Antifungal Drug Development: Challenges, Unmet Clinical ...perspectivesinmedicine.cshlp.org/content/4/5/a019703.full.pdf · antifungal drugs target structures that are unique to fungi

Antifungal Drug Development: Challenges,Unmet Clinical Needs, and New Approaches

Terry Roemer1 and Damian J. Krysan2,3

1Infectious Disease Research, Merck Research Laboratories, Kenilworth, New Jersey 070332Department of Pediatrics, University of Rochester, Rochester, New York 146423Department of Microbiology and Immunology, University of Rochester, Rochester, New York 14642

Correspondence: [email protected]

Invasive, life-threatening fungal infections are an important cause of morbidity and mor-tality, particularly for patients with compromised immune function. The number of ther-apeutic options for the treatment of invasive fungal infections is quite limited whencompared with those available to treat bacterial infections. Indeed, only three classesof molecules are currently used in clinical practice and only one new class of antifungaldrugs has been developed in the last 30 years. Here we summarize the unmet clinicalneeds of current antifungal therapy, discuss challenges inherent to antifungal drug discov-ery and development, and review recent developments aimed at addressing some of thesechallenges.

Over the past 30 years, the importance ofantifungal drugs to the practice of modern

medicine has increased dramatically. Becausethe vast majority of life-threatening fungal in-fections affect people with altered immune func-tion, the increased incidence of invasive fungalinfections can be correlated with an expansionin the number of people living with conditionsor treatments that affect immune function, ex-amples of which include HIV/AIDS, prima-ry immune deficiency, cancer chemotherapy,hematologic and solid organ transplantation,prematurity, and immune-modulatory medica-tions (Richardson 2005). It is, therefore, sober-ing to consider that two of the three classes ofantifungal drugs (azoles and polyenes) in cur-rent use had already been introduced into the

clinics by 1980 and the third class (echinocan-dins) had been discovered (Butts and Krysan2012). Furthermore, even with these newesttherapies, the clinical outcomes for most inva-sive fungal infections are far from ideal. Indeed,infections caused by species of molds for whichthere is no reliable medical therapy are emergingas are strains of the more common organismssuch as Candida albicans and Candida glabratathat are resistant to currently used drugs. Ittherefore seems fairly clear that the tempo ofantifungal drug development has not kept pacewith the clinical needs. In this review, we furtherdiscuss the unmet clinical needs of medical my-cology, the challenges inherent to new antifungaldrug development, and new strategies to meetsome of these challenges (Brown et al. 2012a,b).

Editors: Arturo Casadevall, Aaron P. Mitchell, Judith Berman, Kyung J. Kwon-Chung, John R. Perfect, and Joseph Heitman

Additional Perspectives on Human Fungal Pathogens available at www.perspectivesinmedicine.org

Copyright # 2014 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a019703

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THE CLINICAL PROBLEM

Medically important fungal infections can bebroadly classified into two types (Richardson2005). The first group is mycoses of superficialsurfaces such as skin, skin structures, and mu-cosa. Specific examples include thrush, orophar-yngeal candidiasis, and dermatophyte infec-tions of various regions of the body. Althoughimmunocompromised people may have in-creased rates or severity of disease, superficialmycoses are common among those with intactimmune function. The second major group isinvasive fungal infections, which, by definition,involve sterile body sites such as the blood-stream, central nervous system, or organs in-cluding lung, liver, and kidneys. Many of thefungi that cause invasive disease either infect,or colonize, most human beings but the vastmajority of the clinically significant disease oc-curs in people with compromised immunefunction. For example, C. albicans is a normalpart of the flora of the human gastrointestinaltract and, although it causes superficial diseasein immunocompetent individuals, invasive dis-ease occurs almost exclusively in the setting ofimmune dysfunction or the use of invasive in-terventions to support life (Pfaller et al. 2012).Furthermore, serological evidence of infectionwith Cryptococcus neoformans is common overthe age of 2 yr (Goldman et al. 2001), but thisorganism almost never causes clinically signifi-cant disease unless the patient develops deficitsin cell-mediated immunity.

In resource-rich regions of the world, mostclinically significant invasive fungal disease oc-curs as a complication of prematurity, surgery,chemotherapy, hematopoietic or solid organtransplantation, or immunomodulatory thera-pies. Patients with primary immunodeficienciesare also at risk for fungal infections but theseconditions are quite rare (Lionakis 2012). Ingeneral, resource-rich regions also have goodaccess to combined antiretroviral therapy and,consequently, the rates of opportunistic infec-tions associated with HIV/AIDS have decreasedsignificantly. In the U.S. healthcare setting, C.albicans is the fourth most common cause ofhealthcare-associated bloodstream infections

(Lewis 2009). The incidence of mold infectionsis much lower than candidiasis; however, moldinfections are a significant cause of morbidityand mortality among immunocompromisedpatients. The most common invasive mold in-fections are attributable to Aspergillus fumigatus(Steinbach et al. 2012). Infections caused by dif-ficult-to-treat molds such as Mucor spp. (Lanter-nieret al. 2012) and Fusarium (Muhammed et al.2011), for example, are increasing in incidence.In resource-limited regions, cryptococcosis is amajor health problem and causes more deaths inpeople living with HIV/AIDS than tuberculosis(Park et al. 2009). Because cryptococcal menin-gitis is frequently the first indication that a per-son has HIV/AIDS, many people need to survivecryptococcosis if they are to take advantage ofhighly effective antiretroviral therapy.

CURRENT ANTIFUNGAL THERAPIES

The therapeutic options for invasive fungal in-fections are quite limited and include only threestructural classes of drugs: polyenes, azoles, andechinocandins (Fig. 1). Indeed, there are nowmore classes of antiretroviral drugs than anti-fungals. The oldest class of antifungal drugs isthe polyenes, of which amphotericin B is theonly example used to treat systemic infections.Amphotericin B binds to ergosterol, a mem-brane sterol that is unique to fungi, as part ofits mechanism of action (Gray et al. 2012). Am-photericin B is fungicidal and is the most broadspectrum antifungal available. One of the pri-mary drawbacks of polyenes is their significanttoxicity, although the development of lipid for-mulations of amphotericin B has reduced thisproblem significantly (Hamill 2013); such for-mulations are quite expensive and not availablein some regions. Amphotericin B, in combina-tion with the adjunctive drug 5-flucytosine, isthe treatment of choice for cryptococcal menin-gitis (Dayet al. 2013) as well as for awide range ofless common invasive mycoses. For many of themost common invasive fungal infections, thebetter tolerated azoles and echinocandins haveemerged as first-line agents.

The azoles are the most widely used class ofantifungal drugs (Lass-Florl 2011). Although

T. Roemer and D.J. Krysan

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there are many azoles currently available (Fig. 2),fluconazole, voriconazole, and posaconazole aremost commonly used to treat invasive fungalinfections. Azoles inhibit ergosterol biosynthe-sis and, in general, are fungistatic; an impor-tant exception is that voriconazole is fungicidaltoward A. fumigatus (Meletiadis et al. 2007).Azoles are extremely well tolerated, althoughthey interfere with the metabolism of a numberof other drugs owing to their ability to inhibitcytochrome P450. In general, fluconazole hasbroad activity against clinically relevant yeast

including Candida spp. and Cryptococcus. Manyisolates of C. glabrata and Candida krusei, how-ever, are intrinsically less susceptible. Becauseamphotericin B and 5-flucytosine are not avail-able in many resource-limited regions, flucon-azole is widely used to treat cryptococcal men-ingitis despite the fact that it is less effective.Importantly, fluconazole has essentially no clin-ically relevant activityagainst molds. In contrast,itraconazole, voriconazole, and posaconazole allhave activity against yeast and molds (Lass-Florl2011). Voriconazole is currently the treatment

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Figure 1. Classes and representative examples of antifungal drugs in current use.

Antifungal Drug Development

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of choice for Aspergillus based on its superiorityto amphotericin B in a head-to-head clinicaltrial (Herbrecht et al. 2002). Posaconazole is dis-tinguished from the other azoles by its in vitroactivity against Mucor spp., organisms with re-duced susceptibility to other drugs (Luo et al.2013).

The echinocandins are the most recent ad-dition to the antifungal pharmacopeia (Fig. 1)with the first example, caspofungin, introducedinto clinical use a decade ago (Mukherjee et al.2011). The echinocandins inhibit 1,3-b-glucansynthesis (GS), a key component of the fungalcell wall. The echinocandins have broad fungi-cidal activity against Candida spp. and haveemerged as an important therapeutic optionfor candidiasis. By growth assays and in vivostudies, the echinocandians are fungistatic to-ward Aspergillus; however, vital dye studies in-dicate that caspofungin, for example, kills grow-ing Aspergillus fumigatus (Bowman et al. 2002).At this time, the echinocandins are considereda second-line salvage therapy for those in-fections. The echinocandins do not have clini-cally useful activity against Cryptococcus or Zy-gomycetes.

UNMET CLINICAL NEED

The small number of available antifungal drugs,in and of itself, would not be a problem if theoutcomes for invasive fungal infections weresatisfactory. By and large, however, this is notthe case. For example, 90-day survival followingthe diagnosis of candidemia varies between 55%and 70%, depending on the underlying condi-tion of the patient and the specific species caus-ing infection (Pfaller et al. 2012); it is importantto note that one of the challenges in studyingthe outcomes of fungal infections is separatingmortality attributed to the infection from mor-tality owing to comorbidities. The outcomesare even worse for aspergillosis despite the useof voriconazole (Herbrecht et al. 2002). In re-source-rich regions with access to amphotericinB and 5-flucytosine, the 1-year mortality owingto cryptococcosis is approximately 25% (Brat-ton et al. 2013), whereas in resource-limited re-gions where fluconazole is the only availabletherapy the mortality is much higher (Sloanet al. 2009). As pointed out in an excellent essayon the state of the art for the diagnosis andtreatment of fungal disease, the poor outcomes

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for invasive fungal infections are most likely ow-ing to relatively poor diagnostic methods as wellas the need for more effective therapy (Brownet al. 2012). However, it is important to pointout that the newest class of antifungal drugs, theechinocandins, was discovered in the 1970s andtook 30 years to enter clinical practice (Fig. 2).Similarly, the best therapy for cryptococcosis isbased on two drugs that are more than 50 yearsold. Because medical innovations and the useof immunomodulatory therapies continue toincrease, the numbers of patients at risk for fun-gal infections are almost certain to increase.Thus, the current pace of antifungal drug de-velopment is unlikely to keep up with the clin-ical needs, particularly as resistance to currentagents is being reported more and more fre-quently.

CHALLENGES OF ANTIFUNGAL DRUGDEVELOPMENT

In comparison to the development of new an-timicrobials targeting bacteria, antifungal drugdevelopment faces a key fundamental challengein that fungal pathogens are more closely relatedto the host. For example, the success of Saccha-romyces cerevisiae as a model eukaryotic organ-ism is owing to the fact that many fundamentalbiochemical and cell biological processes areconserved from fungi to humans. Consequent-ly, many small molecules that are toxic to yeastare also toxic to humans. As such, it is thereforenot surprising that the three major classes ofantifungal drugs target structures that areunique to fungi. In addition to scientific chal-lenges affecting the identification of new leadcompounds, the evaluation of new antifungalagents also presents a number of challengeswith respect to clinical trial design that furthercomplicate development (Rex et al. 2001). Un-fortunately, these fundamental challenges arein addition to the well-documented scientific,economic, and regulatory challenges that facethe development of anti-infectives, in general(Boucher et al. 2009). Taken together, it is per-haps not surprising that the pace of new anti-fungal drug development lags considerablywhen compared with other therapeutic areas.

To address this significant gap in the anti-in-fective pipeline, creative approaches to theproblems discussed above will be required.The remaining sections will present work to-ward that goal.

ANTIFUNGAL DRUG DISCOVERY: PROCESSAND NEW APPROACHES

Here, we summarize recent developments in ap-proaches and technologies that have improvedand are likely to further facilitate the discoveryof new antifungal small molecules. Historically,the most common approach to identifying anti-fungal small molecules has been to screen largelibraries of synthetic small molecules or naturalproducts for their ability to inhibit the growthof a selected fungus. In recent years, the impor-tance of the chemical characteristics and originof the molecules within the library has becomebetter understood. As high-throughput screen-ing has emerged as a tool for both drug discoveryand biological investigation, there has also beenan explosion in the number of libraries of syn-thetic small molecules available commercially.The vast majority of the molecules within theselibraries has been designed or collected usingcriteria that maximize their “drug-like” proper-ties with respect to mammalian targets andphysiology. Unfortunately, successful anti-in-fective molecules have physicochemical pro-perties very different from molecules designedforotherclinical indications; this is owing in partto the requirement that the molecule traversemicrobial cell walls (Lewis 2013). Thus, newscreening efforts for antibacterials or antifungalsmay benefit from the use of libraries focused onan alternate set of “drug-like” properties.

Two out of the three majorclasses of current-ly used antifungals are derived from naturalproducts (polyene and echinocandins) (Os-trosky-Zeichner et al. 2010). Indeed, naturalproduct-based screening has led to the discoveryof the majority of clinically used antibiotics aswell. As interest in the discovery of new anti-infectives waned in the pharmaceutical industry,so did natural product-based screening. In ad-dition, many anti-infective screening campaigns



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simply rediscovered the same basic scaffoldsfrom screens of seemingly diverse libraries.More recently, interest in natural product-basedscreening, however, has enjoyed a renaissance.This has been driven not only by the recogni-tion of the valuable features of natural producthits, but by improvements in structure-identi-fication, separation, and target identification(see below). An important survey of antifungalscreening efforts at Merck serves to highlightmany of these issues and provide insights thatmay prove useful for future campaigns (Roemeret al. 2011a). For example, the mechanism ofaction for molecules derived from fungal iso-lates was much more likely to be target specif-ic (e.g., cell wall or ergosterol biosynthesis),whereas those from actinomycete-rich isolateswere more likely to have molecules with non-specific mechanisms of action (i.e., ionophores,mitochondrial respiration inhibitors, DNA in-tercalators, and alkylating agents) (Roemer et al.2011b). Interestingly, the most abundant target-specific antifungals identified from naturalproduct sources in the Merck screening cam-paign corresponded to new and known struc-tural classes of GS inhibitors; similarly, azoleswere the most widely abundant class of target-selective antifungals identified when screeningsynthetic chemical libraries. As such, it is likelynot a coincidence these drug classes were dis-covered early and that much of the antifungal“low hanging fruit” may have already beenpicked. If true, these data emphasize the needfor truly innovative antifungal discovery ap-proaches that holistically address the chemicallibraries, targets, and pathways screened as wellas superior methodologies and technologies torapidly and rigorously determine the precisemechanism of action of those rare but futureantifungal leads.

The assay used most commonly to identifyantifungal and antibacterial small molecule isthe traditional broth or microbroth growth-inhibition assay in which microbial growth ismeasured by optical density of the culture.Like all assays, these growth-inhibition assayshave limitations and a number of these dramat-ically reduce its utility in antifungal drug dis-covery. First, the fact that many pathogenic fun-

gi grow as filaments (e.g., Aspergillus) leads to apoor correlation between organism growth andoptical density (Bowman et al. 2002). Second,traditional growth assays are not useful for iden-tifying molecules active against fungal biofilms,a medically important growth phase of thesepathogens (Pierce et al. 2008; LaFleur et al.2011; Srinivasan et al. 2013). Third, traditionalgrowth assays are unable to distinguish be-tween molecules that inhibit growth and thosethat directly kill the organism, a feature that isparticularly important for the treatment ofsome fungi (e.g., Cryptococcus) (Bicanic et al.2009).

To address the limitations of traditionalgrowth assay screening in antifungal drug dis-covery, a number of screening assays using alter-native readouts have been developed in recentyears. A very productive strategy has been toadapt eukaryotic cell viability assays to fungi.The most widely used approach has been to usereporters of metabolic activity such as AlamarBlue and XTT. These dyes are converted to fluo-rescent molecules when metabolized by viableorganisms. These types of assays were initiallyapplied to antifungal molecules in the contextof alternative methods for susceptibility testing(Pfallerand Barry 1994), but have been extendedto the screening arena by a number of groups.XTT and Alamar Blue–based assays have beenparticularly useful for screening against C. albi-cans biofilms, a growth phase that is not amena-ble to growth-based assay in the context of high-throughput screening. Indeed, straightforwardand reproducible protocols for such screens havebeen developed by the Lopez-Ribot (Pierce et al.2008) and Lewis groups (LaFleur et al. 2011)and have led to the identification of novel mol-ecules with activity against C. albicans biofilms.Screening for molecules active against moldspresents many of the same technical challengesposed by biofilms. Monteiro et al. (2012) have,accordingly, developed an assay for screeningA. fumigatus using the metabolism of the dyeresazurin and applied this to high-throughputscreening. Important characteristics of themetabolic activity-based assays are that they per-form well in the high-throughput screening set-ting with Z0 scores well above 0.5, which is gen-



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erally accepted as the minimum required for auseful assay; are applicable to clinical isolateswithout genetic manipulation; and use readilyavailable low-cost reagents and equipment.

As mentioned above, traditional growth as-says do not differentiate between fungistaticand fungicidal molecules. In principle, fungi-cidal molecules would appear to be preferredover fungistatic agents because most patientswith invasive fungal infections have compro-mised immunity and, thus, are more dependenton the antifungal agent to clear infections. In thesetting of cryptococcal meningitis (Bicanic et al.2009), early fungicidal activity has been shownto correlate with clinical outcome, providinga mechanism for the superiority of fungicidalamphotericin B–based therapy when comparedwith treatment with fungistatic fluconazole. Asecond type of viability assay has been applied tothe specific identification of fungicidal agents:the detection of extracellular adenylate kinaseas a reporter of cell lysis (DiDone et al. 2010).Adenylate kinase is an intracellular enzyme thatconverts two molecules of ADP to ATPand AMP.When the cell loses membrane integrity, adenyl-ate kinase is released into the growth medium;the adenylate kinase activity in the extracellu-lar medium is detected by coupling ATP forma-tion to luciferase activity using a commerciallyavailable assay (DiDone et al. 2010). Fungicidalmolecules such as the echinocandins generaterobust signal, whereas no extracellular adenylatekinase activity is detectable in cultures of flucon-azole-treated cells. The assay has been adaptedto a 384-well format and applied to S. cerevisiae,C. albicans, and C. neoformans. Finally, it is moresensitive than growth assays in that it can detectthe lytic activity of echinocandins at concentra-tions 10-fold lower than the minimum inhibi-tory concentration. In addition to the AK-basedmethod, Rabjohns et al. (2013) have also deviseda useful, Alamar Blue–based protocol to iden-tify molecules with fungicidal activity against C.neoformans that is applicable to high-through-put screening.

Recently, broad interest in identifying mol-ecules that synergize with existing classes of an-tibacterial and antifungal drugs as an approachto improve efficacy has emerged (Mukherjee

et al. 2005; Ejim et al. 2011). In part, this enthu-siasm is based on the shown therapeutic supe-riority of coadministering amphotericin B and5-flucytosine (5-FC) to treat cryptococcal men-ingitis (Day et al. 2013). It is further based onthe extensive network of synthetic lethal geneticinteractions identified between loss-of-function(or hypomorphic) mutations otherwise singlytolerated by S. cerevisiae (Costanzo et al. 2010).Of particular interest are such mutants that ex-acerbate Erg11 or GS activity as they provide agenetic prediction that small molecule inhibi-tors of such targets would display chemical syn-ergy in combination with azoles or echinocan-dins and thus could be developed as adjuvantsto improve the potency and spectrum of exist-ing antifungal agents (Lesage et al. 2004; Co-stanzo et al. 2010). Most of the work in thisregard has focused on either improving activityof, or reversing resistance to, fluconazole. Untilrecently, the majority of published reports de-scribing molecules that interact with flucona-zole involved characterization of a single mole-cule or class. However, the direct screening formolecules that potentiate fluconazole activityhas been reported and led to the identificationof known chemical probes (e.g., brefeldin A),previously approved drugs that synergize withfluconazole as well as novel molecules that over-come fluconazole resistance (Spitzer et al. 2011;Kaneko et al. 2013). For example, a screen per-formed as part of the NIH-Molecular Librariesand Probes Screening Network project identi-fied a class of indole derivatives that restore flu-conazole susceptibility to resistant C. albicansisolates (Youngsaye et al. 2012).

The dedicated search for molecules thatimprove the activity has also included screensof previously approved molecules as potentialagents for repurposing to antifungal indica-tions. For example, Spitzer et al. identified aset of previously approved drugs that synergizewith fluconazole in vitro and used chemical-ge-netic analysis to explore their mode of action. Inaddition, they showed that the antidepressantsertraline combined with fluconazole providesimproved activity relative to either drug alone inan invertebrate model of cryptococcosis (Spitzeret al. 2011). Concurrent work in the Lin labora-



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tory showed similar activity in a mouse modelof disseminated cryptococcosis (Zhai et al.2012). Most intriguingly, Cowen and colleagueshave shown that inhibitors of the molecularchaperone Hsp90, including the natural prod-ucts geldanamycin and radicicol, possess potentin vitro synergy in combination with azoles andechinocandins, that this synergy extends acrossboth C. albicans and A. fumigatus, and that it isobserved in an invertebrate model of candidiasis(Cowen et al. 2009; Singh et al. 2009; Shapiroet al. 2011). Therefore, ongoing chemical mod-ification of geldanamycin as a novel oncologyagent offers opportunities to “repurpose” newgeldanamycin analogs as adjuvants for existingantifungals.

EMERGING TARGETS AND MOLECULARSCAFFOLDS

It is impossible to predict which (if any) of thelead molecules currently being explored willemerge as the next clinically useful antifungal.The science and business of antimicrobial drugdevelopment is an unpredictable and fickleworld, as highlighted by those responsible forthe discovery and development of Cancidas, thefirst echinocandin antifungal drug to reach theclinic (Mukherjee et al. 2011). In this section, wepresent a selection of novel antifungal moleculesthat have emerged from a variety of researchprograms.

From first principles, one of the most at-tractive antifungal drug targets is the cell wall;this structure is absent from host cells and,in a sense, represents a histological commondenominator between the eukaryotic fungiand prokaryotic bacteria. Because bacterial cellwall-targeted molecules (penicillins, cephla-sporins, carbapenam, and glycopepties such asvancomycin) are staples of our antibacterialpharmacopeia, it follows that cell wall-targetedantifungal drugs should be similarly useful. Ad-ditionally, the success of the echinocandins fur-ther emphasizes the potential of molecules thattarget cell wall-related processes and significanteffort has been directed to identifying and de-veloping new antifungal drug leads targetingGS. Enfumifungin represents a structurally dis-

tinct natural product class of GS inhibitors.Originally discovered in Merck by screening nat-ural product extracts to which a S. cerevisiae fks1heterozygote deletion mutant displayed hyper-sensitivity, enfumafungin and several relatedacidic terpenoids (ascosterocide, arundifungin,and ergokonin A) were identified (Onishi et al.2000). The current development candidate MK-3118 (Fig. 3) is an orally active, semisyntheticderivative of enfumafungin with potent in vitroGS activity (Heasley et al. 2012) with potentin vivo activity against Candida and Aspergillusspp. (Pfaller et al. 2013a,b). Importantly, al-though echinocandins and enfumafungin bothtarget the GS enzyme (encoded by the Fks1-encoding catalytic subunit and GTPase regula-tory subunit Rho1), drug-resistant mutationsto each GS inhibitor class map to fks1 but donot display cross-resistance, emphasizing thatthe two molecules have distinct mechanismsof GS inhibition. Schering-Plough, taking asimilar S. cerevisiae whole cell screening ap-proach but reliant on a synthetic chemical li-brary and a sensitized strain deleted of majorefflux pumps and certain cell wall biosyntheticgenes identified a series of piperazinyl-pyrida-zinones (SCH A–D) also shown to inhibit GSactivity (Walker et al. 2011). Although not aclinical development candidate, SCH C possess-es potent in vitro activity against Candida andAspergillus spp. as well as anti-Cryptococcus andantidermatophyte activity. Significant oral effi-cacy was achieved in a murine infection modelof C. glabrata when treated with SCH B (Fig. 3).Again, drug resistance mutants to these inhibi-tors correspond to distinct regions of Fks1 andno cross-resistance was observed between thesefks1 mutants and echinocandin or enfumafun-gin class GS inhibitors.

Efforts to identify new antifungal drug leadstargeting other essential processes critical tofungal cell wall biogenesis have also yielded earlysuccess. Recently, two groups have reportedclasses of molecules that inhibit glycosylphos-phatidylinositol (GPI) biosynthesis. GPI-mod-ified proteins are essential for the constructionof the yeast cell wall as well as for proper mem-brane homeostasis. The first GPI-anchor bio-synthesis inhibitors were discovered by Tsukuba



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Laboratories and emerged from a screen formolecules that interfered with the cell wall local-ization of a GPI-anchored reporter protein: anongrowth assay (Tsukahara et al. 2003). Theinitial hit from this screen was 1-[4-butylben-zyl]isoquinoline (BIQ)and mechanismofactionstudies identified the acyl transferase Gwt1 as thetarget. Medicinal chemical-based optimizationof this initial hit led to the pyridine-2-amine-based molecule E1210 (Fig. 3), an oral, broad-spectrum antifungal molecule with broad activ-ity against both yeast and mold infections (Hata

et al. 2011). E1210 has activity at ng/mL con-centrations against Candida spp., Aspergillusspp., and the difficult-to-treat molds Fusariumand Scedosporium. Importantly, the molecule iswell tolerated and proved efficacious in murinemodels of oropharyngeal candidiasis, dissemi-nated candidiasis, aspergillosis, and fusariosis.It is also active against echinocandin-resistantC. albicans. In 2012, a second chemical scaffoldwith activity against Gwt1 was identified in thecourse of a high-throughput screening cam-paign (McLellan et al. 2012). The molecule is a

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Figure 3. Examples of antifungal small molecules in development. The structure, method of identification,mechanism of action, and spectrum of antifungal activity are listed for each molecule.



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phenyoxyacetanilide and the target was identi-fied by screening an ordered library of S. cerevi-siae strains overexpressing each open readingframe (ORF) and comparing the data to a chem-ically induced haploinsufficiency screen of a setof S. cerevisiae heterozygous deletion mutants;GWT1 was the only ORF that hit in both screens.Subsequently, the target was confirmed by bio-chemical assays. These two efforts nicely illus-trate the use of nontraditional screening ap-proaches followed by chemical genetics-basedtarget identification leading to novel targetsand scaffolds.

A second class of cell wall–targeted mole-cules, b-1,6-glucan synthesis inhibitors, hasalso been identified by specifically screeningfor molecules that interfere with cell wall con-struction (Kitamura et al. 2009a). GPI-linkedcell wall proteins frequently are cross-linkedwithin the cell wall through b-1,6-glucan; how-ever, development of specific inhibitors ofb-1,6-glucan synthesis has been hampered bythe fact that no specific protein or catalytic ac-tivity has been directly linked to b-1,6-glucansynthesis. Similar to the screening assay de-scribed above, the group at Daiichi Sankyoidentified the pyridobenzimidazole (Fig. 3)scaffold by initially screening for agents thatdisrupted cell wall localization of a reporterconstruct. The target for this class was identifiedby traditional screening for UV-generated resis-tant mutants followed by cloning. A mutationin the KRE6 gene, a gene known to be involvedin b-1,6-glucan synthesis, was isolated. In addi-tion, biochemical analysis of the cell wall mate-rial isolated from drug-treated cells showed thatb-1,6-glucan levels were reduced. The spectrumof activity of this class is not as broad as E1210and is largely limited to Candida spp.; it has noactivity against A. fumigatus and neither E1210nor the pyridobenzimidazoles have activityagainst C. neoformans. One example of this scaf-fold, D21-6076, displayed weak in vitro activityagainst C. albicans but good in vitro activityagainst C. glabrata. However, the molecule wasequally effective in murine models of dissemi-nated C. albicans and C. glabrata infection. Itsactivity against the former species seems to beowing to its ability to inhibit C. albicans hyphal

morphogenesis and tissue invasion (Kitamuraet al. 2009b).

Although GS (and presumably additionalaspects of cell wall biogenesis) is clinically vali-dated as a therapeutic target suitable for antifun-gal development, other essential processesof fungal growth and cell viability should notbe ignored. The clinical reliance of azoles withpotent and highly specific inhibition of ergoster-ol biosynthesis, targeting lanosterol 14-a de-methylase (Erg11) over its human ortholog, em-phasizes this point. Natural product-derivedparnafungins (Fig. 3), which inhibit poly(A) po-lymerase, serve as a salient example (Bills et al.2009). Parnafungins display potent broad spec-trum activity against all clinically relevant Can-dida spp. (including azole and echinocandin-re-sistant isolates), anti-Aspergillus activity (albeitbest detected underconditions in which poly(A)polymerase enzyme activity is partially depletedby genetic means), and, most importantly, sig-nificant therapeutic efficacy in a murine infec-tion model of candidiasis without any obviousindication of cytotoxicity in mice or human celllines tested (Jiang et al. 2008). Similarly, theleucyl tRNA synthase inhibitor AN2690 (Fig.3) shows high selectivity against Trichophytonspp. (Rock et al. 2007; Seiradake et al. 2009)and is in clinical development to treat onycho-mycoses, commonly referred to as toenail fungalinfections. Finally, a broadening set of additionalantifungal inhibitors targeting the 26S protea-some (fellutamides), translational elongation(yefafungin), cAMP homeostasis (campafun-gin), microtubule dynamics (12-deoxy-hami-gerone), and other basic eukaryotic processesincluding fatty acid, ergosterol, and ribosomebiosynthesis highlight the largely unexploitedopportunities to identify fungal-specific agents(Roemeret al. 2011b; Xu et al. 2011). Such agentsmay fortuitously possess fungal specificity byinhibiting fungal-specific protein domainsand/or target unique ligand-binding sites; dif-ferences in substrate specificities between fungaland human enzymes may also result in fungalspecificity of such agents. Finally, issues of dif-ferential cell permeability or prodrug activation(as in the case with 5-FC) may maximize anti-fungal activity while mitigating host toxicity.



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FUTURE DIRECTIONS

The global burden of fungal disease is signif-icant and, as a number of investigators haveemphasized, relatively underappreciated andunderfunded relative to other diseases (Brownet al. 2012a,b). Currently, the gold standardtherapy for cryptococcosis, one of the mostprevalent invasive life-threatening fungal infec-tions on the planet, is based on drugs that weredeveloped in the 1950s, when penicillin was astate-of-the-art anti-infective. Since the intro-duction of amphotericin, only two additionalclasses of antifungals have been developed. Thisrate of antifungal drug discovery is unlikely tobe sufficient for future demands. This is partic-ularly true because the number of patients atrisk for fungal infections is increasing as immu-nomodulatory therapies continue to expandand our ability to support highly immunocom-promised patients improves. Consequently, weare faced with the challenge of an expanding setof at-risk patients, increasing the prevalence ofdifficult-to-treat organisms, and a slow pace ofnew drug development.

To meet this challenge, a renewed and reso-lute commitment by the pharmaceutical indus-try partnering with academic laboratories, com-bining innovative screening strategies and novelchemical libraries is required to achieve success.As has been shown and universally acceptedwithin the antibacterial discovery community,in vitro–based high-throughput screening ofindividual antibacterial targets has been unsuc-cessful and is unlikely to provide a different out-come for antifungal lead discovery (Payne et al.2007). Rather, the traditionally successful “com-pound-centric” approach of empiric screeningfor small molecules with desirable whole cellbioactivity, cidal activity, and requisite spec-trum against clinically relevant pathogens re-mains warranted (Roemer and Boone 2013;Walsh and Wencewicz 2013). However, combin-ing this classic approach with genomics-eratechnologies that accelerate discovery time linesis essential. For example, forward genetics plat-forms such as the S. cerevisiae haploinsufficiencyprofiling (HOP) (Shoemakeret al. 1996; Giaeveret al. 1999, 2002, 2004; Roemer et al. 2011a) or

C. albicans fitness test (Xu et al. 2007; Jiang et al.2008; Roemer et al. 2011b) offer whole cell tar-get-specific assays for essentially all possibledrug targets in yeast and has proven enormouslysuccessful in the discovery and mechanism ofaction (MOA) determination of novel antifun-gal agents (Roemer et al. 2011a, 2012). As rou-tinely performed in antibacterial discovery(Mann et al. 2013; Roemer and Boone 2013;Wang et al. 2013), next-generation sequencingalso offers greater speed and resolution in deter-mining the MOA of potential antifungal leads.Whole genomes of drug-resistant mutants de-rived from haploid S. cerevisiae, C. glabrata,C. neoformans, or newly derived haploid C. al-bicans strains (Hickman et al. 2013), and evenA. fumigatus are now (or soon to be) routinelysequenced to map causal mutations, therebydefinitively identifying the drug target by genet-ic means. Genetic strategies based on systems-level synthetic lethality networks (Costanzo etal. 2010; Roemer and Boone 2013) also offerimportant opportunities to identify antifungaladjuvants targeting nonessential proteins thatmay be paired with existing antifungals to en-hance their spectrum or restore their therapeu-tic effects against drug-resistant strains. Shortof completely “new and improved” chemical li-braries to screen—an important but challeng-ing request—future antifungal discovery successwill require these and other innovative ap-proaches to screening existing chemical matter.

ACKNOWLEDGMENTS

We thank Melanie Wellington (University ofRochester) for assistance with the figures. Thiswork is supported in part by the following Na-tional Institutes of Health grants to D.J.K.:1R01AI091422-03 and 1R01AI097142-02.

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2014; doi: 10.1101/cshperspect.a019703Cold Spring Harb Perspect Med Terry Roemer and Damian J. Krysan New ApproachesAntifungal Drug Development: Challenges, Unmet Clinical Needs, and

Subject Collection Human Fungal Pathogens

PathogensEvolutionary Perspectives on Human Fungal

John W. Taylor Pathogenicity TraitPolyphyletic Pathogens with a Convergent

−−Thermally Dimorphic Human Fungal Pathogens

Anita Sil and Alex Andrianopoulos

HumansBlack Molds and Melanized Yeasts Pathogenic to

de HoogAnuradha Chowdhary, John Perfect and G. Sybren

Mechanisms of Antifungal Drug Resistance

Howard, et al.Leah E. Cowen, Dominique Sanglard, Susan J.

within MacrophagesFungal Pathogens: Survival and Replication

MayAndrew S. Gilbert, Robert T. Wheeler and Robin C.

Cryptococcus and CandidaTreatment Principles for

Laura C. Whitney and Tihana Bicanic

Innate Defense against Fungal Pathogens

Hise, et al.Rebecca A. Drummond, Sarah L. Gaffen, Amy G.

The Human MycobiomePatrick C. Seed

PharmacodynamicsAntifungal Pharmacokinetics and

Alexander J. Lepak and David R. AndesInfectionsTreatment Principles for the Management of Mold

Dimitrios P. Kontoyiannis and Russell E. Lewis

EntomophthoralesHuman Fungal Pathogens of Mucorales and

al.Leonel Mendoza, Raquel Vilela, Kerstin Voelz, et

Adaptive Immunity to Fungi

al.Akash Verma, Marcel Wüthrich, George Deepe, et

GenomesFunctional Profiling of Human Fungal Pathogen

Alexi I. Goranov and Hiten D. Madhani

Pathogenic Species ComplexCandidaThe Siobhán A. Turner and Geraldine Butler

and Related SpeciesAspergillus fumigatus

R. Juvvadi, et al.Janyce A. Sugui, Kyung J. Kwon-Chung, Praveen

Fungal Morphogenesis

al.Xiaorong Lin, J. Andrew Alspaugh, Haoping Liu, et

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