynthesis,the combina-tionpartnercouldbeanothersterolbiosynthesisinhibitor [15,18]. Here, we propose as an additional strategy the partnership betweenposaconazoleandsphingolipid inhi-bitors.Thisisbasedonthehypothesisthatsucha combi-nation will be most effective in disrupting membrane integrityandfunctions,whichiscriticalalsoforquiescent cells.Explorationofthechemotherapeuticpotentialofthis proposedpartnershipwillrequire:(i)systemsknowledgeof T. cruzi lipid physiology; (ii) sphingolipid biosynthesis inhibitors; and(iii) atestfor100%sterile cidalityonthe relevantT. cruzistages.

Currently, there is no evidence that sterols and sphingolipids functionally interact in T. cruzi. The ad-vancingtechnologiesforsystem-scaleanalyses ofgenes, transcripts, proteins,and metabolites (includinglipids) accompanied by high throughput genetic and chemical screening,willrevolutionizeourunderstandingofT.cruzi biology and identify possible pathways for combination therapies.Concomitantchemotherapeuticattackofsterol

Table1.Sphingolipidbiosynthesisinhibitorsinclinicaltrialsoronthemarket

Compound name Clinical phase or drug name (if on the market)

Mechanism of action Indication Refs

N-butyldeoxynojirimycin Miglustat,Zavesca1 Glucosylceramidesynthaseinhibitor Gaucherdisease

FTY720 Gilenya Sphingosine-1-phosphatereceptorinhibitor Multiplesclerosis [49]

Safingol PhaseI Sphingosinekinaseinhibitor Cancer [50]

Phenoxodiol PhaseIII Sphingosinekinaseinhibitor Cancer [51]

ABC294640 PhaseI Sphingosinekinaseinhibitor Cancer [52]

Sphingomab Preclinical Anti-sphingosine-1-phosphateantibody Cancer [53]

Fenretinide PhaseI Ceramidedesaturaseinhibitor Cancer [54]

Desipramine Treyzafagit,Norpramin,

andPertofrane

Acidsphingomyelinaseinhibitor Antidepressant

Imiglucerase Cerezyme b-glucocerebrosidasereplacement Gaucherdisease

Amitriptyline PhaseIIb Acidsphingomyelinaseinhibitor CysticFibrosis [55]

Elavil,Endep,andVanatrip Antidepressant

Analgesic

Fluoxetine ROzac,PROzacWeekly,

Sarafem,Rapiflux,Selfemra, andPROzacPulvules

Acidsphingomyelinaseinhibitor Antidepressant

AureobasidinAa PhaseI Inositolphosphorylceramidesynthaseinhibitor Antifungal [56]

a_{FailedinclinicalPhaseI.}

Table2.Sterolbiosynthesisinhibitorsa

Class Target and/or mechanism of action Indication Refs

Statins CompetitiveinhibitorsofHMG-CoAreductase,preventingtheformationof mevalonatefromHMG-CoA;theyoccupytheHMG-bindingpocketandpartof thebindingsurfaceforCoA

Usedas cholesterol-loweringdrugsinhumans

[57,58]

Bisphosphonates(BPs) Potentinhibitorsofboneresorption.Theselectiveactiononboneisbasedon thebindingoftheBPmoietytothebonemineral;nitrogen-containingBPsbind to,andinhibittheactivityof,farnesyldiphosphatesynthase

Usedtotreatosteoporosis andotherboneresorption diseases

[59–61]

Quinuclidinesand/ orzaragozicacids

Inhibitionofsqualenesynthase(SQS);quinuclidinesmayinhibitSQSby actingascarbocationmimicsforFPPtosqualeneconversion.Thearylunits mayactasisosteresfortheisoprenylsubunitsinthefarnesylchain.

Notinclinicaluse [62]

Allylamines Specificinhibitionoffungalsqualenemono-oxygenase Usedfortopicaltreatment

offungalinfections

[63,64] Azoles Bindasthesixthligandtothehaeminlanosterol14_{a-demethylase}(=CYP51),

thusoccupyingtheactivesiteandactingasnoncompetitiveinhibitors; blockingthesynthesisofergosterolleadstotheaccumulationofmethylated sterolprecursors

Usedtotreatfungal infections

[65,66]

Azasterols EvidencefromyeastshowsthatazasterolsinhibittheenzymeC24-sterol methyltransferase

Notinclinicaluse [67]

a_{Compoundclassesofmoleculeknowntointerferewithsterolmetabolism.Targetenzymesandmechanismsofactionareindicated,aswellasclinicalindicationswhere}

moleculesarealreadyonthemarket.

andsphingolipidbiosynthesisis facilitatedbythe avail-abilityofsphingolipidinhibitors[48],owingtothe inter-estsintheirfunctionsinhumanhealth.Aswitheverydrug candidate,consideration must begiven tothe potential toxicityofsphingolipidbiosynthesisinhibitors.Toxicityis onereasonwhymostofthenumerousexisting sphingoli-pidinhibitorsremainexperimentalcompounds[48]. None-theless, this class of compounds is promising, because thereare severalinclinicaluseorinclinicaltrialsfora spectrumofhumandiseases(Table1)and,giventheroleof sphingolipidsin many other human diseases, efforts to discovernovelcompoundsareongoing.Thesameapplies forsterolbiosynthesisinhibitors(Table 2).A crucial re-quirement for R&D of next-generation anti-chagasic agentswillbeaninvitrotestthatisamenabletomedium throughput and that can demonstrate 100% cidality against nonproliferating intracellular amastigote T. cruzi.Suchanassaymustbeabletopredict the lackof sterilecidalityofposaconazoleandravuconazole.

In summary,wearguethat thepotentialof posacona-zolemustbefurtherexploredwithaviewofrationaltarget identificationandachievingcombinationtherapythrough systems-scale approaches. Based on evidence in model organisms,particularly the buddingyeast, the matching ofsphingolipidsynthesisinhibitorsaspartnersoftriazoles can impair membrane functionality and, thus, may kill proliferatingaswellasdormantparasites.

Acknowledgments

X.L.G. was supported by the Swiss National Science Foundation Ambizione(PZ00P3_136738).TheauthorsthanktheDrugsforNeglected Diseasesinitiative(DNDi)andProfessorHowardRiezman(Universityof Geneva)forhelpfuldiscussions.

References

1 Crompton,D.W. (ed.)(2013) Sustaining theDriveto Overcome the Global Impact of Neglected Tropical Diseases, World Health Organization

2 Coura,J.R.andVin˜as,P.A.(2010)Chagasdisease:anewworldwide challenge.Nature465,S6–S7

3 Ehrlich, P. (1907) Chemotherapeutische Trypanosomen-Studien, BerlinerklinischeWochenschrift

4 Guan,X.L.etal.(2009)Functionalinteractionsbetweensphingolipids andsterolsinbiologicalmembranesregulatingcellphysiology.Mol. Biol.Cell20,2083–2095

5 Eisenkolb, M. et al. (2002) A specific structural requirement for ergosterolin long-chainfatty acidsynthesismutants importantfor maintainingraftdomainsinyeast.Mol.Biol.Cell13,4414–4428 6 Chagas, C. (1909) Nova tripanozomiaze humana: estudos sobre a

morfolojiaeocicloevolutivodoSchizotrypanumcruzin.gen.,n.sp., ajente etiolojico de novaentidade morbida do homem. Mem. Inst. OswaldoCruz1,159–218

7 Urbina, J.A. (2010) Specific chemotherapy of Chagas disease: relevance,currentlimitationsandnewapproaches.ActaTrop.115, 55–68

8 Filardi,L.S.andBrener,Z.(1987)Susceptibilityandnaturalresistance of Trypanosoma cruzi strains to drugs used clinically in Chagas disease.Trans.R.Soc.Trop.Med.Hyg.81,755–759

9 Molina, I. et al. (2014) Randomized trial of posaconazole and benznidazole for chronic Chagas’ disease. N. Engl. J. Med. 370, 1899–1908

10 Urbina,J.A.(2014)Recentclinicaltrialsfortheetiologicaltreatmentof chronic Chagas disease: advances, challenges and perspectives. J. Eukaryot. Microbiol. Published online October 5, 2014, (http:// dx.doi.org/10.1111/jeu.12184)

11 Girijavallabhan,V.M.etal.(1995)Sch56592,anovel orallyactive broad spectrumantifungalagent.Abstr.Intersci. Conf.Antimicrob. AgentsChemother.35,123

12 Schering-Plough(2006)KenilworthSchering-PloughAnnouncesFDA Approval of Noxafil for Treatment of Oropharyngeal Candidiasis, MedNews

13 Munayyer,H.K.etal.(2004)Posaconazoleisapotentinhibitorofsterol 14alpha-demethylation in yeasts and molds. Antimicrob. Agents Chemother.48,3690–3696

14 Sheehan,D.J.etal.(1999) Currentand emergingazole antifungal agents.Clin.Microbiol.Rev.12,40–79

15 Maldonado, R.A. et al. (1993) Experimental chemotherapy with combinationsofergosterolbiosynthesisinhibitorsinmurinemodels ofChagas’disease.Antimicrob.AgentsChemother.37,1353–1359 16 Urbina,J.A.etal.(1998)Antiproliferativeeffectsandmechanismof

actionofSCH56592againstTrypanosoma(Schizotrypanum)cruzi:in vitroandinvivostudies.Antimicrob.AgentsChemother.42,1771–1777 17 Molina,J.etal.(2000)ActivitiesofthetriazolederivativeSCH56592 (posaconazole)againstdrug-resistantstrainsoftheprotozoanparasite

Trypanosoma (Schizotrypanum) cruzi in immunocompetent and

immunosuppressed murine hosts. Antimicrob. Agents Chemother. 44,150–155

18 Urbina, J.A. et al. (1993) Mevinolin (lovastatin) potentiates the antiproliferative effects of ketoconazole and terbinafine against Trypanosoma (Schizotrypanum) cruzi: invitro and invivo studies. Antimicrob.AgentsChemother.37,580–591

19 Mo, C. and Bard, M. (2005) A systematic study of yeast sterol biosynthetic protein-protein interactions using the split-ubiquitin system.Biochim.Biophys.Acta1737,152–160

20 Urbina, J.A. et al. (1993) Inhibition of phosphatidylcholine biosynthesisandcellproliferation inTrypanosomacruzibyajoene, anantiplateletcompoundisolatedfromgarlic.Biochem.Pharmacol. 45,2381–2387

21 Lira, R. et al. (2001) Mechanism of action of anti-proliferative

lysophospholipid analogues against the protozoan parasite

Trypanosoma cruzi: potentiation of in vitro activity by the sterol biosynthesis inhibitor ketoconazole. J. Antimicrob. Chemother. 47, 537–546

22 Santa-Rita, R.M. et al. (2005) Anti-proliferative synergy of lysophospholipid analoguesand ketoconazoleagainstTrypanosoma cruzi(Kinetoplastida:Trypanosomatidae):cellularandultrastructural analysis.J.Antimicrob.Chemother.55,780–784

23 Stark,C.etal.(2006)BioGRID:ageneralrepositoryforinteraction datasets.NucleicAcidsRes.34,D535–D539

24 Bretscher, M.S. and Munro, S. (1993) Cholesterol and the Golgi apparatus.Science261,1280–1281

25 Simons,K.andIkonen,E.(1997)Functionalraftsincellmembranes. Nature387,569–572

26 Jacobson,K.etal.(2007)Lipidrafts:atacrossroadbetweencellbiology andphysics.Nat.CellBiol.9,7–14

27 Kusumi, A.et al. (2005) Paradigmshift of theplasma membrane conceptfromthetwo-dimensionalcontinuumfluidtothepartitioned fluid:high-speed single-molecule tracking of membrane molecules. Annu.Rev.Biophys.Biomol.Struct.34,351–378

28 Ahmed,S.N.etal.(1997)Ontheoriginofsphingolipid/cholesterol-rich detergent-insolublecell membranes: physiologicalconcentrationsof cholesterolandsphingolipidinduceformationofadetergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry 36, 10944–10953

29 Baudry, K.etal.(2001)Theeffectof theerg26-1mutationon the regulationoflipidmetabolisminSaccharomycescerevisiae.J.Biol. Chem.276,12702–12711

30 Jin, H.etal. (2008)Ergosterolpromotes pheromone signalingand plasmamembranefusioninmatingyeast.J.CellBiol.180,813–826 31 Parsons,A.B.etal.(2004)Integrationofchemical-geneticandgenetic interactiondatalinksbioactivecompoundstocellulartargetpathways. Nat.Biotechnol.22,62–69

32 Umebayashi, K.and Nakano, A. (2003) Ergosterolis requiredfor targetingoftryptophanpermeasetotheyeastplasmamembrane.J. CellBiol.161,1117–1131

33 Chung, N. et al. (2000) Sphingolipids signal heat stress-induced ubiquitin-dependentproteolysis.J.Biol.Chem.275,17229–17232

34 Proszynski,T.J.etal.(2005)Agenome-widevisualscreenrevealsarole forsphingolipidsandergosterolincellsurfacedeliveryinyeast.Proc. Natl.Acad.Sci.U.S.A.102,17981–17986

35 Gaigg, B.et al.(2006) Verylong-chainfatty acid-containing lipids rather than sphingolipids per seare required for raft association

and stable surface transport of newly synthesized plasma

membraneATPaseinyeast.J.Biol.Chem.281,34135–34145 36 Carvalho,M.etal.(2010)Survivalstrategiesofasterolauxotroph.

Development137,3675–3685

37 Escriba´, P.V. (2006) Membrane-lipid therapy: a new approach in molecularmedicine.TrendsMol.Med.12,34–43

38 Urbina,J.A.(2002)ChemotherapyofChagasdisease.Curr.Pharm. Des.8,287–295

39 Urbina,J.A.andDocampo,R.(2003)SpecificchemotherapyofChagas disease:controversiesandadvances.TrendsParasitol.19,495–501 40 Sutterwala,S.S.etal.(2007)Denovosphingolipidsynthesisisessential

for viability, but not for transport of glycosylphosphatidylinositol-anchoredproteins,inAfricantrypanosomes.Eukaryot.Cell6,454–464 41 Fridberg, A. et al. (2008) Sphingolipid synthesis is necessary for kinetoplast segregationand cytokinesis in Trypanosoma brucei. J. CellSci.121,522–535

42 De Lederkremer, R.M. and Agusti, R. (2009) Glycobiology of

Trypanosomacruzi.Adv.Carbohydr.Chem.Biochem.62,311–366

43Salto, M.L. et al. (2003) Formation and remodeling of

inositolphosphoceramide during differentiation of Trypanosoma cruzi fromtrypomastigotetoamastigote.Eukaryot.Cell2,756–768

44 Cencig,S.etal.(2012)Evaluationofbenznidazoletreatmentcombined withnifurtimox,posaconazoleorAmBisome1inmiceinfectedwith Trypanosomacruzistrains.Int.J.Antimicrob.Agents40,527–532 45 Diniz,L.andde,F.etal.(2013)Benznidazoleandposaconazolein

experimentalChagasdisease:positiveinteractioninconcomitantand sequentialtreatments.PLoSNegl.Trop.Dis.7,e2367

46 Planer, J.D. et al. (2014) Synergy testing of FDA-approved drugs identifies potent drug combinations against Trypanosoma cruzi. PLoSNegl.Trop.Dis.8,e2977

47 Benaim,G.etal.(2006)Amiodaronehasintrinsicanti-Trypanosoma cruziactivityandactssynergisticallywithposaconazole.J.Med.Chem. 49,892–899

48 Delgado, A. et al. (2006) Inhibitors of sphingolipid metabolism enzymes.Biochim.Biophys.Acta1758,1957–1977

49 Brinkmann, V. et al. (2010) Fingolimod (FTY720): discovery and developmentof an oral drugto treat multiplesclerosis.Nat. Rev. DrugDiscov.9,883–897

50 Dickson, M.A. et al. (2011) A phase I clinical trial of safingol in combination withcisplatin in advanced solid tumors.Clin. Cancer Res.17,2484–2492

51 Fotopoulou,C. etal. (2014)Weekly AUC2 carboplatinin acquired platinum-resistantovariancancerwithorwithoutoralphenoxodiol, a sensitizer of platinum cytotoxicity: the phase III OVATURE multicenterrandomizedstudy.Ann.Oncol.25,160–165

52 KummethaVenkata,J.etal.(2014)Inhibitionofsphingosinekinase 2 down-regulates the expression of c-Myc and Mcl-1 and induces apoptosisinmultiplemyeloma.Blood PublishedonlineAugust14, 2014, (http://dx.doi.org/10.1182/blood-2014-03-559385)

53 Sabbadini, R.A. (2011) Sphingosine-1-phosphate antibodies as potentialagentsinthetreatmentofcancerandage-relatedmacular degeneration.Br.J.Pharmacol.162,1225–1238

54 Maurer,B.J.etal.(2013)PhaseItrialoffenretinidedeliveredorallyin anovelorganizedlipidcomplexinpatientswithrelapsed/refractory neuroblastoma:areportfromtheNewApproachestoNeuroblastoma Therapy(NANT)consortium.Pediatr.BloodCancer60,1801–1808 55 Na¨hrlich,L.etal.(2013)TherapyofCF-patientswithamitriptyline

andplacebo:arandomised,double-blind,placebo-controlledphaseIIb multicenter,cohort-study.Cell.Physiol.Biochem.31,505–512 56 Takesako,K.etal.(1993)BiologicalpropertiesofaureobasidinA,a

cyclicdepsipeptideantifungalantibiotic.J.Antibiot.(Tokyo)46,1414– 1420

57 Endo, A. et al. (1976) Competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by ML-236A and ML-236B fungalmetabolites,havinghypocholesterolemicactivity.FEBSLett. 72,323–326

58 Istvan,E.S.andDeisenhofer,J.(2001)Structuralmechanismforstatin inhibitionofHMG-CoAreductase.Science292,1160–1164

59 Rodan,G.A.(1998)Mechanismsofactionofbisphosphonates.Annu. Rev.Pharmacol.Toxicol.38,375–388

60 Dunford, J.E. et al. (2001) Structure-activity relationships for inhibitionoffarnesyldiphosphatesynthaseinvitroandinhibitionof bone resorptionin vivoby nitrogen-containing bisphosphonates.J. Pharmacol.Exp.Ther.296,235–242

61 Kavanagh,K.L.etal.(2006)Themolecularmechanismof nitrogen-containing bisphosphonates as antiosteoporosis drugs. Proc. Natl. Acad.Sci.U.S.A.103,7829–7834

62 Brown,G.R.etal.(1996)Synthesisandactivityofanovelseriesof 3-biarylquinuclidinesqualenesynthaseinhibitors.J.Med. Chem.39, 2971–2979

63 Ryder,N.S.(1992)Terbinafine:modeofactionandpropertiesofthe squaleneepoxidaseinhibition.Br.J.Dermatol.126(Suppl39),2–7 64 Nowosielski, M. et al. (2011) Detailed mechanism of squalene

epoxidaseinhibitionbyterbinafine.J.Chem.Inf.Model.51,455–462 65 Jefcoate,C.R.(1978)Measurementofsubstrateandinhibitorbinding to microsomalcytochrome P-450by optical-differencespectroscopy. MethodsEnzymol.52,258–279

66 Monk,B.C.etal.(2014)Architectureofasinglemembranespanning cytochromeP450suggestsconstraintsthatorientthecatalyticdomain relativetoabilayer.Proc.Natl.Acad.Sci.U.S.A.111,3865–3870 67 Pierce,H.D.etal.(1978)Azasterolinhibitorsinyeast.Inhibitionofthe

24-methylene sterol delta24(28)-reductase and delta24-sterol methyltransferaseofSaccharomycescerevisiaeby23-azacholesterol. Biochim.Biophys.Acta529,429–437