The Fungal Kingdom

Edited by J. Heitman, B. J. Howlett, P. W. Crous, E. H. Stukenbrock, T. Y. James, and N. A. R. Gow

© 2018 American Society for Microbiology, Washington, DC
doi:10.1128/microbiolspec.FUNK-0002-2016

Antifungal Drugs: The Current

Armamentarium and Development
of New Agents
Nicole Robbins,1 Gerard D. Wright,1 and Leah E. Cowen2 44
INTRODUCTION and sub-Saharan Africa (2). Recently, C. gattii was as-
sociated with outbreaks of cryptococcosis in healthy
Importance of Fungal Pathogens individuals on Vancouver Island (5) and other regions
Disease and death caused by fungal infections have
of the Pacific Northwest (6), making Cryptococcus
transitioned from a rare curiosity to a major global
a public health threat to immunocompromised and im-
health problem. Because the vast majority of fungi ca-
munocompetent individuals. Finally, A. fumigatus is a
pable of causing life-threatening infections target indi-
significant cause of invasive infections in individuals
viduals with impaired immunity, recent decades have
with impaired immune function, including those with
witnessed a stark rise in fungal infections due to medi-
neutropenia, solid organ transplant recipients, and pa-
cal interventions such as chemotherapy for cancer and
tients on immunosuppressive therapies such as high-
immunosuppression for organ transplantation and due
dose corticosteroids (2). It is estimated that more than
to the prevalence of HIV infection (1). Pathogenic fungi
200,000 cases of invasive aspergillosis occur each year,
are the causative agents of billions of infections annually,
with staggering mortality rates of up to 50% with treat-
with ∼1.5 million attributable mortalities (2). The public
ment and 100% if left undiagnosed (2). Thus, these
health burden is comparable to that observed with more
chief opportunistic invaders are a leading cause of hu-
notable infectious diseases such as tuberculosis and ma-
man mortality and impose a dire need for effective anti-
laria, yet the impact of fungal infections on human
fungal therapeutics to combat these infections.
health has been largely overlooked (3).
In this article we aim to describe the current arma-
The predominant fungal pathogens of humans include
mentarium of antifungal agents and the most promising
Candida albicans, Cryptococcus neoformans, and Asper-
emerging therapies in the antifungal development pipe-
gillus fumigatus. C. albicans is a common component
line. We also highlight the innovative strategies being
of the human mucosal microbiota and is responsible
leveraged to identify novel antifungal targets and un-
for millions of superficial infections in immunocompe-
cover new therapeutic strategies to combat fungal in-
tent individuals, such as thrush and oropharyngeal can-
fectious disease.
didiasis. It is also the most prevalent fungal etiological
agent of life-threatening invasive infections, with mor-
tality rates approaching 40% despite treatment (1). Challenges for Treatment of Fungal Infections
The CDC has classified Candida species as a serious Fungi are eukaryotic, like their human hosts, limiting
threat to human health due to the dramatic rise in resis- the number of molecular targets that can be selectively
tance to antifungal drugs (4). Cryptococcosis caused by exploited for drug development without the risk of
C. neoformans and the closely related species Crypto- cross-target toxicity. The current arsenal of antifungal
coccus gattii affects over a million individuals annually, compounds approved for the treatment of invasive infec-
with mortality rates as high as 70% in Latin America tions consists of only three classes of antifungal agents

1
Michael G. DeGroote Institute for Infectious Disease Research and Department of Biochemistry and Biomedical Sciences, McMaster University,
Hamilton, ON L8N 3Z5, Canada; 2Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.

903
904
directed against a limited number of cellular processes.
Perhaps of greater concern is that there are few discov-
ery programs devoted to the development of new anti-
fungal therapeutics because of the expectation of limited
financial return for pharmaceutical companies (2). Most
antifungals target ergosterol, the functional fungal ana-
logue of cholesterol; the biosynthesis of ergosterol; or
the biosynthesis of (1,3)-β-D-glucan, a major component
of the fungal cell wall (Fig. 1). This paucity of fungus-
specific targets is problematic because of the prevalence
of cross-resistance to all drugs with a common target.
Resistance to all widely deployed antifungals has been
documented in both the laboratory and the clinic (7)
and continues to be a growing problem in the medical
community. There is a pressing need to bolster the anti-
fungal pipeline, as highlighted by the Generating Anti-
biotic Incentives Now (GAIN) Act (H.R. 2182), which
promises a major effort to reduce the incidence of drug-
resistant fungi and bacteria in the United States through
antimicrobial discovery.
CLASSES OF ANTIFUNGAL
DRUGS IN CLINICAL USE
Polyenes
Since the 1950s, the fungicidal polyenes have served
as a powerful but highly toxic last line of defense for
combating invasive fungal infections. Polyenes are am-
phipathic, consisting of a hydrophobic polyene hydro-
carbon chain and a hydrophilic polyhydroxyl chain.
For decades the prevailing theory was that these mole-
cules elicited their toxic effects by intercalating into
ergosterol-containing membranes to form membrane-
spanning channels that cause leakage of cellular com-
ponents and, ultimately, cell death (8, 9). However,
recent detailed structural and biophysical studies high-
lighted that polyenes act more like an “ergosterol-
sponge,” forming large extramembranous aggregates
that extract the essential membrane-lipid ergosterol
from the plasma membrane (Fig. 1) (10). Given that
polyfunctional lipids such as ergosterol serve as central
molecular nodes in cellular physiology and function, re-
sistance to the polyenes is strongly evaded and comes at
a significant fitness cost to the fungus (11). The differ-
ential binding affinity of amphotericin B for ergosterol
over cholesterol, the mammalian membrane sterol, ac-
counts for its selectivity; however, amphotericin B does
affect mammalian cells, and nephrotoxicity is a com-
mon side effect of clinical usage that can be mitigated
somewhat by liposomal formulations (8). Despite these
drawbacks, amphotericin B is still used for the treat-
FUNGI AND THE HUMAN HOST
ment of the most serious fungal infections, in large part
due to its broad spectrum of activity.
Pyrimidines
Pyrimidines such as flucytosine (5-fluorocytosine) were
first used to treat fungal infections in the 1960s. How-
ever, the rapid onset of resistance precludes the use of
flucytosine as a monotherapy, and consequently it is
only used in combination with other antifungals such
as amphotericin B. This drug combination remains the
“gold standard” for treating Cryptococcus infections
and other less common invasive mycoses (12). Flucyto-
sine is a synthetic fluorinated analogue of cytosine. Upon
entry into the cytosol, flucytosine becomes rapidly de-
aminated to generate 5-fluorouracil by fungal specific
cytosine deaminases (13). 5-Fluorouracil acts as a potent
antimetabolite that causes RNA miscoding and inhibits
DNA synthesis (Fig. 1).
Azoles
The azole antifungals encompass compounds with both
imidazole and triazole moieties. They are the most
widely deployed class of antifungal in clinical use be-
cause of their impressive safety profile and availability
in both oral and intravenous formulations. For exam-
ple, oral azoles are more broadly accessible in resource-
limited settings such that oral fluconazole is widely
used to treat cryptococcal meningitis in these regions
despite treatment outcomes being much better with the
i.v.-administered combination of amphotericin B and
flucytosine (14). Furthermore, voriconazole is the drug
of choice for aspergillosis based on its superiority
to amphotericin B in a head-to-head clinical trial (15,
16). The azoles consist of a five-membered nitrogen-
containing heterocyclic moiety, which binds to an iron
atom in the heme group located in the active site of the
drug target lanosterol 14-α demethylase (also referred
to as cytochrome P45014DM and Erg11) (Fig. 1). Azole
binding blocks the demethylation of lanosterol, result-
ing in ergosterol depletion and the accumulation of
toxic sterol intermediates, which ultimately impedes cel-
lular growth and division and induces severe membrane
stress (7, 8). Most of the antifungal imidazoles are for-
mulated for topical use, as a consequence of toxicity
or bioavailability problems that limit their potential as
systemic agents. The only agents licensed for treatment
of invasive fungal disease are triazoles and include
fluconazole, itraconazole, posaconazole, voriconazole,
and isavuconazole (8, 17). The azoles are typically fun-
gistatic, with the notable exception of voriconazole,
which has fungicidal activity against A. fumigatus. The
fungistatic nature of azoles imposes strong directional
44. ANTIFUNGAL DRUGS: CURRENT ARMAMENTARIUM AND NEW AGENTS 905

Figure 1 Structures and mechanisms of action of clinically relevant antifungal drugs.

(A) The azoles function by targeting the ergosterol biosynthetic enzyme lanosterol de-
methylase, encoded by ERG11 (C. albicans and C. neoformans) or cyp51A and cyp51B
(A. fumigatus), causing a block in the production of ergosterol and the accumulation of a
toxic sterol produced by Erg3. This toxic sterol exerts a severe membrane stress on the cell.
(B) Polyenes such as amphotericin B primarily exist in the form of large extramembranous
aggregates that extract ergosterol from lipid bilayers. (C) Fungal cell walls are composed
of (1,3)-β-D-glucans covalently linked to (1,6)-β-D-glucans as well as chitin, mannans, and
cell wall proteins. The echinocandins act as noncompetitive inhibitors of (1,3)-β-D-glucan
synthase (encoded by FKS1 in C. albicans, C. neoformans, and A. fumigatus) and thereby
cause a loss of cell wall integrity and severe cell wall stress. (D) Pyrimidines such as
flucytosine become rapidly deaminated in the cytosol to generate 5-fluorouracil (5-FU) by
fungal-specific cytosine deaminases. 5-FU acts as a potent antimetabolite that causes RNA
miscoding and inhibits DNA synthesis. Adapted from reference 185 with permission.
906
selection for the evolution of resistance. Moreover,
several Candida species such as Candida glabrata and
Candida krusei are intrinsically less susceptible to this
class of drugs.
Echinocandins
The echinocandins are the only new class of antifungal
to reach the clinic in decades, with three echinocandins
currently available for clinical use: caspofungin, mica-
fungin, and anidulafungin. These compounds are cyclic
hexapeptides that act as noncompetitive inhibitors of
(1,3)-β-D-glucan synthase, an enzyme involved in cell
wall synthesis (Fig. 1). (1,3)-β-D-Glucan is an essential
cell wall polysaccharide that provides structural in-
tegrity. Inhibition of (1,3)-β-D-glucan synthesis culmi-
nates in a loss of cell wall integrity and severe cell wall
stress (18). The safety profile of these compounds is
impressive and likely attributable to a fungal-specific
target that is not conserved in mammals. Despite
their potent activity against Candida and Aspergillus,
these compounds are completely ineffective at combat-
ing Cryptococcus infections (18). Echinocandins have
emerged as an important treatment option for candidi-
asis, especially in the context of the increasing preva-
lence of azole resistance.
DEVELOPMENT OF IMPROVED AGENTS
WITHIN EXISTING CLASSES
Polyenes
In an attempt to improve the therapeutic index of am-
photericin B, alternative lipid-associated formulations
have been developed and incorporated into clinical
use since the 1990s (19). Amphotericin B lipid complex
(ABLC) consists of amphotericin B in a complex with
two lipids: L-α-dimyristoyl phosphatidylcholine and
L-α-dimyristoyl phosphatidylglycerol (19). The large
molecular size of the compound results in rapid uptake
of the drug by macrophages, which distribute the com-
pound throughout the mononuclear phagocyte sys-
tem including target organs such as the liver and spleen
(19). Consequently, compared with the conventional
formulation, there are lower circulating amphotericin B
serum concentrations and reduced accumulation in
the kidneys. Also of benefit, ABLC fails to stimulate
myriad proinflammatory signaling molecules in vitro
(20). Liposomal amphotericin B (L-AmB), marketed as
AmBisome, is composed of a small unilamellar vesicle
formulation consisting of hydrogenated soy phosphati-
dylcholine, cholesterol, and distearoyl phosphatidylglyc-
erol. Amphotericin B is tightly bound to the liposome
FUNGI AND THE HUMAN HOST
through charge pairing between the amino group of the
amphotericin B and the phosphate group of distearoyl
phosphatidylglycerol. Tissue concentrations in patients
receiving L-AmB tend to be highest in the liver and
spleen and much lower in the kidneys and lung. In a
rabbit model of hematogenous C. albicans meningo-
encephalitis, L-AmB accumulates in the brain at higher
levels than standard amphotericin B, significantly de-
creasing fungal burden in this tissue (21). This supports
the preference for L-AmB in treatment of invasive cen-
tral nervous system infections. Amphotericin B colloi-
dal dispersion, marketed as Amphocil and Amphotec,
encompasses two molecules of amphotericin B bound
to two molecules of cholesteryl sulfate. Similar to the
other liposomal formulations, there is reduced drug ac-
cumulation in the kidneys. However, unlike the other
liposomal formulations, amphotericin B colloidal dis-
persion induces a high degree of inflammatory gene
upregulation (20), which was likely a contributing fac-
tor related to the adverse side effects observed in sev-
eral clinical studies (22, 23).
In addition to these lipid polyene formulations,
alternative preparations continue to be developed. A
cochleate formulation of amphotericin B displayed
efficacy in experimental models of candidiasis and as-
pergillosis (24–26). Cochleates are a novel lipid-based
delivery vehicle consisting of crystalline phospholipid-
cation structures that form spiral lipid sheets. They
represent a new technology platform for oral delivery
of clinically important drugs that possess poor oral
bioavailability. Initial biodistribution studies of coch-
leate amphotericin B administered orally in mice found
that cochleates deliver significant levels of ampho-
tericin B to target organs (24, 26). Despite these suc-
cesses in animal models, clinical trials conducted by
BioDelivery Sciences International appear to have been
discontinued.
For the treatment of most invasive fungal infections,
an amphotericin B lipid formulation provides a safer
alternative than conventional amphotericin B, especially
in the context of nephrotoxicity. Unfortunately, despite
their use in the clinic for close to 20 years, it is difficult
to conclude which (if any) of these lipid formulations
possesses superior in vivo antifungal activity compared
to amphotericin B or compared to each other due to a
paucity of well-designed, randomized trials and also
due to the multitude of invasive infections caused by
evolutionarily diverse fungal pathogens.
Due to the pharmacological importance of ampho-
tericin B in treating systemic mycoses, medicinal chemists
have made extensive efforts to generate amphoteri-
cin B derivatives with more favorable pharmacological
44. ANTIFUNGAL DRUGS: CURRENT ARMAMENTARIUM AND NEW AGENTS
properties and reduced toxicity. Modification of the
mycosamine appendage through a double reductive alkyl-
ation generated a semisynthetic molecule with improved
efficacy against amphotericin B-resistant C. albicans
in vitro and reduced hemotoxicity (27). Improvements
to the therapeutic index of amphotericin have also
been made in a C2´deoxyamphotericin B derivative
(28). Often the major limitation of these derivatives
is the limited synthetic access, which precludes future
development. Recently, synthetic chemists reported
urea derivatives of amphotericin B that are easily acces-
sible from the natural product by using a three-step
synthesis reaction (29). These derivatives bind ergos-
terol with far greater selectivity than amphotericin B
in vitro and maintain potent antifungal activity with
reduced toxicity in mouse models of disseminated
candidiasis (29). Moreover, urea-containing amphoteri-
cin B derivatives strongly evade resistance (29). The
promising biological activities coupled with favorable
synthetic properties suggest bright prospects for these
amphotericin B derivatives in combating invasive fun-
gal disease.
Azoles
Given that azoles are the most widely deployed class of
antifungal, there is a long history of developing novel
analogues with improved efficacy and safety. Isavucon-
azole was developed as an orally active, water-soluble
prodrug that is also amenable to intravenous formula-
tions (Fig. 2). The prodrug, termed BAL-8557, becomes
cleaved in a reaction catalyzed by plasma esterases in
mammalian cells (30). It exhibits a long elimination
half-life, has broad-spectrum activity against Candida
and Aspergillus species, and has demonstrated good ef-
ficacy in several clinical trials (31). One such study sup-
ported the safety and tolerability of isavuconazole as
a prophylactic treatment in immunosuppressed patients
at high risk of fungal infections (32). This compound
has undergone phase III clinical trials to evaluate effi-
cacy in the treatment of Aspergillus infections in renally
impaired patients and those infected with other rare
fungi (clinicaltrials.gov NCT00634049), as well as to
compare efficacy to a caspofungin-voriconazole cother-
apy against Candida infections (NCT00444366 and
NCT00413218). In 2015, isavuconazole was approved
by the U.S. Food and Drug Administration (FDA) for
the treatment of invasive Aspergillosis and invasive
mucormycosis.
Albaconazole is another triazole currently under
development with potent broad-spectrum antifungal
activity, good pharmacokinetics, and excellent oral bio-
availability (Fig. 2). A phase II clinical study evaluated
907
the therapeutic effects of a single dose of albaconazole
in women with acute Candida vulvovaginitis and found
that doses of ≥40 mg had greater therapeutic effects
than fluconazole at 150 mg (30). Despite the efficacy in
treatment of vulvovaginitis, it remains unclear whether
albaconazole will have utility in treatment of invasive
fungal infections.
Echinocandins
The echinocandins were first introduced into the clinic
in 2001. Since this time there has been incentive to de-
velop novel analogues in this class, primarily to over-
come the current limitation of echinocandins to daily
intravenous dosing regimens. Aminocandin (Fig. 2),
also referred to as IP960 or HMR3270, is a semisyn-
thetic fermentation product from Aspergillus sydowii.
Its long half-life makes infrequent dosing a feasible op-
tion, and it has shown promising in vitro and in vivo
activity against Candida and Aspergillus species (33,
34). Similarly, Cidara Therapeutics has developed an
echinocandin derivative, CD 101 IV, which shows a
dramatically extended half-life in animal models of
C. albicans infection (35, 36). Preclinical evaluations
in rats uncovered no hepatotoxicity at doses up to
20 mg/kg for 2 weeks (37). Currently, this compound is
in phase I clinical trials (NCT02551549) and has been
designated a qualified infectious disease product with
fast track status from the FDA.
Enfumafungin is a structurally distinct natural prod-
uct class that is similar to the echinocandins in tar-
geting glucan synthase (38). The current development
candidate MK-3118 is an orally active, semisynthetic
derivative of enfumafungin with potent in vitro activity
against glucan synthase and potent in vivo activity
against Candida and Aspergillus species (Fig. 2) (39,
40). Thus, novel echinocandin derivatives and distinct
antifungals targeting glucan synthase activity represent
promising new treatment options to combat Candida
and Aspergillus infections.
WHAT CHEMICAL MATTER SHOULD WE BE
THINKING ABOUT FOR NEW DRUGS?
The optimal chemical space for antifungal compounds
is poorly defined. Unlike antibacterial drugs that are
dominated by microbial natural products, antifungals
include both synthetic and natural product compounds
in equal proportion (Fig. 1). In fact, synthetic com-
pounds dominate the fungicides used in agriculture.
This should be good news for antifungal drug discovery
because, in contrast to antibacterial campaigns, exist-
ing libraries of synthetic compounds in pharmaceutical
908 FUNGI AND THE HUMAN HOST

Figure 2 Structures of compounds with antifungal activity. Chemical structures of anti-

fungal molecules highlighted throughout the review. The description in brackets describes
the molecular target of the chemical compound.
44. ANTIFUNGAL DRUGS: CURRENT ARMAMENTARIUM AND NEW AGENTS
companies are likely to be good starting points for
antifungal screens. There are challenges in using these
compounds, however. For example, there are fungal-
specific PAIN (pan assay interference) compounds such
as the rhodanines that are promiscuous in antifungal
campaigns but have a poor likelihood of generating
good leads in drug development (41). Additionally, the
synthetic compounds that dominate pharmaceutical
company libraries are enriched in molecules with phys-
icochemico characteristics such as the Lipinski rule of
five (42), which emphasize human targeted drugs. As a
result, compounds identified in antifungal screens from
such libraries often overlap with compounds that have
bioactivity against human cellular targets. This reflects
the relatively narrow unique target space between fungi
and host biology (human, animal, or plant). Threading
the needle between uniquely antifungal targets and the
host is significantly challenging, especially if screened
compounds are biased toward human biology. Indeed,
as noted below, many highly useful drugs for human
diseases, such as rapamycin, were first identified in anti-
fungal screens. Not surprisingly then, most antifungal
drugs have significant off-target effects that are reflected
in host toxicity.
Expanding screens of synthetic molecules to libraries
that typically have not yielded bioactivity in human
targets (so-called chemical dark matter) can also offer
an entry point into a selective antifungal target space.
A group at Novartis specifically mined such chemical
dark matter in a screen for antifungal compounds that
identified a new compound and a new target, heme
biosynthesis (43).
Another option is a return to screens of natural
products to identify new leads in antifungal drug dis-
covery and development. Screens of microbial natural
product extracts often return polyenes such as ampho-
tericin, but these can often be quickly dereplicated
based on their characteristic absorption spectra. The
Merck group has been especially active in this area over
the past decade, developing new screening technologies
(44) and reporting a series of novel natural products
with antifungal activity that modulate new targets
such as poly(A) polymerase (45) and the fungal protea-
some (46).
The narrow target space for antifungal drugs, the
diversity of fungal physiology, and the absence of sys-
tematic physicochemico rules for nontoxic antifungal
agents complicate antifungal drug discovery. Neverthe-
less, clever screens for new targets and judicious mining
of natural products and synthetic molecule libraries con-
tinue to yield new antifungal leads and offer encourage-
ment for future success.
909
NEW ANTIFUNGAL DRUG
TARGETS IN DEVELOPMENT
Our current arsenal of antifungal drugs target either
ergosterol synthesis (azoles), ergosterol itself (polyenes),
or cell wall synthesis (echinocandins). These targets ac-
count for a minor fraction of the potential drug targets
encoded by genomes of fungal pathogens (47). Here we
highlight some of the most promising new antifungal
drug targets currently being explored for development
of novel therapeutics.
Fungal Sphingolipids
Sphingolipids are ubiquitous eukaryotic membrane
components that play a central role in signal transduc-
tion, cell regulation, and virulence in pathogenic fungi
(48). There are a variety of fungal-specific sphingolipids
present in the cell membrane and cell wall including
glucosylceramides (GlcCer), which are glycosphingo-
lipids composed of a glucose unit bound via a glycosyl
linkage to a ceramide moiety. As proof-of-principle,
genetic studies have established that targeting sphin-
golipid biosynthesis holds promise for blocking fun-
gal virulence. In C. neoformans, strains lacking GlcCer
synthase 1 are avirulent in a mouse inhalation infec-
tion model (49). Similarly, deletion of genes required
for sphingolipid biosynthesis in C. albicans renders the
pathogen avirulent (50). These genetic studies predict
that small molecules targeting sphingolipid biosynthesis
and/or function may serve as novel antifungal agents.
High-throughput screening efforts have been fruit-
ful in identifying small molecules that target sphingo-
lipid biosynthesis. A screen of 49,120 small molecules
to identify inhibitors of fungal sphingolipid GlcCer
synthesis identified two compounds: [N´-(3-bromo-4-
hydroxybenzylidene)-2-methylbenzohydrazide (BHBM)
(Fig. 2) and 3-bromo-N´-(3-bromo- 4-hydroxybenzyl-
idene) benzohydrazide (D0)] (51). BHBM and D0
were efficacious against C. neoformans in vitro, in an
intranasal infection model, and in an invasive infection
model of the central nervous system (51). Paradoxically,
although BHBM and D0 did not show potent anti-
fungal activity against C. albicans in vitro, they were
efficacious in a model of invasive candidiasis (51). Ge-
nome sequencing of drug-resistant mutants identified
four candidate targets of BHBM: Apl5, Cos111, Mkk1,
and Ste2, which are involved in vesicular transport and
cell cycle progression (51). Thus, this study has identi-
fied promising compounds that inhibit fungal GlcCer.
Beyond small molecules, additional strategies to tar-
get sphingolipid biosynthesis show considerable prom-
ise. Monoclonal antibodies specific to C. neoformans
GlcCer provide protection against C. neoformans infec-
910
tion, likely due to downregulation of the inflammatory
response (52). Moreover, treatment with Cerezyme, a
recombinant enzyme that metabolizes GlcCer, hydro-
lyzes fungal GlcCer in growing C. neoformans cultures,
disrupts membrane integrity, and exhibits in vivo effi-
cacy in an inhalation murine model of cryptococcosis
(53). Notably, sphingolipid biosynthesis is also impor-
tant for the host. Compromised sphingolipid biosyn-
thesis in mice confers hypersensitivity to C. albicans
infection due to the inability of immune cells to phago-
cytose yeast and clear the infection (54). These studies
reinforce the potential of sphingolipids as an antifungal
drug target and highlight the importance of specifically
targeting fungal, and not mammalian, sphingolipids.
GPI Anchor Biosynthesis
Many cell surface proteins are modified at their
C-terminus by the addition of a glycosylphosphatidyl-
inositol (GPI) anchor, which consists of a lipid group,
myoinositol, glucosamine, several mannose residues,
and a phosphoethanolamine group that connects the
GPI anchor to the protein through an amide bond (55).
This posttranslational modification results in proteins
being transported from the endoplasmic reticulum (ER;
the site of modification) to the plasma membrane and
cell wall, where they are directly or indirectly involved
in cell wall organization. GPI anchoring is a conserved
posttranslational modification in eukaryotes, although
the number of mannose groups and the position of side
chains on the GPI anchors vary widely between species.
In C. albicans, deletion of GPI7, a gene with important
roles in GPI anchor synthesis, results in abnormal mor-
phogenesis and reduced virulence (56). In A. fumigatus,
transcriptional repression of genes involved in GPI-
anchor biosynthesis results in cell wall defects, abnor-
mal morphology, attenuated virulence, and increased
cell death through autophagy and necrosis (57, 58).
Further, the GPI-anchored protein Ecm33 has important
roles in A. fumigatus morphogenesis, cell wall stress re-
sponse, and virulence (59).
Multiple small molecules that inhibit GPI-anchor
biosynthesis have been discovered. The first was iden-
tified by a screen for molecules that interfere with the
cell wall localization of a GPI-anchored reporter pro-
tein (60). The initial hit was 1-[4-butylbenzyl] isoquin-
olone, a molecule shown to target the acyl transferase
Gwt1 (60). Medicinal chemical-based optimization led
to development of the pyridine-2-amine-based molecule
E1210, a compound with potent antifungal activity
against numerous pathogenic fungi (61). Subsequently,
a small molecule originally identified as an inducer of
ER stress, gepinacin, was determined to be a specific
FUNGI AND THE HUMAN HOST
inhibitor of Gwt1, a critical acyltransferase required for
the biosynthesis of fungal GPI anchors (62). Sublethal
concentrations of gepinacin or genetic disruption of
GPI-anchored proteins exposes elevated levels of (1,3)-
β-D-glucan on the cell surface, enhancing the host im-
mune response against C. albicans (62, 63).
Farnesyltransferase
Farnesyltranferase is a ubiquitous eukaryotic enzyme
that catalyzes posttranslational lipidation on the C-
terminus of more than 120 important signaling pro-
teins in mammalian cells (64). This acts as a signal,
which culminates in the translocation of proteins from
the ER to the plasma membrane. Protein substrates of
farnesyltransferase bear a C-terminal CAAX sequence
motif: cysteine (C), two aliphatic residues (AA), and a
variable (X) residue. Ras proteins are key modulators
of diverse signaling cascades in eukaryotes and are an
example of proteins modified by farnesyltransferases.
In pathogenic fungi, Ras signaling pathways regulate
virulence, morphogenesis, and cell cycle control (65,
66). In the model yeast Saccharomyces cerevisiae, dele-
tion of the gene RAM1 encoding farnesyltransferase
results in reduced fitness, but it is not essential for via-
bility (67). However, farnesyltransferases are essential
for growth and virulence in C. neoformans, C. albi-
cans, and A. fumigatus (68–70). Farnesyltransferase
inhibitors such as manumycin A (Fig. 2), originally de-
veloped as an anticancer therapeutic, have antifungal
activity against C. neoformans and block localization
of C. neoformans Ras1 to the cell membrane (71). Man-
umycin A also displays some activity against A. fumi-
gatus and C. albicans (72). Additional farnesyltransferase
inhibitors, such as ethylenediamine inhibitor #2 and
tipifarnib, also show some antifungal activity against
C. neoformans, particularly in a mutant deficient in
capsule production (71). Finally, farnesyltransferase in-
hibitor III blocks the C. albicans morphological transi-
tion from yeast to hyphae, a virulence trait regulated by
Ras signaling (73).
Key physical interactions between farnesyltransfer-
ases and their inhibitors have been illuminated through
structural studies comparing fungal farnesyltransferases
with their human homologs. Using X-ray crystallogra-
phy, conformational differences between these enzymes
from humans and C. neoformans have been identified,
supporting feasibility of the development of fungal-
selective farnesyltransferase inhibitors (71). Similarly,
structural studies of farnesyltransferase enzymes from
A. fumigatus confirmed structural differences from the
human enzyme that are sufficient for species-specific
targeting (74). Although much of the work with
44. ANTIFUNGAL DRUGS: CURRENT ARMAMENTARIUM AND NEW AGENTS
farnesyltransferase inhibitors has focused on their im-
pacts on Ras localization and signaling, mapping the
fungal farnesylome may also uncover novel drug tar-
gets for future antifungal development. One example
is the Ras-related protein RhbA in A. fumigatus, which
is important for fungal pathogenesis and vegetative
growth in vitro and for virulence in an in vivo model of
infection (75).
Calcineurin
A principal regulator of cellular stress responses in
all eukaryotes is the Ca2+-calmodulin-activated protein
phosphatase calcineurin (76, 77). In pathogenic fungi,
calcineurin regulates myriad physiological processes in-
cluding cell cycle progression, cation homeostasis, mor-
phogenesis, virulence, and antifungal drug responses
(76, 77). Calcineurin is required for survival at physio-
logical temperatures in C. neoformans but is dispens-
able for growth in C. albicans and A. fumigatus (77).
Hence, the calcineurin signaling pathway has a conserved
role in fungal biology and virulence yet also possesses
unique specializations across the fungal genera.
Pharmacological inhibitors of calcineurin function,
including the natural products cyclosporin A and tac-
rolimus, have potential therapeutic indications. For
example, these agents are deployed in the clinic as im-
munosuppressants to prevent graft rejection in trans-
plant recipients (77). Their immunosuppressive activity
is due to inhibition of calcineurin-dependent activation
of a key transcriptional regulator termed the nuclear fac-
tor of activated T cells (NFAT), which is involved in
T cell activation. In fungi, calcineurin inhibitors have
diverse effects on cellular stress responses and virulence.
Pharmacological inhibition of calcineurin in C. neofor-
mans abrogates growth at 37˚C (78), potentiates the
activity of both azoles and echinocandins in C. albicans
(79–83), and renders the fungistatic azoles fungicidal
against multiple Candida species (84). Further, cyclo-
sporin A and tacrolimus act synergistically with azoles
against C. albicans biofilms both in vitro and in vivo,
implicating calcineurin as a key modulator of azole
resistance during biofilm growth (85). In A. fumigatus,
calcineurin inhibitors enhance echinocandin activity
and transform the fungistatic activity of caspofungin to
fungicidal (86, 87). Calcineurin inhibitors also show po-
tent activity against azole- and echinocandin-resistant
strains of A. fumigatus (88). Recently, it was shown
that calcineurin orchestrates dimorphic transitions,
antifungal drug responses, and virulence of the fungal
pathogen Mucor circinelloides (89, 90). Thus, targeting
calcineurin provides a logical strategy for combating
fungal disease caused by evolutionarily diverse fungi.
911
Therapeutic exploitation of calcineurin as a tar-
get for antifungal drug development requires fungal-
selective agents. Nonimmunosuppressive cyclosporin A
and tacrolimus derivatives retain antifungal activity
against C. neoformans, including azole-resistant strains
(91). These nonimmunosuppressive analogues also re-
tain synergistic activity with azoles against C. albicans
(84). Chromofungin, a synthetic vasostatin-I-related
peptide, acts as a calcineurin inhibitor and also dis-
plays in vitro activity against A. fumigatus (92). Devel-
opment of effective antifungal therapeutic agents that
target calcineurin signaling will require crystal struc-
tures of calcineurin from fungal pathogens coupled
with structure-guided drug design to enhance potency
and fungal-selectivity of calcineurin inhibitors or devel-
opment of agents targeting fungal-specific calcineurin
effectors (77).
Hsp90
Hsp90 is an essential molecular chaperone that regu-
lates the form and function of diverse client proteins
(93, 94). As a consequence of its role in stabilizing key
signal transducers, Hsp90 modulates the relationship
between genotype and phenotype by acting as both a
capacitor and a potentiator for the storage and release
of genetic variation (95–97). In pathogenic fungi, tar-
geting Hsp90 function as an antifungal strategy holds
considerable promise given that it orchestrates crucial
cellular responses to drug-induced stress (98–100). In
S. cerevisiae and C. albicans, genetic depletion or phar-
macological inhibition of Hsp90 impairs the evolution
of azole resistance and abrogates resistance that has
been acquired through diverse molecular mechanisms
(82, 83). Compromise of Hsp90 function also trans-
forms azoles from fungistatic to fungicidal (82). Phar-
macological inhibition of Hsp90 in C. neoformans and
C. gattii inhibits growth and potentiates azole activity
in vitro (101). Moreover, in C. albicans, C. glabrata,
and A. fumigatus, Hsp90 enables basal tolerance and
resistance to the echinocandins. Thus, Hsp90 has a pro-
found impact on resistance to multiple antifungal clas-
ses in diverse fungal pathogens (81, 102–104).
Hsp90 regulates responses to antifungal-induced
stress through its downstream effectors calcineurin and
protein kinase C signaling (81, 105). In a series of clini-
cal isolates recovered over time from an HIV-infected
patient treated with fluconazole, the basal fluconazole
resistance phenotypes of isolates recovered early during
treatment were dependent on Hsp90, calcineurin, and
Pkc1 function (82, 105). This dependence gradually
evolved toward independence of Hsp90, calcineurin,
and Pkc1, corresponding with upregulation of multi-
912
drug transporters (82, 105, 106). Biochemical analysis
confirmed that Hsp90 stabilizes the catalytic sub-
unit of calcineurin and the terminal MAP kinase of
the Pkc1 cell wall integrity pathway, Mkc1 (81, 105).
Hsp90 is one of the most highly connected hubs in
cellular networks, interacting with an estimated 10%
of the yeast proteome (107, 108). Chemical genomic
studies in C. albicans identified a multitude of Hsp90
genetic interactors important for cellular responses to
azoles and echinocandins (109), suggesting that Hsp90
may have pleiotropic effects on circuitry governing
drug resistance.
Hsp90 is also a powerful therapeutic target given
its function in orchestrating central virulence traits
such as fungal morphogenesis. In C. albicans the ability
to transition between distinct morphological states is
correlated with virulence (50) and is tightly regulated
by diverse environmental cues coupled with elevated
temperatures of at least 37˚C (7). Genetic or pharmaco-
logical inhibition of Hsp90 function induces filamenta-
tion by relieving its repression on Ras1-protein kinase
A signaling (110, 111). Elevated temperatures similarly
relieve Hsp90-mediated repression of filamentation as
the chaperone becomes overwhelmed with global pro-
tein misfolding in the cell (110, 112). To add additional
complexity, Hsp90 also regulates C. albicans morpho-
genesis through the cyclin Pcl1, the cyclin-dependent
kinases Pho85 and Cdc28, and the transcription fac-
tor Hms1 (113, 114). Intriguingly, genetic depletion of
Hsp90 in A. fumigatus has the opposite effect in that it
decreases hyphal growth and leads to severe defects in
germination and conidiation (102). Given its intimate
and complex role in governing fungal morphogenesis in
multiple species, targeting Hsp90 holds promise as an
antivirulence strategy to combat fungal infection.
The impact of Hsp90 inhibition on fungal virulence
and antifungal efficacy has been explored using a vari-
ety of in vivo infection models. In Galleria mellonella
models of fungal pathogenesis, combination therapy
with Hsp90 inhibitors that have been in clinical devel-
opment for cancer, such as 17-AAG (Fig. 2), with either
caspofungin or fluconazole improves survival upon in-
fection with A. fumigatus or C. albicans, respectively
(115). Furthermore, in murine models of C. albicans dis-
seminated disease, genetic compromise of fungal HSP90
expression reduces virulence (110) and enhances the
therapeutic efficacy of fluconazole and caspofungin
(81, 115). Similarly, genetic compromise of A. fumi-
gatus Hsp90 abrogates virulence in a murine model of
invasive aspergillosis (104). In a Caenorhabditis elegans
model of cryptococcal infection, Hsp90 inhibition en-
hances the activity of fluconazole (101). Finally, in an
FUNGI AND THE HUMAN HOST
in vivo rat catheter model of infection, 17-AAG trans-
forms fluconazole from ineffective to highly efficacious
without host toxicity in the context of this localized
infection and drug delivery (116). In the context of sys-
temic fungal infections, toxicity associated with inhibi-
tion of host Hsp90 precludes the use of current Hsp90
inhibitors as antifungals and demands the development
of fungal-selective Hsp90 inhibitors or agents that tar-
get fungal-specific regulators of Hsp90 function.
Acetyltransferases and Deacetylases
Lysine deacetylases (KDACs) and lysine acetyltrans-
ferases (KATs) catalyze the removal or addition of ace-
tyl groups from the ε-amino group of lysines. Such
modifications in core histones result in changes in chro-
matin structure and gene expression (117, 118). Con-
sequently, KDACs and KATs have pleiotropic effects
on cellular signaling and stress responses in fungi. The
broad-spectrum KDAC inhibitor trichostatin A poten-
tiates azole activity in C. albicans (119, 120). Further,
C. albicans clinical isolates are sensitive to nicotin-
amide, an inhibitor of NAD+-dependent KDAC com-
plexes, and nicotinamide reduces fungal kidney burden
in a mouse model of disseminated candidiasis (121).
Nicotinamide is also active against other Candida spe-
cies and against A. fumigatus and Aspergillus nidulans,
suggesting broad antifungal properties (121). Fur-
ther, a molecule thought to inhibit the KDAC Hos2,
MGCD290 (Mirati Therapeutics, San Diego, CA), dis-
plays synergistic activity with azoles and echinocandins
against diverse C. albicans drug-resistant clinical isolates
(122, 123). These studies were restricted to in vitro anal-
ysis, but in vivo studies coupled with preliminary clini-
cal trials have supported the use of MGCD290 in
combination with fluconazole to treat C. albicans infec-
tions (124). Thus, these findings suggest considerable
promise for the use of KDAC inhibitors in combination
with current antifungals in combating invasive fungal
disease.
In addition to their roles in modifying histones,
KDACs also deacetylate a spectrum of nonhistone pro-
teins, such as Hsp90. Acetylation of Hsp90 provides a
mechanism of posttranslational control that governs
the emergence and maintenance of Hsp90-dependent
azole resistance (120). In S. cerevisiae, Hsp90 is acety-
lated on lysine 27 and 270, residues that are regulated
by KDACs Rpd3 and Hda1 (120). Genetic or pharma-
cological inhibition of both Hda1 and Rpd3 blocks the
physical interaction between Hsp90 and calcineurin,
thereby impeding calcineurin function (120). Deletion
of both HDA1 and RPD3 does not increase global
Hsp90 acetylation levels, suggesting that additional
44. ANTIFUNGAL DRUGS: CURRENT ARMAMENTARIUM AND NEW AGENTS
KDACs orchestrate Hsp90 deacetylation. One candi-
date KDAC is Hos2, which interacts genetically with
Hsp90 under diverse environmental conditions in C. albi-
cans (109). In A. fumigatus, Hsp90 lysine residues im-
portant for drug resistance and virulence are conserved
with those identified in S. cerevisiae (120, 125). Thus,
by influencing gene expression through histone modifi-
cation and controlling the function of key signal trans-
ducers, KDAC inhibitors hold promise as antifungal
therapeutics.
KATs also modulate azole resistance. Genetic impair-
ment of C. albicans ADA2, which encodes a component
of the Spt-Ada-Gcn5-acetyltransferase (SAGA) coactiva-
tor complex, confers hypersensitivity to fluconazole due
to impaired upregulation of the efflux pumps Cdr1 and
Mdr1 (126). Further, deletion of the C. albicans KAT
gene RTT109 confers hypersusceptibility to macrophages,
altered metabolic gene expression, and induction of a
weaker inflammatory response, culminating in attenuated
virulence in a murine infection model (121, 127). Deletion
of RTT109 also confers increased sensitivity to echino-
candins and to other genotoxic stresses such as hydroxy-
urea and methyl methane sulfonate (121).
Other Antifungal Targets
The TOR protein kinases are global regulators of cellu-
lar growth in response to nutrient cues, including amino
acids (128). In S. cerevisiae, inhibition of TOR signal-
ing has a multitude of effects including induction of
autophagy, inhibition of translation, and repression
of ribosomal gene expression, as well as upregulation
of genes involved in the retrograde response, nitrogen
catabolite response, and stress response (128–130).
TOR can be inhibited pharmacologically by the immu-
nosuppressant rapamycin (sirolimus). Rapamycin is a
natural product of the bacterium Streptomyces hygro-
scopicus, originally identified in a screen for antimi-
crobial activity against C. albicans and later found to
have potent immunosuppressive activity (131). Pioneer-
ing genetic studies in S. cerevisiae identified FKBP12,
Tor1, and Tor2 as the direct targets of rapamycin
(132). Rapamycin inhibits growth of diverse fungi, in-
cluding C. neoformans, C. albicans, A. fumigatus, Fusa-
rium oxysporum, and Penicillium spp. (133, 134) and
abrogates erg3-mediated azole resistance (135). Ana-
logues of rapamycin with reduced immunosuppressive
activity retain antifungal activity in vitro (136).
There are several other antifungal agents in develop-
ment that target a variety of other cellular processes.
Parnafungins inhibit poly(A) polymerase and display po-
tent broad-spectrum activity against clinically relevant
Candida species and therapeutic efficacy in murine
913
models of candidiasis (Fig. 2) (45). Inhibitors of leucine
tRNA synthases, such as tavaborole (AN2690), are
highly selective against Trichophyton species (137, 138)
and are in clinical development to treat onychomycosis
(14). Finally, inhibitors targeting the 26S proteasome
(fellutamides, Fig. 2), translation elongation (yefa-
fungin), cyclic AMP homeostasis (campafungin), and
microtubule dynamics (12-deoxy-hamigerone) are all
unexploited areas that may represent the next break-
through in novel antifungal scaffold (14, 44, 46). While
it is impossible to predict which, if any, of the targets
highlighted above will prove to be the next clinically
exploited antifungal target, the portfolio of distinct
prospects provides renewed hope for the development
of novel antifungal treatments.
COMBINATION THERAPY TO EXPAND
THE REPERTOIRE OF DRUG TARGETS
Although advances are being made to improve our
current antifungal arsenal and to identify novel drug
targets, the rate at which antimicrobials are being pro-
duced is vastly outpaced by the rate at which resis-
tance is emerging (139, 140). A promising strategy for
combating drug-resistant fungi is through combination
therapy. Drug combination therapy has benefits such as
enhanced efficacy and specificity when compared to in-
dividual drug treatments and can impede the evolution
of resistance (141, 142). Further, by carefully selecting
specific drug combinations, microbial drug resistance
may be not only neutralized but also reversed through
a process called selection inversion (143). Combina-
tion therapy provides the foundation for treatment of
other infectious diseases such as HIV (144), tuberculo-
sis (145), and malaria (146). Despite the therapeutic
potential, there is only one combination that has been
well validated in clinical trials for fungal infections: the
combination of amphotericin B and flucytocine as the
gold standard to treat cryptococcosis (12). Combina-
tion therapy also has the potential to unveil myriad
additional antifungal targets given that fungal biology
is controlled by a highly interconnected and function-
ally redundant network of biological interactions (147,
148). The yeast deletion project revealed that only
∼1,000 of the ∼6,000 genes in the S. cerevisiae genome
are essential for viability (149). However, a tour de
force systematic analysis of ∼5.4 million pairs of gene
deletions identified ∼170,000 negative genetic interac-
tions, including 10,000 synthetic lethal interactions
(148). Synthetic lethality represents an extreme nega-
tive genetic interaction in which two mutations, causing
little to no fitness defect individually, result in a lethal
914
double mutant phenotype (150). The potential for
harnessing synthetic lethality provides a new avenue for
antimicrobial drug discovery through the use of combi-
nation agents.
There is precedent for leveraging the power of ge-
netic interactions to predict combination drug therapy.
In S. cerevisiae, FKS1 and its paralog FKS2 encode the
biosynthetic enzyme for (1,3)-β-D-glucan synthesis and
the molecular target of the echinocandins. FKS1 and
FKS2 are synthetically lethal, in keeping with echino-
candin efficacy. FKS1 is also synthetically lethal with
CHS3, a chitin synthase required for the synthesis of
the cell wall component chitin (148), and inhibitors of
chitin synthases, such as nikkomycin, are synergistic
with caspofungin against numerous fungal pathogens
(151, 152). FKS1 is also synthetically lethal with CNB1,
the regulatory subunit with calcineurin, and as high-
lighted above, calcineurin inhibitors exert potent syn-
ergy with echinocandins against diverse fungi (77).
While these examples support the fact that genetic in-
teractions underpin the network response to compound
combinations, the complexities of chemical action and
genetic network density seem to preclude the predic-
tion of synergism on a genome-wide scale (153). Thus,
additional factors must be employed to identify effec-
tive antifungal combinations.
In addition to leveraging genetic interaction land-
scapes to predict chemical synergy, target-specific in-
hibitors may be screened ad hoc for synergistic activity
against specific antifungal agents. We explored many
of these examples above, as with the capacity of inhibi-
tors of Hsp90 (98, 99), calcineurin (77), KDACs (120,
123), and TOR (136), to potentiate antifungals against
fungal pathogens. Other notable examples include po-
tentiation of azole activity by pharmacological inhibi-
tion of ADP-ribosylation factors (154), protein kinase
C (105), and protein translation (155). Further, tamoxi-
fen and other triphenylethylene-based estrogen recep-
tors have antifungal activity against C. neoformans
(156) and potentiate the activity of azoles, polyenes,
and echinocandins in diverse fungal species (157).
Tamoxifen inhibits the C. neoformans calmodulin pro-
tein Cam1, which activates calcineurin (158). Given that
Cam1 is fungal-specific, optimization of the triphenyl-
ethylene scaffold provides a promising path for anti-
fungal drug development.
Small molecules that target resistance determinants
can be effectively paired with currently available anti-
microbials to combat drug-resistant isolates. Examples
of such drug combinations in the clinic include drugs
such as amoxicillin, a β-lactam antibiotic, with clavu-
lanic acid, an inhibitor of β-lactamase drug-resistant
FUNGI AND THE HUMAN HOST
enzymes (140). A recent study with C. glabrata iden-
tified a small molecule (iKIX1) (Fig. 2) that inhibits
azole efflux pump expression by disrupting the interac-
tion between the mediator complex and the transcrip-
tional activator Pdr1 (159). Follow-up in vivo studies
supported the use of iKIX1 to potentiate fluconazole ef-
ficacy against fluconazole-resistant C. glabrata in both
metazoan and mammalian models of infection (159).
A complementary approach to identify synergistic
drug interactions employed in both academia and in-
dustry utilizes high-throughput screening of large com-
pound collections. Molecules that enhance the activity
of established antifungals against S. cerevisiae have
been identified by chemical biology screens (160, 161)
and computational methods (162–164). Recent large-
scale screening efforts have extended the strategy of
chemical synthetic lethality to the fungal pathogens
C. albicans and C. neoformans. Two such studies ex-
amined the potential of drugs approved for other ther-
apeutic indications to potentiate current antifungal
agents (157, 165). Compounds that increase azole effi-
cacy were found to often target membrane function or
sphingolipid biosynthesis (165). One such example is
the antimycobacterium compound clofazimine, which
induces cell membrane stress in fungi (157). A combi-
nation of clofazimine and caspofungin was efficacious
in vivo against C. albicans in a G. mellonella model
of infection (157). These studies illustrate the potential
of combining current antifungal treatments with repur-
posed medications.
CHEMICAL GENOMICS TO LINK
BIOACTIVES TO CELLULAR TARGETS
A variety of genetic and genomic approaches have been
developed to uncover the molecular target for a com-
pound whose mechanism of action remains elusive. Tra-
ditionally, forward chemical-genetic approaches have
been used to identify targets by selection of resistant
mutants coupled with plasmid-based library comple-
mentation, as well as gene and whole-genome sequenc-
ing (166).
More recently, reverse chemical-genetic approaches
have become widely used to identify the mechanism
of action of bioactive agents. In S. cerevisiae, haplo-
insufficiency profiling screens were developed to iden-
tify targets of bioactive compounds de novo based on
the principle that reducing the dosage of a drug tar-
get confers hypersensitivity to the drug. In these assays,
a genome-scale collection of heterozygous deletion
strains are pooled, grown in the presence of compound,
and sampled over time (167, 168). Each heterozygous
44. ANTIFUNGAL DRUGS: CURRENT ARMAMENTARIUM AND NEW AGENTS
strain has a unique barcode sequence that enables de-
convolution of strain identity in pooled screens (169).
Quantitative analysis of strain fitness can be achieved by
next-generation sequencing or microarray technology to
monitor barcode abundance (167, 168). Homozygous
deletion profiling (HOP) provides a complementary as-
say to investigate the mode of action of compounds that
lack an essential protein target (for example, DNA or
lipids) or for those compounds with redundant protein
targets (166). Further, HOP reveals the genes important
for buffering the cellular response to a drug. For exam-
ple, compounds that target DNA generate HOP profiles
that identify nonessential DNA-repair genes and com-
plexes required for surviving DNA damage (170).
Large chemogenomic screening efforts have unveiled
chemical-genetic interaction maps for thousands of
small molecules and revealed how eukaryotic cells
respond to cellular perturbations. An intensive study
with 1,144 haploinsufficiency profiling and 418 HOP
experiments identified growth phenotypes for mutants
covering 97% of the S. cerevisiae genome (171). This
expands our knowledge of yeast physiology and sug-
gests that most genes are conditionally essential with
functions that can be probed by small molecules. Che-
mogenomic profiles generated for 3,250 compounds
in S. cerevisiae revealed that the cellular response to
small molecules is largely restricted to a network of
45 chemogenomic signatures (172). These signatures
highlight the fundamental small molecule response sys-
tem in eukaryotic cells and provide a resource for
the exploration of relationships between genes, biolo-
gical processes, and chemical structures (172). Using
chemical-genomic datasets, researchers recently devel-
oped a machine-learning-based prediction algorithm
capable of predicting chemical synergies from large
chemical libraries (153). This algorithm achieves a
6-fold enrichment in predicting synergistic chemical
pairs with activity against fungi compared with chance
selection alone, providing an effective platform for
identifying antifungal chemical combinations.
Enabled by the development of powerful functional
genomics resources, analogous tools for chemical biology
are now available for the fungal pathogens C. albicans
(173, 174) and C. neoformans (175). It is becoming
abundantly clear that gene essentiality and chemical-
genetic responses diverge rapidly over evolutionary time
(175, 176), underscoring the importance of performing
chemical genomic analyses directly in fungal pathogens.
In addition to chemogenomic profiling, the develop-
ment of a functional variomics tool provides a com-
plementary approach to elucidate drug mode of action
(177). This technology applies plasmid-based, high-
915
complexity random mutagenesis (or variomic) libraries
covering ∼90% of the S. cerevisiae genome. Screening
this library against test compounds allows for systematic
discovery of resistance-conferring mutations, thereby de-
fining residues critical for drug binding and identifying
potential mechanisms of resistance (177). Research com-
bining chemogenomic and variomic tools has led to the
identification of two novel antifungal geranylgeranyl-
transferase I inhibitors, highlighting the importance of an
integrated platform for drug target identification (178).
OUTLOOK
It is clear that creative and focused efforts are urgently
needed to bolster the antifungal pipeline and improve
clinical outcome for the millions of people with life-
threatening fungal infections worldwide. There are new
strategies on the horizon to expand the development
opportunities beyond targeting essential genes or cellu-
lar functions. A complementary approach is to target
proteins that are required for pathogen growth in the
host or for virulence. Identifying nonessential virulence
factors opens new opportunities for chemical diversity
in therapeutic drugs, because the cognate inhibitors
are not typically explored by conventional screening
approaches. Although the potential utility of targeting
nonessential genes has only recently been appreciated,
there are already examples of antibiotics that target es-
sential structures rather than essential proteins, as with
daptomycin and the Gram-positive cell wall (179). Fur-
ther, the diphtheria and tetanus vaccines, as well as the
antitoxin for botulinum toxin, are examples of life-
saving treatments targeting nonessential virulence fac-
tors (180, 181). Targeting virulence factors offers many
benefits including expanding the repertoire of antifun-
gal targets, minimizing effects on the host mycobiome,
and reducing selection pressure for the evolution of
drug resistance (182, 183). With advances in genomics
technologies and structure-guided drug design, as well
as having antifungals now granted “orphan status” by
the FDA and a priority in the GAIN act (H.R. 2182)
to facilitate clinical trials (184), there is great potential
to foster stronger relationships between academia and
industry to accelerate antifungal drug discovery.
Citation. Robbins N, Wright GD, Cowen LE. 2016. Antifun-
gal drugs: the current armamentarium and development of
new agents. Microbiol Spectrum 4(5):FUNK-0002-2016.
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