Vitamin Biosynthesis as an Antifungal Target

14 Pages·2017·1.74 MB·English

Journal of
Fungi
Review
Vitamin Biosynthesis as an Antifungal Target
ZoharMeirandNirOsherov*
DepartmentofClinicalMicrobiologyandImmunology,SacklerSchoolofMedicine,Tel-AvivUniversity,
Ramat-Aviv,Tel-Aviv69978,Israel;[email protected]
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:29May2018;Accepted:13June2018;Published:17June2018
Abstract: The large increase in the population of immunosuppressed patients, coupled with
the limited efficacy of existing antifungals and rising resistance toward them, have dramatically
highlighted the need to develop novel drugs for the treatment of invasive fungal infections.
Anattractivepossibilityistheidentificationofpossibledrugtargetswithinessentialfungalmetabolic
pathwaysnotsharedwithhumans. Here,wereviewthevitaminbiosyntheticpathways(vitamins
A–E,K)ascandidatesforthedevelopmentofantifungals. Wepresentasetofrankingcriteriathat
identifythevitaminB2(riboflavin),B5(pantothenicacid),andB9(folate)biosynthesispathwaysas
beingparticularlyrichinnewantifungaltargets. Weproposethatrecentscientificadvancesinthe
fieldsofdrugdesignandfungalgenomicshavedevelopedsufficientlytomeritarenewedlookat
thesepathwaysaspromisingsourcesforthedevelopmentofnovelclassesofantifungals.
Keywords: antifungals;fungalvitaminmetabolism;drugtarget;essentialgenes
1. Introduction
The number of life-threatening fungal infections has risen dramatically over the last twenty
years. Recentestimateshaveidentifiedaglobalburdenofalmosttwomillionpatientswithsystemic
andinvasivefungalinfections, including~700,000casesofinvasivecandidiasis, ~500,000casesof
Pneumocystisjiroveciipneumonia,~250,000casesofinvasiveaspergillosis,~220,000casesofcryptococcal
meningitis,and~100,000casesofdisseminatedhistoplasmosis[1,2]. Theprimaryreasonforthisisthe
rapidriseinthenumberofchronicallyimmunosuppressedanddebilitatedpatients. Thisisdueto
aggressivechemotherapytotreatleukemiaandotherhematologicalmalignancies,theriseinbone
marrowtransplantations(BMTs),andAIDS.
Treatments for invasive fungal infections remain unsatisfactory. There are only four classes
of established antifungal drugs on the market—polyenes (e.g., amphotericin B formulations),
triazoles(e.g.,voriconazole),thenewlyintroducedechinocandins(e.g.,caspofungin),andallylamines
(e.g., terbinafine). Of these, only the first three classes are currently used to treat systemic fungal
infections[3]. Nevertheless,despitetreatment,thereremainsanunacceptablyhighmortalityratein
high-riskpatients. Inaddition,someofthecurrentantifungaltreatmentsinteractunfavorablywith
other medications, have resistance problems, a low spectrum of activity, limited formulation, are
fungistaticasopposedtofungicidal,andarefrequentlytoxic[3]. Thisisprimarilybecausefungiare
eukaryotesandsharemanybiochemicalpathwaysandsubcellularstructureswithmammaliancells.
Consequently,mostcurrentlyusedantifungalsarenottrulyfungal-specific. Onlytheechinocandins
inhibit a specific target of the fungal cell-wall, and indeed exhibit an excellent safety profile and
clinicalefficacy[4]. However,theyarenotorallyavailable,haveanarrowtherapeuticrange,andare
fungistaticagainstmolds[4].
Becauseofdownsizing,consolidation,andlowprofitability,mostlargepharmaceuticalcompanies
haveconsiderablyreducedorevenhaltedtheireffortstodevelopnovelantifungals,evenasresistance
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totheexistingdrugsrapidlyemergesamongstclinicalisolates[5]. Thus,thereisanurgentandunmet
needtodevelopadditionalandnovelantifungaldrugsthatinhibitessentialfungal-specificcellular
targetsandpathways[6].
During the last two decades, extensive molecular studies have helped identify several
fungal-specificdrugtargetssharedbythemostimportanthumanpathogenicfungi,Candidaalbicans,
Aspergillus fumigatus, and Cryptococcus neoformans, and not found in higher eukaryotes including
humans[7–10]. Theyincludeessentialgenesthatsynthesize,maintain,andcontrolthestructureofthe
fungalcellwall(e.g.,FKS1glucansynthase,PKCproteinkinaseC,CHS1chitinsynthase)[11],unique
pathways participating in the uptake of iron (e.g., SIT1 siderophore transporter, sidA siderophore
biosynthesis, FTR1 iron permease) [12,13], zinc and copper (e.g., zrfA-C zinc transporters, crpA
coppertransporter)[14],andthetransportandsynthesisofessentialaromaticaminoacids,metabolic
precursors,andvitamins(e.g.,ARO1-9,HisB,SNO1/SNZ1,ABZ1-2,andPabaA,respectively)[15–18].
Importantly, deletion of genes participating in these pathways, in one or more of the main
systemic human fungal pathogens (C. albicans, A. fumigatus, and C. neoformans), abolishes their
virulence[9,19–23],suggestingthattheyarepromisingdrugtargets.
This review focuses exclusively on the essential vitamin biosynthetic pathways (A, B1–12, C,
D,E,andK)andtheirsuitabilityasantifungaldrugtargets. Amongthecriteriaqualifyingthemas
validdrugtargetsaretheirabsenceinthehumanhost,conservationamongfungi,well-understood
biochemistry/structuraldata,andessentialityforfungalgrowthintheinfectedhost. Animportant
caveatisthatthehostnicheshouldnotsupplytheinfectingfunguswiththosenutrients,asitwould
allowittobypasspathwayinhibition. Anotablelimitationinouranalysisisthatthevirulenceofthe
fungalvitaminauxotrophsdescribedherewastestedindiverseinfectionmodels,usingdifferentmouse
strains,immunosuppressiveregimens(neutropenic,non-neutropenic),infectionroutes(disseminated,
lungs),andreadouts(mortality,fungalload)thatcanresultindifferentoutcomes. Thus,ourapproach
hasbeentofocusonpathwaysinwhichauxotrophiesresultinavirulenceinthemaximalnumberof
infectiousmodels,givingthemostrobustresults.
Thereareseveraloverlookedadvantagesintargetingfungal-uniqueenzymesparticipatingin
vitaminbiosynthesis: (i)Thesubstratesandproductsoftheseenzymesarewell-characterizedsmall
molecules with high potential druggability. This has already been exploited in the generation of
inhibitorsofvitaminB5(pantothenicacid)andB9(folate)biosynthesis(seerelevantsectionsbelow)[11];
(ii) competitive inhibitors of vitamin biosynthesis can be identified relatively easily because their
activityisblockeduponadditionofexcessproduct. Thispropertycanbeexploitedtorapidlyidentify
pathway-specific inhibitors from large compound libraries [24,25]. Note that this approach can
effectively identify metabolic inhibitors in general (e.g., essential amino acids, nucleotides, metal
uptake, etc.); (iii) several of the vitamin biosynthetic pathways, including vitamin B2 (riboflavin),
vitaminB5(pantothenicacid),andvitaminB9(folate)biosynthesis,supplycofactorsforhundredsof
enzymesinvolvedinessentialmetabolicprocesses. Therefore,inhibitionofthesevitaminbiosynthetic
pathwayswillhaveaverylargeinhibitorycascadeeffectonmanymetabolicprocesses,potentially
leadingtolethalcellulardamage;(iv)someofthevitaminpathwayssupplyprecursorsneededforthe
biosynthesisofknownvirulencefactors. Asaresult,inhibitingthesepathwayswillalsoinhibitthe
productionofthedependentvirulencefactors. Forexample,riboflavin/vitaminB2servesasacofactor
forornithine-N5-monooxygenaseSidA,whichcatalysestheinitialstepinsiderophorebiosynthesisin
A.fumigatus. Siderophoresareessentialvirulencefactors,allowingthefungustoovercomesevereiron
limitationinthehost[26]. Inhibitionofriboflavinbiosynthesisstrongly(>80%)reducestheproduction
ofsiderophores[27],deliveringanunexpectedadvantagebyinhibitingfungalironacquisitionand
growthduringinfection.
Despite their potential, to date there are few antifungals targeting the vitamin biosynthetic
pathways. Pneumocystis pneumonia, caused by the yeast P. jirovecii, is treated with a synergistic
combination of sulfamethoxazole and trimethoprim, which inhibit two key vitamin B9/folate
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biosynthetic enzymes. A similar combinatorial approach is used to treat malaria caused by
Plasmodiumfalciparum,aswellasmanytypesofbacterialinfections(seebelow).
While we have limited our target search to include only fungal-specific enzymes not found
in humans, there are many examples of existing antimicrobial drugs (e.g., azole and allylamine
antifungals, antimicrobial DHFR inhibitors, etc.) and pipeline antifungals (AX001 inositol
acyltransferaseinhibitor,F901318dihydroorotatedehydrogenaseinhibitor)thatinhibittargetsshared
withhumans[6]. Theantifungalspecificityofthesedrugswasachievedbypainstakinglyoptimizing
theirstructuretobindmoretightlyandselectivelytothemicrobialenzyme.
2. TheVitaminA,C,D,E,andKPathwaysAreNotSuitableasAntifungalTargets
Thefirstpartofthisreviewbrieflydescribestheessentialvitaminbiosyntheticpathwaysthatare
unsuitable,inouropinion,forthedevelopmentofnewantifungals. TheyincludethevitaminA,C,D,
E,andKpathways.
Vitamin A compounds (retinol, retinal, retinoic acid, and their precursors, the carotenoids)
areimportantforgrowthanddevelopment, forthemaintenanceoftheimmunesystemandgood
vision[28]. AnimalslackthevitaminAbiosyntheticpathwayandrelyonexogenoussourcessuch
as plants. Carotenoids are organic pigments that are found in the chloroplasts and chromoplasts
ofplantsandsomeotherphotosyntheticorganisms, includingsomebacteriaandfungi. Themost
common carotenoids include lycopene and the vitamin A precursor β-carotene. β-carotene is an
intensered-orangepigmentabundantinplantsandfruits. Fungiproducecarotenoidsfordifferent
non-essentialfunctions,includingstresstoleranceandsynthesisofphysiologicallyactiveby-products.
However,inhumanpathogenicfungithatproducecarotenoids(RhizopusandAspergillusspp.),mutants
unable to produce them do not display phenotypic alterations in the laboratory, apart from lack
of pigmentation [29]. Many fungi do not produce carotenoids, including pathogenic species of
CandidaandCryptococcus[29]. Thus,thecarotenoidpathwayisnotagoodtargetforthedevelopment
ofantifungals.
VitaminCorascorbicacidisacofactorforanumberofenzymesandanimportantantioxidant.
Itisproducedinallhigherplantsandmostanimals. Humansandanthropoidapescannotsynthesize
ascorbate,becauseofmutationsintheL-gulono-γ-lactoneoxidase(GLO)genecatalyzingthelaststep
initsbiosynthesis[30].InthepathogenicfungusC.albicans,deletionoftheGLOhomologalo1increases
sensitivitytooxidativestressinvitroandattenuatesvirulenceininfectedimmunocompetentmice[31].
Nevertheless,deletionoftheA.fumigatusGLOhomologAfu1g14950doesnotaffectoxidativestress
sensitivityorvirulence(ourunpublishedresults). Collectively, theseresultssuggestthateventhe
mostpotentinhibitorofAlo1wouldonlypartiallyreduceC.albicansvirulenceandwouldprobablybe
ineffectiveagainstA.fumigatus.
Finally, vitamin D (vitamin D /ergocalciferol) is produced by fungi from ergosterol in a
2
non-enzymaticphotochemicalreactioncatalyzedbyUV-Brays[32]. Itthuslacksadruggabletarget,
whilevitaminsEandKareonlysynthesizedbyplantsandnotfungi.
3. TheBvitaminsasAntifungalTargets
B vitamins are a chemically diverse group of water-soluble compounds that are important
cofactors in cell metabolism. They include vitamins B1 (thiamine), B2 (riboflavin), B3 (niacin),
B5(pantothenicacid),B6(pyridoxine),B7(biotin),B9(folate),andB12(cobalamin). Wewillbriefly
describetheirbiosyntheticpathwaysandfocusonthosecontainingtargetssuitableforthedevelopment
ofnewantifungals. AsummaryisprovidedinTable1.
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Table1.EvaluationofthesuitabilityoffungalvitaminBbiosyntheticgenesforantifungaldevelopment.
Essentialfor FUNGALCRYSTAL FungalInhibitors
Pathway Gene*
FungalVirulence STRUCTURE Developed
THI4 No[33] Yes[34] No
THI5 ND† Yes[35] No
VitaminB1thiamine
THI6 No[27] Yes[36] No
THI20 ND Yes[37] No
RIB1 Yes[27,33] No No
RIB2 Yes[9,38] No No
VitaminB2riboflavin RIB3 ND Yes[39] No
RIB4 ND Yes[40] Yes[42]‡
RIB5 ND Yes[41] No
ECM31 Yes[9] No No
PAN2 ND Yes[43] No
VitaminB5pantothenate
PAN5 ND No No
PAN6 Yes[27,38] No No
VitaminB6pyridoxine SNZ1 Partially[27,33] Yes[44] No
VitaminB7biotin BIO2 No[33,38,45] No No
ABZ1 Yes[19,46] No No
VitaminB9folate ABZ2 ND Yes[47] No
FOL1 Essentialgene Yes[48] Yes[49]‡
*Saccharomycescerevisiae;†ND=notdetermined;‡Onlyactiveagainstrecombinantfungalenzyme.
3.1. VitaminB1(Thiamine)
Thiamine is synthesized by bacteria, plants, and fungi but not by animals. It is a cofactor in
carbohydrateandaminoacidmetabolism,glycolysis,TCAcycle,andthepentoseshunt[50]. Thiamine
is generated by the Thi6p-dependant coupling of thiazole and pyrimidine, each synthesized by
a separate pathway (Figure 1A). Fungi can also obtain thiamine from their environment, using a
dedicatedtransporter,Thi7p. Theimportanceoffungalthiaminebiosynthesisduringanimalinfection
hasonlybeendeterminedintheAspergilli. InAspergillusnidulans,athiamineauxotrophgeneratedby
pointmutationofthi4wasfullyvirulentinamurinemodelofsystemicinfection[33]. InA.fumigatus,
a thiamine auxotroph generated by thi6/thiB gene deletion showed attenuated virulence in both
pulmonaryandsystemicmodelsofinfection. Therewasnodifferencebetweenwild-typeandmutant
infungalloadandhistopathology,suggestingthatthereissufficientthiamineintheinfectedhostto
allowstronggrowthofthethi6/thiBmutant[27]. Therefore,inhibitingthiaminebiosynthesismaynot
beagoodstrategyforantifungaldevelopment,unlessperhapsifcombinedwiththedevelopment
ofinhibitorsofthiamineuptake. Interestingly,thiaminebiosynthesisisessentialforvirulenceinthe
plant pathogenic fungus Verticillium dahlia, suggesting that this pathway could be suitable for the
developmentofagriculturalfungicides[51].
3.2. VitaminB2(Riboflavin)
Riboflavin in its active forms, flavin adenine dinucleotide (FAD) and flavin mononucleotide
(FMN),functionsasacofactorfornumerousflavocoenzyme-catalyzedreactions,includingelectron
transport, fatty acid oxidation, and vitamin B3/B6/B9 synthesis. Riboflavin is synthesized from
onemoleculeofGTPandtwomoleculesofribulose5phosphatebysixenzymes(Rib1–Rib5pand
Rib7p)[52](Figure1B).InS.cerevisiae,deletionofRIB1-RIB5resultsinriboflavinauxotrophy.
ThevirulenceofriboflavinauxotrophshasbeenassessedinthepathogenicfungiC.albicans[9],
Histoplasmacapsulatum[38],A.nidulans[33],andA.fumigatus[27]. InC.albicans,conditionalrepression
ofRib2resultsinavirulenceinsystemicallyinfectedimmunocompetentmice[9]. InH.capsulatum,
disruptionofRib2preventsfungalproliferationinsidemacrophagesinvitro. Virulenceinintranasally
infected immunocompetent mice is severely attenuated as evidenced by an inability to replicate
in the lungs or disseminate [38]. Rib2 may be unsuitable as a drug target because it has high
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homologytoanuncharacterizedpseudouridylatesynthasedomain-containingprotein(NP689473)in
humans. MutationalinactivationofRib1inA.nidulansattenuatesvirulenceinsystemicallyinfected
immunocompetentmice[33]. DeletionofRib1(riboB)inA.fumigatusabolishesvirulenceinintranasally
infected cortisone-compromised mice and systemically infected neutropenic mice, and attenuates
virulenceinintranasallyneutropenicmice[27]. Underriboflavinlimitation,the∆riboBstrainismore
sensitive to nitric oxide and produces less siderophores, further reducing its ability to survive in
thehost[27]. Together, thesefindingsindicatethatriboflavinbiosynthesisoffersattractivetargets
for the development of novel antifungals. The crystal structure of the last two enzymes in the
pathway, Rib4 lumazine synthase and Rib5 riboflavin synthase, has been elucidated in the yeast
Schizosaccharomycespombe,C.albicans,andCandidaglabrata[40]. Small-moleculescreenshaveidentified
severalcompoundsthatinhibittheactivityofpurifiedS.pombelumazinesynthaseRib4p,although
they lack antifungal activity because of poor cell penetration [42]. To identify compounds that
inhibitriboflavinbiosynthesisandcanenterthefungus,wescreenedasmall-moleculelibraryagainst
A.fumigatusandidentifiedtwocompoundsthatlosetheirantifungalactivitywhenexcessriboflavinis
addedtothegrowthmedium,bypassingtheneedfordenovosynthesis[24]. Wepresumethatthese
compoundsinhibitakeystepinfungalriboflavinbiosynthesis. Ongoingworkwilldeterminetheir
precisetargets.
Interestingly, many species of bacteria, including Mycobacterium tuberculosis, lack exogenous
riboflavinuptakesystemsandarecompletelyreliantonendogenousbiosynthesis. Severalsubstrate
analogs or antimetabolites with moderate activity against purified Rib4p or Rib5p and weak
activity against M. tuberculosis bacteria in culture have been described. Taken together, these
findingsdemonstratethefeasibilityofdevelopingriboflavinbiosynthesisinhibitorsasantimicrobial
drugs[53,54].
3.3. VitaminB3(Niacin/nicotinicAcid)
Vitamin B3 or niacin is a precursor of NAD and NADP, which are coenzymes of many
dehydrogenases. Three lines of evidence suggest that the niacin biosynthetic pathway is not a
good target for antifungal development: (i) Niacin is not strictly an essential vitamin, as it can be
synthesizeddenovofromtryptophan(throughtheconservedkynureninepathway)bymostorganisms,
includingplants,bacteria,fungi,andanimals[55];(ii)niacinauxotrophydoesnotaffectvirulencein
A.nidulans[33];and(iii)C.glabrata,whichisanaturalniacinauxotroph,isneverthelessaneffective
pathogen[56].
3.4. VitaminB5(PantothenicAcid)
PantothenicacidisaprecursoroftheessentialcofactorCoA,whichfunctionsinmanycentral
cellularmetabolicpathways,includingfattyacidandcarbohydratemetabolism,aswellaspolyketide
and nonribosomal peptide biosynthesis [57]. Four enzymes, encoded by the genes panB-E, are
responsibleforthebiosynthesisofpantothenicacidfromaspartate/spermineandketoisovalerateina
pathwayfoundinmostbacteria,fungi,andplants,butnotinmammals(Figure1C).Inallorganisms,
exogenouslyavailablepantothenicacidcanbeimportedintothecellbyapantothenatetransporter.
In S. cerevisiae, deletion of ECM31 and PAN6, homologous to bacterial panB and panC, results in
pantothenicacidauxotrophy. InC.albicans,downregulationofECM31expressionstronglyreduces
fungalburden[9]. InthepathogenicyeastH.capsulatum,auxotropicpan6RNAiknockdownsshow
reducedlungandspleenfungalloadsininfectedmice,indicatingreducedvirulence[38].InA.nidulans,
deletionofpantoA/pan6andpantoB/ECM31resultsinpantothenicacidauxotrophy[58]. InA.fumigatus,
panA/pan6deletionalsoresultsinpantothenicacidauxotrophy. Invitro,growthofthismutantunder
limitingpantothenicacidandironisstronglyreduced,probablybecausepantothenicacidisneeded
for siderophore biosynthesis. The A. fumigatus ∆pan6 mutant is avirulent in systemically infected
neutropenicmiceandstronglyreducedinvirulenceinlung-infectedneutropenicmice[27].Importantly,
pantothenicacidbiosynthesishasbeenstudiedasapossibleantibacterialtargetsincethe1940s. The
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enzymesencodingpanBwerepurifiedandcrystalizedinE.coliandM.tuberculosis,enablingaccurate
determinationoftheirstructureandthedevelopmentofspecificinhibitors[57].Amajorefforthasbeen
madetodeveloppantothenatesynthase(panC)inhibitorsactiveagainstM.tuberculosis,asthisenzyme
isessentialforvirulence[59]. Whiletheseapproachesyieldedeffectiveinhibitors(submicromolar
range)ofthepurifiedenzyme,theiractivitywasstronglyreduced(~twoordersofmagnitude)against
theintactorganismandnonewastestedinvivo. Itwouldbeofgreatinteresttoelucidatethecrystal
structureofafungalpantothenatesynthaseandtotesttheactivityofavailablebacterialinhibitors
andspecificallydesignedinhibitorsagainstthepurifiedfungalenzymeandtheintactfungusinthe
presence/absenceofexogenouspantothenicacid.
3.5. VitaminB6(Pyridoxine)
Vitamin B6, in the form of pyridoxal 5(cid:48)-phosphate (PLP), is a cofactor for a large number of
essentialenzymestakingpartincarbohydrate,aminoacid,andfattyacidmetabolism. PLPcanalso
actdirectlyasaprotectiveagentagainstreactiveoxygenspecies[60]. Bacteria,fungi,andplantsare
abletosynthesizevitaminB6,whereasmostanimals,includinghumans,lackthisabilityandacquire
itfromtheirfood. Fungiandplantsusethedeoxy-xylose5(cid:48)-phosphate(DXP)-independentvitamin
B6biosynthesispathwaytogeneratePLPdenovo. Twosynthaseproteins,encodedbytheSNZ1and
SNO1genesinS.cerevisiae,directlysynthesizePLPfromribose5(cid:48)-phosphateorribulose5(cid:48)-phosphate,
in combination with glyceraldehyde 3(cid:48)-phosphate and glutamine (Figure 1D) [61]. Baker’s yeast
also use the high-affinity transporter Tpn1p to uptake pyridoxine from their environment [62].
Amongthepathogenicfungi,pyridoxineauxotrophshaveonlybeengeneratedinA.nidulansand
A. fumigatus. In A. nidulans, mutational inactivation of pyroA, the homolog of S. cerevisiae SNZ1,
resultsinauxotrophyandstronglyattenuatesvirulenceinsystemicallyinfectedmice[33]. Similarly,
deletionofA.fumigatuspyroAresultsinauxotrophyandstronglyattenuatesvirulence(70%survival)in
neutropeniclung-infectedmice,althoughvirulenceisonlypartiallyattenuatedinsystemicallyinfected
neutropenicmice(100%mortality10dayspost-infection)[27]. Therefore,inhibitingPLPbiosynthesis
maynotbeanoptimalantifungalstrategyunlesspyridoxineuptakeisalsoblocked. Interestingly,
deletionofPDX1, thehomologofS.cerevisiaeSNZ1inthepathogenicbacteriaM.tuberculosisand
Helicobacter pylori, results in pyridoxine auxotrophy and severely attenuates colonization in mice,
suggestingthatthispathwaycontainspotentantibacterialdrugtargets[63,64]. Thepdx1geneofthe
malariaparasiteP.falciparumisalsoessentialforvirulence,andinhibitorswithactivityagainstintact
plasmodiumhavebeendescribed[65,66].
3.6. VitaminB7(Biotin)
Biotin,aprostheticgroupincarboxylases/decarboxylases,isproducedbyplants,bacteria,and
mostfungi. Infungi, biotinissequentiallysynthesizedfrompimeloyl-CoAbythreeBioenzymes:
Bio6p,aKAPAsynthase;Bio3/4p,achimericproteincomposedofDTBandDAPAsynthases;and
Bio2p,abiotinsynthase(Figure1E)[67]. Severallinesofevidencesuggestthatthebiotinbiosynthetic
pathwayisnotsuitableasanantifungaltarget: C.albicanslacksBIO6andisanaturalbiotinauxotroph
invitro. Despite this, it is a successful pathogen and grows well invivo [45]. Likewise, RNAi
knockdown/mutation/gene deletion of bio2/bioB in H. capsulatum, A. nidulans, and A. fumigatus,
respectively,resultsinbiotinauxotrophyinvitro,butdoesnotaffectvirulenceininfectedanimals,
suggestingthereissufficientbiotininthehosttoallownormalfungalgrowthandvirulenceper[33,38]
andourunpublishedfindings.
3.7. VitaminB9(Folate)
Folatesareessentialcofactorsforthebiosynthesisofpurinesandthymidylate,andhenceforDNA
replication. Bacteria,plants,andfungicansynthesizefolatedenovo,generatingdihydrofolate(DHF)
frompara-aminobenzoicacid(PABA;derivedfromchorismateandglutamine)anddihydropteridine
diphosphate (DHPP; derived from GTP). This reaction is sequentially catalyzed by the enzymes
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dihydropteroatesynthase(DHPS)encodedbyFOL1anddihydrofolatesynthase(DHFS)encodedby
FOL3(Figure1F).HumanslackthoseenzymesandmustobtainfolateintheformofDHFfromtheir
food,subsequentlyconvertingitintotetrahydrofolate(THF)inareactioncatalyzedbydihydrofolate
reductase (DHFR). DHFR is highly conserved among all animals, plants, and microorganisms.
In S. cerevisiae, deletion of ABZ1, encoding PABA synthase, and FOL1/DHPS result in PABA and
folate auxotrophy, respectively [68]. The importance of the pathway in fungal virulence has only
beenstudiedinA.nidulansandA.fumigatus. Inbothfungi,deletionofpabA/ABZ1,encodingPABA
synthase,resultsinPABAauxotrophyandtotalavirulence[19,46]. VirulenceofthepabAnullmutant
wasrestoredwhenthemicereceivedPABAintheirdrinkingwater. Importantly,withdrawalofPABA
supplementation during late infection blocked further lethality, suggesting that inhibitors of pabA
willlikelybeactiveagainstestablishedinfection. InhibitorsofbacterialPABAsynthasewererecently
identifiedbuthavenotundergoneinvivoorantifungaltesting[69]. FOL1/DHPSisalsoaviabletarget
forantifungaldevelopment: DeletionofA.fumigatusFOL1/DHPS(Afu2g09840)islethalandcannotbe
complementedbyfolateorfolinicacidsupplementation,suggestingthatthefungusreliesexclusively
onendogenousproduction(ourunpublishedfindings). InhibitorsofbacterialDHPS,betterknownas
sulfadrugs,havebeeninclinicalusesincethe1930s[70]. TheyaresimilartothesubstratePABAand
competitivelyinhibititsbindingtoDHPS.Theyalsoreactwiththesecondsubstrate,DHPP,depleting
itspoolandforminganinhibitorydead-endproduct. Currentsulfadrugsexhibitonlyweakantifungal
activity. DerivativeswithhigheractivityagainstpurifiedC.albicansDHPSwereidentifiedbuttheyare
ineffectiveagainstthewholeorganism,possiblybecauseofefflux[49]. TheS.cerevisiaeDHPScrystal
structureisavailableandcouldbeusedtofacilitatethediscoveryofimprovedfungalDHPSinhibitors.
AnattractiveoptionistodevelopbisubstrateinhibitorsmimickingbothPABAandDHPP.Thiscan
increasetheiraffinityandspecificitycomparedwiththeexistingsulfamonosubstrateinhibitors[48].
TheenzymeDHFRishighlyconservedbetweenhumansandmicroorganisms, andseemsan
unlikelycandidateforthedevelopmentofantimicrobials. Nevertheless,drugsthatselectivelyinhibit
humanDHFR(e.g.,thechemotherapyagentmethotrexate)andmicrobialDHFR(e.g.,trimethoprim)
weredevelopedandareinwidespreadclinicaluse[16]. Theyarecompetitiveinhibitorsthatmimicthe
naturalsubstrate,DHF.However,forbothantimicrobialDHPSandDHFRdrugs,resistancerapidly
emergesbecauseofpointmutationsintheactivesiteoftheenzyme. Thisproblemhasbeenaddressed
by combination therapy. The simultaneous inhibition of DHPS and DHFR is both synergistic and
highlyeffectiveincombattingresistance.Itisusedtotreatbacterialinfections(e.g.,resprim,containing
sulfamethoxazole and trimethoprim), malaria (e.g., Lapdab, a combination of chlorproguanil and
dapsone),andfungalpneumoniacausedbyP.jorovecii(resprim)[71].
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and cannot be complemented by folate or folinic acid supplementation, suggesting that the fungus
relies exclusively on endogenous production (our unpublished findings). Inhibitors of bacterial
DHPS, better known as sulfa drugs, have been in clinical use since the 1930s [70]. They are similar to
the substrate PABA and competitively inhibit its binding to DHPS. They also react with the second
substrate, DHPP, depleting its pool and forming an inhibitory dead-end product. Current sulfa drugs
exhibit only weak antifungal activity. Derivatives with higher activity against purified C. albicans
DHPS were identified but they are ineffective against the whole organism, possibly because of efflux
[49]. The S. cerevisiae DHPS crystal structure is available and could be used to facilitate the discovery
of improved fungal DHPS inhibitors. An attractive option is to develop bisubstrate inhibitors
mimicking both PABA and DHPP. This can increase their affinity and specificity compared with the
existing sulfa monosubstrate inhibitors [48].
The enzyme DHFR is highly conserved between humans and microorganisms, and seems an
unlikely candidate for the development of antimicrobials. Nevertheless, drugs that selectively inhibit
human DHFR (e.g., the chemotherapy agent methotrexate) and microbial DHFR (e.g., trimethoprim)
were developed and are in widespread clinical use [16]. They are competitive inhibitors that mimic
the natural substrate, DHF. However, for both antimicrobial DHPS and DHFR drugs, resistance
rapidly emerges because of point mutations in the active site of the enzyme. This problem has been
addressed by combination therapy. The simultaneous inhibition of DHPS and DHFR is both
synergistic and highly effective in combatting resistance. It is used to treat bacterial infections (e.g.,
resprim, containing sulfamethoxazole and trimethoprim), malaria (e.g., Lapdab, a combination of
J.Fungi2018,4,72 8of14
chlorproguanil and dapsone), and fungal pneumonia caused by P. jorovecii (resprim) [71].

(a)

(b)

Figure1.Cont.
J. JF.uFnugnig 2i021081,8 4,,4 x, 7F2OR PEER REVIEW 8 9ofo 1f31 4

(c)

(d)
(e)
Figure1.Cont.
J. FJu.nFguin 2g0i1280,1 48,, x4 ,F7O2R PEER REVIEW 9 1o0f 1o3f 14
(f)
Figure 1. Overview of the S. cerevisiae pathways responsible for the biosynthesis of (A) vitamin
Figure 1. Overview of the S. cerevisiae pathways responsible for the biosynthesis of (A) vitamin
B1/thiamine;(B)vitaminB2/riboflavin;(C)vitaminB5/pantothenicacid;(D)vitaminB6/pyridoxine;
B1/thiamine; (B) vitamin B2/riboflavin; (C) vitamin B5/pantothenic acid; (D) vitamin B6/pyridoxine;
(E) vitamin B7/biotin; and (F) vitamin B9/folate. Red box indicates the vitamin product. Genes
(E) vitamin B7/biotin; and (F) vitamin B9/folate. Red box indicates the vitamin product. Genes denoted
denotedwith*havenohumanhomologs.
with * have no human homologs.
3.8. VitaminB12(Cobalamins)
3.8. Vitamin B12 (Cobalamins)
Cobalaminsareorganometalliccofactorsusedbyalimitednumberofessentialenzymes,notably
Cobalamins are organometallic cofactors used by a limited number of essential enzymes, notably
methionine synthase and methylmalonyl-CoA mutase [72]. Only bacteria and archaea have the
methionine synthase and methylmalonyl-CoA mutase [72]. Only bacteria and archaea have the
enzymes required for vitamin B synthesis. Fungi, plants, and animals (including humans) are
enzymes required for vitamin B12 1s2ynthesis. Fungi, plants, and animals (including humans) are
incapableofproducingvitaminB andthusthispathwaydoesnotconstituteaviableantifungaltarget.
incapable of producing vitamin B1122 and thus this pathway does not constitute a viable antifungal
target.
4. MetabolicPathwayInhibitorsandtheGenerationofResistance
4. MetaEbxopleicr iPenatchewhaays tIanuhgibhittuosrst haantda tnhteim Gicernoebriaatlisotna rogfe Rtiensgismtaentcaeb olicpathwaysarehighlyproneto
thedevelopmentofresistance(e.g., antifolates, azoles)becauseofmutationsinthetargetenzyme.
Experience has taught us that antimicrobials targeting metabolic pathways are highly prone to
Nonetheless, many strategies can be used to slow down the development of drug resistance.
the development of resistance (e.g., antifolates, azoles) because of mutations in the target enzyme.
They include: (i) The use of drug combinations (e.g., inhibiting both DHPS and DHFR activity
Nonetheless, many strategies can be used to slow down the development of drug resistance. They
infolatebiosynthesis–seeabove);(ii)generatingdrugswithmultipletargets;(iii)designingdrugsthat
include: (i) The use of drug combinations (e.g., inhibiting both DHPS and DHFR activity in folate
producetoxicendproductsormetabolites(asoccurswithazoles)andwhicharefungicidalrather
biosynthesis–see above); (ii) generating drugs with multiple targets; (iii) designing drugs that
thanfungistatic,givingnotimeforresistancetodevelop(e.g.,amphotericinB);(iv)developingdrugs
produce toxic end products or metabolites (as occurs with azoles) and which are fungicidal rather
targetingconservedsubstratesorproductsratherthaneasilymutableenzymes(likevancomycinand
than fungistatic, giving no time for resistance to develop (e.g., amphotericin B); (iv) developing drugs
amphotericinB);(v)moreprecisetargetingofthedrug(e.g.,localizingazolestotheirtargetenzymein
targeting conserved substrates or products rather than easily mutable enzymes (like vancomycin and
theER[73]);and(vi)judicioususeofnewdrugs,includinglimitingtheirusetohumans.
amphotericin B); (v) more precise targeting of the drug (e.g., localizing azoles to their target enzyme
in 5t.hCe oEnRc [l7u3s]i)o; nasnd (vi) judicious use of new drugs, including limiting their use to humans.
Insummary,thevitaminB2(riboflavin),B3(pantothenicacid),andB9(folate)pathwaysappear
5. Conclusions
toofferthemostattractiveantifungaldrugtargetsamongtheessentialvitaminbiosyntheticpathways.
TheIny scuonmtaminarwye, ltlh-ceh vairtaacmteirni zBe2d (ernibzoyfmlaevsinth),a Bt3a r(epeasnsteontthieanlfioc rafcuidn)g, aalnvdir Bu9le (nfcoela,itne)c lpuadtihnwgaoynse appapthewara y,
to foolfafteer btihosey nmthoesst isa,twtrhacictihvhe asanatlirfeuandgyapl rodvruedg ittsarwgoetrsth aamsoannga ntthime icersosbeniatliaaln dviatnamtifiunn gbaiolsdyrnutghteatrigc et.
paSthciwenatyisfi.c Tkhneyo wcolendtagien iwnetlhl-echfiaerladctoerfizderdu gendzeysmigens tahnadt afuren egsaslegnetinaol mforic fsuhnagsald veivruelleonpceed, isnucflfiucdiienngt ly
one pathway, folate biosynthesis, which has already proved its worth as an antimicrobial and