Microbial natural products as a source of antifungals

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REVIEW
Microbial natural products as a source of antifungals
M.F.Vicente,A.Basilio,A.CabelloandF.Pela´ez
Centro de Investigac´ıon Ba´sica, Merck Research Laboratories, Merck, Sharp and Dohme Espan˜a, S. A.
Josefa Valca´rcel 38, 28027 Madrid, Spain
Thevastnumberandvarietyofchemotherapeuticagentsisolatedfrommicrobialnatural
productsandusedtotreatbacterialinfectionshavegreatlycontributedtotheimprove-
ment of human health during the past century. However, only a limited number of
antifungalagents(polyenesandazoles,plustherecentlyintroducedcaspofunginacetate)
are currently available for the treatment of life-threatening fungal infections. Further-
more,theprevalenceofsystemicfungalinfectionshasincreasedsignificantlyduringthe
pastdecade.Forthisreason,thedevelopmentofnewantifungalagents,preferablywith
novelmechanismsofaction,isanurgentmedicalneed.Aselectionofantifungalagentsin
early stages of development, produced by micro-organisms, is summarized in this
review.Thecompoundsareclassifiedaccordingtotheirmechanismsofaction,covering
inhibitorsofthesynthesisofcellwallcomponents(glucan,chitinandmannoproteins),of
sphingolipid synthesis (serine palmitoyltransferase, ceramide synthase, inositol phos-
phoceramidesynthaseandfattyacidelongation)andofproteinsynthesis(sordarins).In
addition,someconsiderationsrelatedtothechemotaxonomyoftheproducingorganisms
and some issues relevant to antifungal drug discovery are also discussed.
Keywords Secondary metabolites, antifungals, natural products, fungi
Accepted 19 February 2002
Clin Microbiol Infect 2003; 9: 15–32
havebeendescribedsofar,secondarymetabolites
INTRODUCTION
still appear to be an inexhaustible source of lead
The exploration of micro-organisms as sources of structures for new antimicrobials, antivirals, anti-
therapeutically useful compounds has a much tumour drugs, and agricultural and pharmacolo-
shorter and less well-known history than the use gical agents. In addition, numerous secondary
of plants and plant extracts in human medicine. metabolites, such as benzylpenicillin, cephalos-
Secondary metabolites are defined as naturally porins, erythromycin, strobilurin, etc. were lead
producedsubstanceswhichdonotplayanexplicit structuresthatlaterbecamethebasisforsynthetic
roleintheinternaleconomyoftheorganismsthat and semisynthetic derivatives with improved
produce them. These micro-organisms may have pharmacological properties.
evolved the ability to produce such compounds Fungal infections range from superficial condi-
because of the selection advantages conferred tionsoftheskin(e.g.ringwormandathlete’sfoot)
upon them as a result of the interactions of the and nails (onychomycoses) to disseminated life-
compoundswithspecificreceptorsinotherorgan- threatening diseases. Serious invasive fungal
isms[1,2].Althoughalmost20000microbialmeta- infections caused by Candida spp., Cryptococcus
bolites and approximately 100000 plant products neoformans, Aspergillus spp., Pneumocystis carinii
and Histoplasma capsulatum, represent an increas-
Corresponding author and reprint requests: M. F. Vicente, ing threat to human health. The prevalence of
Centro de Investigacio´n Ba´sica, Merck Research Labora- thesesystemicfungalinfectionshasincreasedsig-
tories, Merck, Sharp and Dohme de Espan˜a, S.A. Josefa nificantly during the past decade.
Valca´rcel38,28027Madrid,Spain
Major factors responsible for this dramatic
Tel:þ34913210720
rise include greater use of broad-spectrum anti-
Fax:þ34913210614
E-mail:[email protected] biotics, marked increases in the numbers of
(cid:1) 2003CopyrightbytheEuropeanSocietyofClinicalMicrobiologyandInfectiousDiseases
16 Clinical Microbiology and Infection, Volume 9 Number 1, January 2003
immunocompromised persons (AIDS, cancer and
Glucan synthesis inhibitors
transplant patients), the use of central venous
catheters, and an aging patient population [3,4]. Atpresent,glucansynthesisistheonlycomponent
Until the 1970s, fungal infections were consid- of the cell wall synthesis machinery that has suc-
ered largely treatable and the demand for new cessfullyledtothedevelopmentofanewdrugon
medicinestotreatthemwasverysmall.Beforethis themarket.Glucanisapolysaccharideconstituted
period, antifungal chemotherapy included only by glucose monomers linked by (1,3)-b or (1,6)-b
twokindsofcompounds:potassiumiodide,effec- bonds,anditisanessentialcomponentofthecell
tive in the treatment of sporotrichosis; and two wall,guaranteeingmanyofitsphysicalproperties
useful polyenes, nystatin and amphotericin B, [8].Inhibitorsofglucansynthesishavebeenshown
which were introduced in the 1950s. Except for topossessantifungalactivityinvitroaswellasin
the development of flucytosine (1964), there was vivo in many different animal models. The most
little progress until the development of the azole classicalexamplesofinhibitorsofglucansynthesis
drugsintheearly1970s.Therefore,onlyalimited are the echinocandins. These are cyclic hexapep-
numberofantifungalagents(polyenesandazoles tides N-acylated with an aliphatic chain of differ-
plus the recently introduced Cancidas) are cur- entlength[9].Thevariousechinocandinsdifferin
rently available for the treatment of life-threaten- having different substituents in the hexapeptide
ing fungal infections. These antifungal agents ring or adistinctfattyacid chain (Figure 1). Since
show some limitations, such as the significant thefirstechinocandinwasdiscoveredintheearly
nephrotoxicityofamphotericinB[5]andemerging 1970s[10],manymembersofthefamilyhavebeen
resistance to the azoles [6], despite several recent discovered in diverse fungi (Table1).
improvements,suchaslipidformulationsofpoly- The pneumocandins in particular have been
enes with lower toxicity and new triazoles (vor- successfully used to develop an antifungal drug
iconazole, rovuconazole and pasaconazole) with thathasbeenrecentlyapprovedbytheFDA.This
a wider spectrum of action, including activity semi-synthetic pneumocandin, caspofungin acet-
againstsomeazole-resistantisolates[7].Thedevel- ate, is an aza-substituted derivative of pneumo-
opmentofnewantifungalagents,preferablynatu- candin B [11]. Pneumocandins are natural
0
rallyoccurringwithnovelmechanismsofaction,is products derived from the fermentation of the
an urgent medical need. fungus Glarea lozoyensis [12,13]. The introduction
This review presents a selection of antifungal of additional amino groups in the peptide ring of
agents produced by micro-organisms, classified pneumocandin B increased the solubility of the
0
accordingtotheirmodeofaction.Someconsidera- molecule and the potency against fungal patho-
tionsrelatedtothechemotaxonomyoftheproduc- gens by two orders of magnitude [14]. The com-
ing organisms and some issues relevant for pound has been shown to be effective in animal
antifungal drug discovery are also discussed. modelsofdisseminatedcandidiasis,aspergillosis,
coccidiomycosis and pneumonia caused by Pneu-
mocystis carinii [15–18]. The clinical trials have
CELL WALL BIOSYNTHESIS
demonstrated good tolerance of the compound
INHIBITORS
and its efficacy in the treatmentof oropharyngeal
One of the targets for novel antifungals under andoesophagealcandidiasis,aswellasininvasive
active investigation is the fungal cell wall. Anti- aspergillosis [19,20]. Cancidas has recently been
fungal agents acting on this target are inherently approved by the FDA for use against invasive
selective.Thischaracteristicandthefactthatthey aspergillosis, refractory to, or intolerant of, other
arefungicidalforCandidamakethemparticularly therapies.
attractive for clinical development. Fungal cell Other echinocandin-like antifungal agents in
wall composition varies among species, but it advanced phases of drug development include
generally has three polymeric components: glu- FK463 (micafungin), an echinocandin derivative
can, chitin and mannoproteins. The subcellular with a sulphate ester moiety in the hexapeptide
mechanismsoftheirsynthesisandassemblyhave nucleus [21], and LY303366 (V-echinocandin, ani-
been used as potential targets to search for new dulafungin), which has a terphenyl head group
antifungals and numerous natural products have and a C5 chain [22]. Other echinocandin deriva-
beenidentifiedasinhibitorsactingattheselevels. tives have also been reported from Fujisawa [23],
(cid:1) 2003CopyrightbytheEuropeanSocietyofClinicalMicrobiologyandInfectiousDiseases,CMI,9,15–32
Vicente et al Microbial natural products as source of antifungals 17
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18 Clinical Microbiology and Infection, Volume 9 Number 1, January 2003
Table1 Naturalproductsinhibitorsofglucansynthesis
Compound Producingspecies Reference
Lipopeptides
EchinocandinB Aspergillusnidulans NyfelerandKeller1974[10]
A.rugulosus
Aculeacin Aspergillusaculeatus Mizunoetal.1977[107]
Mulundocandin Aspergillussydowii Royetal.1987[25]
Sporiofungins Penicilliumarenicola TscherterandDreyfuss1982[108]
Cryptosporiopsissp.
Pneumocandins Glarealozoyensis Schwartzetal.1992[12]
Peziculasp. Billsetal.1999[13]
Cryptosporiopsissp. Nobleetal.1991[109]
Cryptocandin Cryptosporiopsisquercina Strobeletal.1999[110]
WF11899andrelated Coleophomaempetri Hori1999[111]
sulfate-derivatives
Coleophomacrateriformis
Tolypocladiumparasiticum
Chalarasp.
FR901469 Unidentifiedfungus Fujieetal.2000[27]
Arborcandins Unidentifiedfungus Ohyamaetal.2000[26]
Clavariopsins Clavariopsisaquatica Kaidaetal.2001[28]
Glycolipids
Papulacandins Papulariasphaerosperma Traxleretal.1977[29]
Corynecandin Coryneummodonium Gunawardanaetal.1997[112]
Mer-WF3010 Phialophoracyclaminis Kanetoetal.1993[113]
Fusacandin Fusariumsambucinum Yeungetal.1996[31]
BU-4794F Gilmaniellasp. Aokietal.1993[114]
L-687781 Dictyochaetasimplex VanMiddlesworthetal.1991[115]
Acidicterpenoids
Efumafungin Hormonemasp. Pela´ezetal.2000[34]
Arundifungin Arthriniumarundinis Cabelloetal.2001[33]
A.phaeospermum
Leotialesanamorphs
Coelomyceteundetermined
Ascoteroside Ascotrichaamphitricha Onishietal.2000[32]
Mycoleptodiscusatromaculans
ErgokoninA Trichodermalongibrachiatum Vicenteetal.2001[35]
T.koningii
T.viride
Roche (aerothricins) [24] and Aventis (mulundo- lipophilic radical, have also been suggested as
candins) [25]. actingasinhibitorsofglucansynthesis[28].How-
The success of the lipopeptide class of glucan ever, to date only two other types of glucan
synthesis inhibitors has prompted interest from synthesisinhibitorsareknown,besidescycliclipo-
theindustryinthesearchforotherstructuraltypes peptides, the papulacandins and related com-
with improved features over the echinocandins pounds, and the acidic triterpenes.
(especially for their lack of oral absorption). The papulacandins are glycolipids discovered
Besides echinocandins and the like, other cyclic in the late 1970s [29]. A series of related com-
peptides have been described as acting as glucan pounds has been discovered over the years, all
synthesis inhibitors. Arborcandins are recently ofthemproducedbyfungi(Table1andFigure1).
described antifungal agents, containing a 10- Despitemedicinalchemistryefforts,neitherpapu-
amino-acidringandtwolipophilictails[26].Like- lacandins nor any of their relatives have been
wise,thecompoundnamedFR901469isamacro- developedasdrugs,basicallyduetotheirlimited
cyclic lipopeptidolactone composed of 12 amino potency in animal models [30,31].
acids and a 3-hydroxypalmitoyl moiety [27]. Cla- The most recently discovered compound class
variopsins, cyclic depsipeptides lacking a long of glucan synthesis inhibitors are triterpenes
(cid:1) 2003CopyrightbytheEuropeanSocietyofClinicalMicrobiologyandInfectiousDiseases,CMI,9,15–32
Vicente et al Microbial natural products as source of antifungals 19
containing a polar (acidic) moiety [32]. This polar synthase II, an essential enzyme for primary sep-
moiety can be a glycoside (in enfumafungin and tum formation between mother and daughter
ascosteroside), a succinate (in arundifungin) or a cells; and chitin synthase III, which synthesizes
sulphate-derivative amino acid (in ergokonin A) lateral chitin in the cell wall [38].
(Figure 1). The conclusion that these compounds The classical inhibitors of chitin synthesis are
areactingasinhibitorsofglucansynthesisisbased nikkomycins andpolyoxins[39].Bothbelongtoa
onseverallines ofevidence:first,thespectrum of family of peptide-nucleoside antimycotic agents
activity, being active against Aspergillus and Can- that are substrate analogues of UDP-N-acetylglu-
didaspecies,butinactiveagainstCryptococcus;sec- cosamine, the essential building block for chitin
ond, they induced the same alterations in the biosynthesisandwereisolatedfromtwodifferent
micromorphologyofAspergillusfumigatusasother Streptomycesspecies:S.tendae(nikkomycin)andS.
inhibitors of glucan synthesis [33–35]; and third, cacaoi var. asoensis (polyoxin). Candida albicans is
direct measurement of the effect of these com- resistant to polyoxins due to the difficulty that
pounds on synthesis of cell wall macromolecules these agents present in being transported to the
indicatesthatglucanistheonlypolymerofwhich interiorofthecellwall.Nikkomycinsexhibitactiv-
the synthesis is significantly altered upon treat- ity against dimorphic fungi but low activity
mentwiththeseagents[32].Althoughthesecom- against yeast and filamentous fungi. However,
poundswereinactiveoronlyweaklyactiveinthe both demonstrated synergy with the azole com-
invivomousemodel[33–35],theyrepresentanew poundsandwithb-glucaninhibitors[40,41].Nik-
paradigminthesearchforantifungalcompounds komycins and polyoxins are currently used
withthismodeofaction,andtheycouldbeuseful exclusivelyasagriculturalfungicides,duetotheir
asabaseforthedevelopmentofimproveddrugs. modest activity against human pathogens [42].
Clearly,aweakpointinalltheglucansynthesis Recently, as a part of the continuing screening
inhibitors discovered or developed up to date is fornewchitinsynthaseinhibitors,twonovelanti-
their lack of activity against C. neoformans. The fungal compounds were found: phellinsin A and
reasons for this lack of activity are unclear. The arthrichtin. Phellinsin A is a phenolic compound
hypothesis that echinocandins do not inhibit b- whichselectivelyinhibitedchitinsynthaseIandII
(1,6)glucansynthesis,whichseemstobethemain of S. cerevisiae. In addition, this compound exhib-
glucan in C. neoformans cell wall [30], has been ited antifungal activity against human pathogens
recently contradicted experimentally [36]. More- such as Trichophyton mentagrophytes and A. fumi-
over, the FKS1 homologue gene, coding the cata- gatusandveryweakactivityagainstotherhuman
lytic subunit of b-(1,3)-glucan synthase, has been pathogens such as C. neoformans and Coccidioides
shown to be essential in C. neoformans. However, immitis.However,itshowednoactivityagainstC.
the enzyme could be relatively resistant to the albicans, C. lusitaniae, C. krusei, C. tropicalis and
actionofechinocandinsandtherestoftheglucan Fusarium oxysporum [43]. Arthrichitin is a cyclic
synthesis inhibitors [36]. depsipeptidewhichwasisolated fromArthrinium
phaeospermumand(asLL156256g)fromthemarine
fungus Hypoxylon oceanicum [44,45]. This com-
Chitin synthesis inhibitors
pound showed activity against Candida spp.,
Chitin is an insoluble polysaccharide made of b- Trychophyton spp. and several phytopathogens.
(1,4)-linked N-acetylglucosamine units. This bio- Although its in vitro potency is too low for its
logical polymer is one of the structural microfi- use in the clinic, it has been suggested that ana-
brillarcomponentsofthefungalcellwallstructure logueswithimprovedactivitycouldbedeveloped
which maintains the morphological shape of the [44].
cellsandplaysanessentialroleinfungalmorpho-
genesis[37].Inyeasts,chitinaccountsfor1%ofthe
Mannoprotein synthesis inhibitors
cellwallandisdistributeddifferentlyfromglucan.
Chitin also links covalently to the cellular glucan, Mannoproteins are the third main component of
thereby strengthening the wall. There are at least the fungal cell wall. They form the outer layer of
three different chitin synthases in Saccharomyces thecellwallandcontainasmuchas50%carbohy-
cerevisiae:chitinsynthaseI,whichisinvolvedina drate.Themajorityofthecellwallmannoproteins
repair function at the time of cytokinesis; chitin are anchored by b-(1,6)- and b-(1,3)-glucan [46]
(cid:1) 2003CopyrightbytheEuropeanSocietyofClinicalMicrobiologyandInfectiousDiseases,CMI,9,15–32
20 Clinical Microbiology and Infection, Volume 9 Number 1, January 2003
and play several roles in the function of fungal inositol phosphoceramide (IPC) synthase; the lat-
membranes.Therefore,theyhavebeenconsidered terlacksamammaliancounterpartand,asshown
another potential target in fungal membranes for in Figure 2, inhibitors to all three have been dis-
antimycoticagents.Untilnow,anymannoprotein covered from natural sources. Sphingofungins
associated with the cell wall has been considered [51,56], lipoxamycin [57] and viridiofungins [58]
as essential by examining for viability after gene inhibit serine palmitoyltransferase. Fumonisin B1
disruption experiments. Inhibitors of the manno- [54,59] and australifungin [52] inhibit ceramide
proteins function are the pradimicin/benanomy- synthase, and aureobasidins [60], khafrefungin
cin family, whose chemical structure possesses a [61], and rustmicin [62] inhibit IPC synthase.
benzo [a] naphthacenequinone skeleton [47,48]. Furthermore,minimoidin,aninhibitorofthefatty
Thefreecarboxylgroupofthesecompoundsinter- acid elongation pathway, was isolated from a
acts with the saccharide portion of cell-surface coprophilic fungus [63]. The chemical structures
mannoprotein, which is followed by disruption ofthesecompoundsarepresentedinFigure3,and
of the plasma membrane and leakage of intracel- the micro-organisms from which they were iso-
lular potassium. These are antifungal (and anti- lated are shown in Table2.
viral) agents without significant acute toxicities,
produced by Actinomadura (Actinomycetes) spe-
Serine palmitoyltranferase inhibitors
cies.Theseantibioticsexhibitedremarkableinvivo
activity against systemic fungal infections caused SphingofunginsAtoFconstituteafamilyofnovel
by C. albicans, A. fumigatus and C. neoformans in chemical structures that resemble the long-chain
mice. Pradimicin/benanomycin and analogues base intermediates in the sphingolipid pathway.
werestudiedinexperimentalanimalmodelswith They were initially identified as broad spectrum
goodsuccessrates;however,phaseIclinicaltrials antifungalagents,inhibitingthegrowthofvarious
suggested drug-related toxicities and develop- Candida species and showing anespeciallypotent
ment was stopped [49]. activityagainstC.neoformans,buttheyareinactive
against filamentous fungi [56,64].
Lipoxamycinwasdiscoveredin1970asananti-
SPHINGOLIPID SYNTHESIS
fungal compound of unknown mechanism of
INHIBITORS
action [65]. Lipoxamycin and hydroxylipoxamy-
Sphingolipids,althoughpresentinrelativelysmall cin,ananalogueco-producedinthefermentation,
proportion in the fungal cytoplasmic membrane, have a long alkyl chain and an amino-containing
areessentialforcellularfunctions[50],andinhibi- polar head group, but otherwise do not resemble
tion of sphingolipid synthesis results in growth the sphingoid bases as closely as the sphingofun-
inhibition and cell death [51,52]. Ceramide has ginsdo.Bothcompoundshaveantifungalactivity
been implicated as a component of an essential against a panel of human pathogenic fungi, with
cell-signalling pathway in Saccharomyces [53]. better potency against some of the C. neoformans
Sphingoid bases also have a regulatory role in andCandidaspecies.A.fumigatuswasnotinhibited
yeast where they have been shown to inhibit in broth dilution assays, but other filamentous
severalkeyenzymesinphospholipidbiosynthesis fungi were sensitive to the lipoxamycins in disk
[54]. Sphingolipids are also involved in the syn- diffusion assays [57].
thesis of glycosylphosphatidylinositol anchors in Viridiofungins A, B and C comprised a novel
Saccharomyces, and they appear to be the major family of amino alkyl citrates that have potent,
repositoryforverylongchainfattyacids(C24and broad spectrum antifungal activity, inhibiting the
C26 species) in fungi [55]. growthofpathogenicfungisuchasC.neoformans,
Although manysteps inthe human and fungal Candida species and A. fumigatus [66].
sphingolipid biosynthetic pathway are similar, It has been demonstrated by a variety of biolo-
thereareseveralenzymesuniquelyfoundinfungi gicalandbiochemicalmeansthatsphingofungins,
that make sphingolipid synthesis attractive for lipoxamycin,hydroxylipoxamycinandviridiofun-
antifungal therapy (Figure 2). Three key enzymes gins are specific inhibitors of serine palmitoyl-
in the sphingolipid synthesis pathway have been transferase in fungi at nanomolar concentrations
targeted to search for novel antifungals: serine [51,56,58]. The potent in vitro inhibition of serine
palmitoyltransferase, ceramide synthase and palmitoyltransferase and inhibition of whole cell
(cid:1) 2003CopyrightbytheEuropeanSocietyofClinicalMicrobiologyandInfectiousDiseases,CMI,9,15–32
Vicente et al Microbial natural products as source of antifungals 21
Figure2 Sphingolipid biosynthesis pathway in fungal and mammalian cells. The different inhibitors of sphingolipid
synthesisareindicatedinthecorrespondingstepsofthepathwaytheyareblocking.
sphingolipid synthesis at minimum inhibitory Viridiofungins are not as specific for serine
concentrations (MICs) pointed to inhibition of palmitoyltransferase inhibition as the other inhi-
serine palmitoyltransferase as the likely mechan- bitors of the enzyme. They also inhibit squalene
ism of their antifungal activity. synthase and other enzymes that are sensitive to
(cid:1) 2003CopyrightbytheEuropeanSocietyofClinicalMicrobiologyandInfectiousDiseases,CMI,9,15–32
22 Clinical Microbiology and Infection, Volume 9 Number 1, January 2003
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Vicente et al Microbial natural products as source of antifungals 23
Table2 Naturalproductsinhibitorsofshingolipidbiosynthesisandproteinsynthesis
Compound Producingspecies Reference
Sphingolipidbiosynthesis
Sphingofungins Aspergillusfumigatus Zweerinketal.1992[51]
Paecilomycesvariotii Hornetal.1992[56]
Lipoxamycin Streptomycessp. Mandalaetal.1994[57]
Viridiofungins Trichodermaviride Mandalaetal.1997[58]
Myriocin Isariasinclairii Miyakeetal.1995[67]
FumonisinB1 Fusarioummoniliforme Wangetal.1991[59]
Australifungin Sporormiellaaustralis Mandalaetal.1995[52]
AureobasidinA Aureobasidiumpullulans Nagiecetal.1997[60]
Khafrefungin Unidentifiedsterilefungus Mandalaetal.1997[61]
Rustmicin Micromonosporachalcea Takatsuetal.1985[69]
Streptomycesgalbus Fauthetal.1986[70]
Micromonosporasp. Mandalaetal.1998[62]
GalbonolideB Micromonosporasp. Harrisetal.1998[71]
Minimoidin Sporomiellaminimoides Mandalaetal.2001[63]
Proteinsynthesis
Sordarin Sordariaaraneosa Siggetal.1969[79]
Zofimarin Zopfiellamarina Ogitaetal.1987[83]
BE31405 Penicilliumminioluteum Okadaetal.1998[85]
SCH57404 Unidentifiedsterilefungus Covaletal.1995[86]
Xylarin Xylariasp. Schneideretal.1995[87]
Hypoxysordarin Hypoxyloncroceum Daferneretal.1999[88]
GR135402 Graphiumputredinis Kinsmanetal.1998[92]
dicarboxylic acids, although at higher concentra- synthesis and the concomitant accumulation of
tionsthanrequiredforserinepalmitoyltransferase sphingoidbases[59].AlthoughfumonisinB1does
inhibition [66]. inhibit fungal ceramide synthase in vitro [54] the
Unfortunately, the serine palmitoyltransferase fumonisinshave very poor activity against whole
inhibitorsdescribedabovealsohavepotentactiv- cell fungal sphingolipid synthesis or growth;lim-
ityagainstthemammalianenzyme[51,56,58],and itedpenetrationcouldaccountfortheirpooranti-
other studies showed that lipoxamycin is highly fungal activity.
toxicinmicewhenappliedeithersubcutaneously Australifungin is a highly potent, broad-spec-
or topically [65]. Toxicity may be mechanism trum antifungal compound containing a unique
based,sincestudieswithaChinesehamsterovary combination of a-diketone and b-ketoaldehyde
(CHO)cellmutanthaveshownthatthisenzymeis functional groups. It was the first non-sphingo-
essentialinmammaliancells.Anothernaturalpro- sine-basedinhibitordescribedforthesphingolipid
duct isolated from the fungus Isaria sinclairii, the biosyntheticpathway.Australifungininhibitscer-
potent immunosuppressant ISP-1/myriocin, has amide synthase in vitro at nanomolar concentra-
alsobeenreportedtoinhibitserinepalmitoyltrans- tions and the enzyme inhibition accounts for the
ferase at picomolar concentrations in an interleu- arrestofsphingolipidsynthesis.Australifunginol,
kin-2-dependent mouse cytotoxic T-cell line [67]. an analogue isolated from the same fungus, also
blocked the enzyme converting sphinganine to
ceramide, but it was at least 50-fold less potent
Ceramide synthase inhibitors
[52]. Australifungin had MICs of 1mg/L or less
Thefumonisinsaremycotoxinsinitiallycharacter- against all the species tested, with particularly
ized as tumour-promotingagents associated with good activity against Candida pseudotropicalis, C.
severetoxicologicaleffectsinanimals.Theyinhibit tropicalis,andC.neoformans.Muchweakeractivity
de novo sphingolipid biosynthesis blocking the was detected for australifunginol with MICs
reaction catalyzed by ceramide synthase in rat between 8 and 64mg/L [52].
hepatocytes,andtheirtoxicityandcarcinogenicity Althoughtheactivityofaustralifungininmam-
have been attributed to inhibition of ceramide malian systemshas been largely uncharacterized,
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24 Clinical Microbiology and Infection, Volume 9 Number 1, January 2003
itisknowntoinhibitceramidesynthesisinHepG2 essential in this organism, which also contains
cells[58],andthereforeconstitutesamajorlimita- glycosylated sphingolipids.
tiontothetherapeuticuseofaustralifunginforthe Unlikeinhibitorstoearlierstepsinsphingolipid
treatment of fungal infections. synthesis, khafrefungin and rustmicin did not
have any detectable effect on lipid synthesis in
mammaliancells[61].Rustmicinandaureobasidin
IPC synthase inhibitors
A were non-toxic in animal studies [62,72], sup-
Aureobasidins A to R are cyclic depsipeptides porting the idea that inositol phosphoceramide
described as antifungal agents with high in vitro synthase is a fungal selective target, and thus
activity, particularly against C. albicans, aureoba- preferable in the search for novel antifungals
sidins A, B, C and E being the most potent [68]. blocking sphingolipid biosynthesis. Although
Their mechanism of action remained unknown khafrefungin was lytic to washed red blood cells
until it was shown that aureobasidin A inhibits at 12.5–25mg/L, the toxicity may be due to the
theIPCsynthasefrom S. cerevisiaewithanIC of detergent-like properties of the compound.
50
about 0.2nM [60]. Among the three structurally diverse IPC syn-
Khafrefungin is a novel 22-carbon linear poly- thaseinhibitors,rustmicinisuniqueinitsremark-
ketideacidesterifiedtoanaldonicacidthatshows able activity against C. neoformans. However,
abroadantifungalspectrum,withC.albicansbeing althoughencouraging,thelevelofinvivoefficacy
the most susceptible organism in vitro. The com- isfarlessthanexpectedfromtheinvitrosuscept-
pound causes the accumulation of ceramide and ibility of this organism to rustmicin. Poor in vivo
inhibitstheIPCsynthaseofS.cerevisiaeandpatho- efficacy in animals is probably due to chemical
genic fungi atpicomolar to nanomolar concentra- instability. Even at its optimal pH, rustmicin
tions [61]. degrades relatively rapidly in aqueous media,
Rustmicin (also named galbonolide A) is a converting totheinactive g-lactone.Despitethese
macrolide antifungal agent with potent activity limitations, concentrations of rustmicin that are
against wheat stem rust fungus Puccinia graminis required to completely inhibit phosphosphingoli-
[69],Botrytiscinereaandseveralotherphytopatho- pid synthesis and accumulate ceramide in fungi
gens [70]. The mechanism of its fungal growth are easily achieved [62].
inhibitionwasnotdetermineduntil13yearslater,
whenitsextraordinarilypotentantifungalactivity
Fatty acid elongation inhibitors
against several human pathogens, especially C.
neoformans was observed, and it was found that Minimoidin is a novel compound that indirectly
its antifungal activity was due to inhibition of inhibits sphingolipid synthesis by blocking the
sphingolipid synthesis at the IPC synthase level fatty acid elongation pathway, thus depriving
at picomolar to low nanomolar concentrations of theceramidesynthaseofsubstrate[63].Inexperi-
the compound [62]. The rustmicin-related macro- ments of 14C-acetate uptake, minimoidin blocked
lidegalbonolideBwasalsoreportedtoinhibitIPC radioactivityuptakeintoC26fattyacids,whilethe
synthase, but with less potency [71]. patternofincorporationintootherfattyacidswas
A.fumigatusisoneofthefewhumanpathogens notmodified.Moreover,minimoidininhibitedthe
that is insensitive to any of the known IPC incorporationof14C-malonyl-CoAintolong-chain
synthase inhibitors, although some inhibitors of fatty acids.
earlierstepsinthesphingolipidbiosyntheticpath-
waydoinhibitthegrowthofthisorganism[52,66].
PROTEIN SYNTHESIS INHIBITORS
The reason for this resistance is unknown. Sphin-
golipids are thought to be essential in Aspergillus Proteinsynthesishasalwaysbeenconsideredone
[52,66] and A. fumigatus does synthesize alkali- ofthemoreattractivetargetsinthedevelopmentof
stable inositol lipids, and their synthesis is inhib- antimicrobialagents[73].However,applicationof
ited by khafrefungin, but at much higher concen- thisideatothefieldofantifungaltherapyisnotan
trations than those required for the other fungi. easytask,duetotheeukaryoticnatureoffungiand
Lackofgrowthinhibitionmaybeduetoaresistant therefore the great degree of similarity between
enzymeorpooruptakeofthedrug.Alternatively, the fungal and mammalian protein synthesis
the inositol-containing sphingolipids may not be machineries.Twosolubleelongationfactorsshow
(cid:1) 2003CopyrightbytheEuropeanSocietyofClinicalMicrobiologyandInfectiousDiseases,CMI,9,15–32