Journal of

Fungi
Review
Sources of Antifungal Drugs
Giel Vanreppelen † , Jurgen Wuyts † , Patrick Van Dijck * and Paul Vandecruys

KU Leuven Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Kasteelpark Arenberg 31,
B-3001 Leuven, Belgium
* Correspondence: [email protected]; Tel.: +32-16-32-15-12
† These authors contributed equally to this work.

Abstract: Due to their eukaryotic heritage, the differences between a fungal pathogen’s molecular
makeup and its human host are small. Therefore, the discovery and subsequent development of
novel antifungal drugs are extremely challenging. Nevertheless, since the 1940s, researchers have
successfully uncovered potent candidates from natural or synthetic sources. Analogs and novel
formulations of these drugs enhanced the pharmacological parameters and improved overall drug
efficiency. These compounds ultimately became the founding members of novel drug classes and
were successfully applied in clinical settings, offering valuable and efficient treatment of mycosis for
decades. Currently, only five different antifungal drug classes exist, all characterized by a unique
mode of action; these are polyenes, pyrimidine analogs, azoles, allylamines, and echinocandins. The
latter, being the latest addition to the antifungal armamentarium, was introduced over two decades
ago. As a result of this limited arsenal, antifungal resistance development has exponentially increased
and, with it, a growing healthcare crisis. In this review, we discuss the original sources of antifungal
compounds, either natural or synthetic. Additionally, we summarize the existing drug classes,
potential novel candidates in the clinical pipeline, and emerging non-traditional treatment options.

Keywords: antifungal; natural products; synthetic compound library; antifungal pipeline

1. Introduction
Citation: Vanreppelen, G.; Wuyts, J.; From the foxfire bioluminescent mushrooms to the largest organisms on earth, fungi
Van Dijck, P.; Vandecruys, P. Sources are diverse, ubiquitous cornerstone members of various ecosystems. Whereas many fungi
of Antifungal Drugs. J. Fungi 2023, 9, are beneficial to humans, e.g., for cheese and alcohol production, a number of them also
171. https://doi.org/10.3390/ display pathogenic characteristics. Fungal infections pose a continuous global threat to
jof9020171 human and animal health, jeopardize entire ecosystems, and place a tremendous burden
Academic Editors: Maria
on food production [1]. Fungi cause a range of infections in humans, from harmless, super-
Elisa Rodrigues and Sónia Silva ficial maladies to life-threatening invasive mycoses. The global acquired immunodeficiency
syndrome (AIDS) crisis, the increased use of implants, and the overall improved survival
Received: 31 December 2022 rates of immunocompromised patients have resulted in a steady increase in fungal infec-
Revised: 22 January 2023 tions [2,3]. These are associated with relatively high incidence, high mortality rates, and
Accepted: 26 January 2023
high hospitalisation costs [4]. This is particularly the case for tenacious biofilm-associated
Published: 28 January 2023
infections [5]. Biofilms are complex three-dimensional structures with a typical micro-
colony architecture characterized by extensive spatial heterogeneity and an extracellular
matrix material associated with increased resistance to host immune factors and antifun-
Copyright: © 2023 by the authors.
gals [6]. Due to these characteristics, biofilms frequently allow infections to re-establish after
Licensee MDPI, Basel, Switzerland.
treatment. Therefore, it is no surprise that fungal infections are responsible for 1.4 million
This article is an open access article deaths on a global scale each year [7].
distributed under the terms and Treatment options for human fungal infections are currently limited to five different
conditions of the Creative Commons classes of antifungals, of which just three are regularly used as standalone treatments for
Attribution (CC BY) license (https:// mycosis. Figure 1 provides a chronological overview of their point of origin and both the
creativecommons.org/licenses/by/ discovery and introduction to the market of their most established member. As discussed
4.0/). further in this review, recently, a number of novel classes of antifungals reached the market

J. Fungi 2023, 9, 171. https://doi.org/10.3390/jof9020171 https://www.mdpi.com/journal/jof

J. Fungi 2023, 9, x FOR PEER REVIEW 2 of 21

J. Fungi 2023, 9, 171 mycosis. Figure 1 provides a chronological overview of their point of origin and both 2 ofthe
20
discovery and introduction to the market of their most established member. As discussed
further in this review, recently, a number of novel classes of antifungals reached the mar-
ketare
or orin
are in advanced
advanced clinical
clinical trials.trials.
Here,Here, weprovide
we will will provide a summary
a summary of theof the currently
currently used
used drug classes.
drug classes.

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Figure Timelineofofthe
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andtheir
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(1) The
(1) The polyenes
polyenes are are the
the oldest
oldest antifungal
antifungal drugdrug classclass inin clinical
clinical use
use today
today andand hall-
hall-
markedaamajor
marked majorclinical
clinical advancement
advancement in mycosis
in mycosis treatment.
treatment. AlthoughAlthough the boasts
the class class boasts
some
some
of of the
the most mostantifungal
potent potent antifungal
compounds, compounds, they arewith
they are associated associated with severe ne-
severe nephrotoxicity [8]
phrotoxicity [8] and low water solubility [9], which limits their
and low water solubility [9], which limits their use as antifungal drugs. Amphotericin B use as antifungal drugs.
Amphotericin
(AmB) B (AmB)
is the most is the member
well-known most well-known
of this class.member
It was of this class.
purified in 1953It was
as a purified
secondary in
1953 as a secondary
metabolite metabolitebroth
from the secretion fromofthethesecretion broth of the
actinobacterium actinobacterium
Streptomyces nosodusStreptomy-
by Eliza-
ces nosodus
beth L. Hazen by Elizabeth
and Rachel L.F. Hazen
Brown and Rachel
[10]. F. Brown
Additional [10]. Additional
representatives representatives
of the polyene drug of
class currently
the polyene in use
drug classfor antifungal
currently therapy
in use are natamycin
for antifungal therapyand arenystatin.
natamycin Theandlatter was
nystatin.
unearthed
The latter by wasHazen and Brown
unearthed by Hazenbeforeandthey discovered
Brown before AmB. Amphotericin
they discovered AmB. B, and its lipid
Amphoteri-
formulations with enhanced activity, were and are still predominantly
cin B, and its lipid formulations with enhanced activity, were and are still predominantly used in the clinical
context
used in[11]. Polyenes
the clinical exert their
context [11]. effect by binding
Polyenes sterols,
exert their effect primarily
by binding ergosterol,
sterols,which
primarilyare
crucial components
ergosterol, which are forcrucial
the stability of the fungal
components for themembrane.
stability ofAlthough
the fungal discussion
membrane. on Alt-
the
exact
hough mode of action
discussion on of
thepolyenes
exact mode is ongoing,
of actionit ofis generally
polyenes is accepted
ongoing, that the
it is antifungal
generally ac-
effect is caused by sequestration of ergosterol from the fungal cell
cepted that the antifungal effect is caused by sequestration of ergosterol from the fungal membrane, by acting as
acell
sterol-sponge [11]. Therefore, fungal exposure to the polyenes
membrane, by acting as a sterol-sponge [11]. Therefore, fungal exposure to the poly- causes membrane pore
formation,
enes causessterol rafts, and
membrane pore reactive oxygen
formation, species
sterol rafts, (ROS) formation,
and reactive oxygenwhich induces
species (ROS) apop-
for-
tosis.
mation, which induces apoptosis. Hence, polyenes are strong fungicidal compounds[12].
Hence, polyenes are strong fungicidal compounds for most fungal pathogens for
Although
most fungal nephrotoxicity
pathogens [12]. limits the practicality
Although of polyenes
nephrotoxicity limitsin thefavor of newer
practicality ofantifungals,
polyenes in
due
favortooftheir potent
newer fungicidal
antifungals, effect
due and low
to their resistance
potent fungicidal rate,effect
they and
havelow remained
resistancethe rate,
last
bastion for patients with life-threatening invasive fungal infections
they have remained the last bastion for patients with life-threatening invasive fungal in- for over 70 years.
Initially
fections synthesized
for over 70 years.as an anti-cancer agent in 1957, flucytosine is the sole member of
the (2)Initially
pyrimidine analogsas
synthesized antifungal drug class
an anti-cancer agentand was introduced
in 1957, flucytosinein is 1971.
the soleThese pyri-
member
dine analogs are compounds that mimic the structure of pyrimidine
of the (2) pyrimidine analogs antifungal drug class and was introduced in 1971. These [13]. 5-fluorocytosine
(5-FC) is a fluorinated analog of the nucleoside cytosine. The 5-FC drug is administered as
a pro-drug and taken up by susceptible fungi through cytosine permease. Upon entry in
the cytosol, it is rapidly deaminated by cytosine deaminase to yield 5-fluorouracil (5-FU).
This compound exerts its antifungal activity via two distinct mechanisms. First, from 5-FU,
J. Fungi 2023, 9, 171 3 of 20

fluorodeoxyuridine monophosphate (FdUMP) can be generated through the action of uri-

dine monophosphate pyrophosphorylase. This FdUMP inhibits fungal DNA biosynthesis
by acting on thymidylate synthase. Second, 5-FU can also yield fluorouridine triphos-
phate (FUTP), which can serve as a substitute for uridylic acid. Its integration into fungal
RNA inhibits protein synthesis, which disturbs the amino acid pool and amino-acetylation
of tRNA [14]. Depending on the fungal pathogen, its effect can either be fungicidal or
fungistatic [14]. Although it interferes with fundamental molecular mechanisms, resis-
tance against 5-FC is widespread and has barred the drug from monotherapy treatment.
However, it is administered in combination with other drugs, acting synergistically with
AmB and with members of the azole drug class [15]. The 5-FC drug is recommended at
the early stages of cryptococcal pneumonia and meningoencephalitis. Less frequently, this
combination therapy is used for invasive fungal infections in favor of monotherapy [16,17].
However, in regions where access to AmB is limited, 5-FC combined with fluconazole is
administered for cryptococcal meningoencephalitis.
Due to the toxic nature of polyene compounds, and the intrinsic tolerance of pathogens
to 5-FC, researchers continued to search for novel and safer compounds. This ultimately
led to the introduction of the azole drug class in the 1960s, although the antifungal effect
of the azole compound benzimidazole had been described in 1944 by Woolley [18,19].
Currently, (3) the azoles are the most widely used antifungals. Initially discovered during a
synthetic screening, members of the drug class can be divided into two groups, namely
the imidazoles (miconazole and ketoconazole) and the triazoles (fluconazole, itracona-
zole, voriconazole, posaconazole, and isavuconazole). Due to lower host toxicity, overall
better pharmacokinetics, and increased efficacy, the triazoles have replaced the earlier
imidazoles starting with fluconazole, which was introduced in 1990 [20]. All azoles act by
preventing the biosynthesis of ergosterol, a key membrane component. More specifically,
they inhibit cytochrome P450-dependent lanosterol 14-α-demethylase, a crucial step in
the ergosterol biosynthesis pathway, by competitively binding its heme group within the
substrate pocket [21,22]. Depletion of ergosterol results in the inability of fungal cells to
develop mature cell membranes. Additionally, with lanosterol demethylase inhibited, the
fungal cell will accumulate toxic methylated sterol intermediates. As a result, the azoles
inhibit fungal growth and are in general fungistatic drugs. Although they function through
an identical mode of action, there is a distinct difference between different triazoles in
efficacy, spectrum of activity, and overall pharmacological parameters. As mentioned
before, azoles suffer from rapid resistance development and overall intrinsic tolerance of
multiple species, their widespread use in clinical and agricultural settings has exacerbated
this trend. Additionally, azoles are subjects of unfavorable drug–drug interactions, which
causes significant problems with patients undergoing polypharmacy. If no innovative adap-
tation or next-generation compounds are found for this chemical drug class, the resistance
issue might result in a continuous downfall in applicability.
(4) Allylamine antifungals were discovered by chance in 1974 during efforts to syn-
thesize novel compounds for the treatment of central nervous system disorders. Its most
successful member, terbinafine, was approved for topical use in 1992 and subsequently
as an oral antifungal in 1996. [23] The drug is commonly used for treating dermatophyte
infections. Allylamines work similarly to the azoles by inhibiting the ergosterol pathway.
However, they target squalene oxidase, an enzyme upstream in the ergosterol pathway [24].
This causes intracellular squalene accumulation and ergosterol deficiency in the fungal
cell membrane. Interestingly, it was reported very recently that researchers purified and
identified allylamine compounds from secretion extracts of a Lysinibacillus isolate, making
this class the only one that has both a synthetic and natural origin [25].
(5) The echinocandins drug class is the newest member in the clinical antifungal
armory, introduced in 2001 [26]. They are semisynthetic lipopeptides whose bioactive
precursor compounds were purified from fungal secretion extracts [27], and later chemi-
cally adjusted to improve solubility and overall pharmacokinetic characteristics. Its first
member approved for systemic use, caspofungin, was derived from the fungus Glarea
J. Fungi 2023, 9, 171 4 of 20

Lozoyensis, which secreted pneumocandin B0 , a lipophilic cyclic peptide. Currently, three

echinocandins have been approved for clinical use, namely caspofungin, anidulafungin,
and micafungin. More recently a new member, rezafungin from Cidara Therapeutics,
was determined a QIDP with fast-track status by the FDA and has obtained an orphan
drug designation in the US and EU [28]. Echinocandins inhibit β-(1,3)-glucan synthase,
a crucial enzyme in glucan biosynthesis [29], by binding non-competitively to the Fks1
subunit. In general, the fungal cell wall relies heavily on an interlinked polysaccharide
skeleton of β-(1,3)-glucan and chitin for structural integrity. Disruption of β-(1,3)-glucan
synthesis results, therefore, in compromised cell walls. This causes osmotic instability,
lysis of the cell, and ultimately cell death. Echinocandins are restricted to IV use only and
are expensive relative to other antifungals. These antifungal drugs are associated with a
paradoxical growth pattern, with growth at supra-MIC concentrations, referred to as “the
Eagle effect” [30,31]. Depending on the targeted organism, echinocandins are considered
as either fungistatic or fungicidal [32].
Several antifungal candidates as representatives of known and potentially novel drug
classes are currently in the clinical pipeline and have been extensively reviewed elsewhere.
The three compounds belonging to potential novel drug classes will be briefly discussed
here, as well as a novel azole formulation [33–35].
First, ibrexafungerp (formerly known as SCY-078 and MK-3118) is an antifungal
developed by Scynexis. It is currently in phase 3 clinical trials to treat recurrent vulvo-
vaginal candidiasis (RVVC) and invasive fungal infections (NCT05399641) (NCT05178862).
Ibrexafungerp received FDA approval on June 1, 2021. The compound employs a similar
mechanism of action as the echinocandins; however, as a triterpenoid it has a completely
different chemical structure from the echinocandins. Second, olorofim, formerly known
as F901318, is a synthetic compound developed by F2G, representing the orotomide drug
class [36]. Olorofim inhibits dihydroorotate dehydrogenase [37], a novel target, and an
essential part of pyrimidine biosynthesis of molds, such as the Aspergillus species. At the
time of writing, olorofim is undergoing phase 3 clinical trials for the treatment of inva-
sive aspergillosis (NCT05101187). Third, fosmanogepix, formerly known as APX001, is a
synthetic compound discovered in a target-based screening assay [38,39]. It is currently
in phase 2 clinical trials for the treatment of non-neutropenic patients with candidemia
(NCT03604705) and invasive mold infections (NCT04240886). Fosmanogepix is currently
being developed by Pfizer after the company acquired Amplyx Pharmaceuticals. Fosman-
ogepix is a N-phosphonooxymethylene prodrug that metabolizes into manogepix after
administration. It acts by targeting the fungal enzyme GPI-anchored wall transfer protein 1
(Gwt1), which is necessary for the acylation of inositol in the glycosylphosphatidylinositol
(GPI) anchor biosynthesis pathway. Since this pathway is vital for proper mannoprotein
anchoring to the cell wall, its interruption compromises the structural integrity of the fungal
cell wall, which will ultimately lead to growth cessation. Finally, oteseconazole (formerly
VT-1161) developed by Mycovia Pharmaceuticals, is a tetrazole, and was granted FDA
approval for the treatment of RVVC in April 2022 [40]; it is undergoing phase 3 clinical
trials (NCT03562156). The tetrazole has an improved affinity for fungal CYP51 compared
to triazoles but an overall lower affinity for heme iron [41]. Tetrazoles might introduce a
new, improved trend for the azole drug class.
Despite the availability of several different classes of antifungals, there are still unmet
needs that require the development of novel antifungals [33]. Antifungal use is widespread
to treat human infections, for crop protection, timber preservation, and treatment of animal
infections. This excessive use is leading to a global increase in resistance, primarily to
the azole antifungals [42]. Moreover, the emergence of species that are able to acquire
multidrug, or even, pan-resistance, such as Candida auris [43], complicates treatment options
and highlights the need for novel antifungal drug candidates. Given this unmet need, we
review potential sources for novel antifungals. Here, we focused on the sources that can
be used to screen for novel antifungals and what the implications are when using these
sources.
J. Fungi 2023, 9, 171 5 of 20

2. Natural Products
Historically, natural products have been a rich source of antimicrobials [44]. It all
started when Alexander Fleming accidentally discovered penicillin [45]. He observed
a mold contamination that visibly inhibited the growth of his staphylococci. Selman
Waksman (1944) applied the same principle on a larger scale to screen for antimicrobials
produced by Actinobacteria. This approach is also referred to as the Waksman platform.
As a result, he and his team discovered streptomycin, the first antibiotic active against
Gram-negative pathogens that could be used as a drug [46]. Using this same approach, they
also identified several antifungal compounds (e.g., candicidin) [47]. Still, it was Hazen and
Brown (1951) that discovered nystatin, the first antifungal compound from Actinobacteria,
that would be developed as an antifungal drug [48].
It comes as no surprise that a large proportion of the antimicrobials that are currently
applied are derived from natural products. Consider how millions of years of evolution
shaped the continuous arms race between microorganisms, for which the production of
antimicrobials offered a competitive edge to survive or even thrive in a certain niche. The
further development of these compounds boomed, a success caused by the effectiveness of
the Waksman platform, but also due to the inefficiency of synthetic screening campaigns
and target-based approaches. Even though hit rates for antifungals from natural product
libraries sometimes exceed synthetic screening campaigns up to 200-fold (see Table 1),
they tend to be challenging drug candidates. An overall downside of natural products is
that they are often large and complex, making de novo synthesis or production of analogs
challenging and, consequently, making it harder to establish them as treatment options in
the clinical context [49].

Table 1. An overview of what to expect when setting up a screening to find (novel) antifungals.

Natural Products Synthetic Compounds

Fungi-Derived Bacteria-Derived
B-glucan synthase inhibitors Polyenes
Most common hits Azoles (15% of hits) [50]
(12% of hits) [50] (95% of hits) [47]
Hit rates (%) ~20% [50] ~9% [50] ~0.1–2% [51–53]
Common PAINs Mycotoxins [50] Ionophores [50] Rhodanines [54]

It is recognized that microorganisms have a complex life cycle [55]. They often reside in
multicellular structures, such as biofilms, and this preferred lifestyle is also reflected in the
clinical context. These biofilms are up to 100-fold more resistant to antifungals compared
to planktonic cultures and it has been well-estimated that most infections originate from
biofilms [56–58]. Strikingly, natural product antifungals appear to have higher anti-biofilm
activity, compared to synthetic antifungals. Echinocandins and polyenes, both derived
from natural products, are associated with strong anti-biofilm activity. In contrast, azoles,
allylamines, and pyrimidine analogs are synthetic in origin and exert poor anti-biofilm
properties [56,59–61]. The reduced efficacy of these antifungals on biofilms is attributed
to their sequestration by the extracellular matrix containing β-glucan, which reduces the
antifungal concentration able to reach the target cells, resulting in increased tolerance of
cells within the biofilm [62,63].
Large-throughput screening campaigns by biotech and pharma companies, but also
academia, are well-suited for lead compound discovery [64]. To fill in the gaps, over the
last decade, compound libraries containing pure or semi-pure natural products have been
composed. A prime example is the compound library of the National Cancer Institute’s
Natural Products Branch (NPB). With over 320,000 fractions available for large-scale screen-
ing, it holds one of the largest collections of publicly available pre-fractionated natural
product libraries [65]. These natural products can be derived from plants, fungi, or bacteria.
Plant-derived antifungals. Plants live in timescales that cannot be compared to those
of most (micro)organisms. Combined with their sessile lifestyle, plants need defense mecha-
J. Fungi 2023, 9, 171 6 of 20

nisms that trigger little to no resistance development, ensuring their usefulness throughout
their lifespan. Preferably, these active compounds address various challenges that plants
may experience at a given moment, such as predation by rodents or insects, and infection
by microorganisms. As a result, plant-derived compounds appear to be mainly toxic
with lower specificity, restricting their application scope to anti-cancer or anti-parasitic
drugs [66]. Prime examples of aspecific plant-derived natural compounds are curcumin
and resveratrol. These compounds have been reported to have antiviral, antibacterial,
and antifungal properties (among others) [67–70]. Despite several hundreds of clinical
trials and thousands of publications, these compounds are now regarded as pan-assay
interference compounds (PAINs). These molecules are frequent hitters in (phenotypic)
screening campaigns and often share structural features that show promiscuous biological
activity. Therefore, clinical applications for most of these molecules are unlikely [54]. No-
table exceptions here are anti-malaria compounds, such as artemisinin and quinine, which
act on malarial mitochondria and purine nucleoside phosphorylase as their specific targets,
respectively [71,72]. Interestingly, some anticancer compounds, such as camptothecin and
podophyllotoxin, identified in plant extracts, are now assumed to be produced by fungal
endosymbionts [73].
Fungal-derived antifungals. Remarkably, fungi are among the best producers of
antifungals. As with some bacteria, fungi have multidomain non-ribosomal peptide syn-
thetases (NRPS) that can produce peptides without the aid of ribosomes. Although the
principle of NRPS is the same, clear differences between fungal and bacterial non-ribosomal
peptides exist, such as peptide size distribution and monomer composition. Aside from the
final peptide itself, the enzymatic synthesis methodology can strongly differ [74]. Fungal-
derived natural products are often unique to a fungal genus or species, since horizontal
gene transfer in fungi is rather rare compared to bacteria [75]. Therefore, the isolation
of rare fungi is associated with increased chances of isolating novel natural antifungal
products. Several medically useful antifungals derived from natural products produced
by fungi include the echinocandins and the novel ibrexafungerp [26,47,76]. Both classes
of antifungals target the catalytic subunit of β-glucan synthase. These β-glucan synthase
inhibitors are the most frequently isolated compounds from fungal extracts but have never
been isolated from bacterial sources [50]. The fungi that produce the natural precursors
of these drugs all belong to the family of Trichocomaceae. They are aggressive colonizers
and probably produce antifungals to maximize their potential as saprobes. Generally,
they themselves are less susceptible to the antifungals they produce. For example, the
echinocandins have strong concentration-dependent fungicidal activity against Candida but
are only static against Aspergillus, a member of the Trichocomaceae family. Due to the diver-
sity of the fungal kingdom, these family feuds should be considered when the antifungal
development program focuses on different pathogenic lineages, for example, on Aspergillus
or other members of the Trichocomaceae family.
Bacteria-derived antifungals. Antifungals derived from bacterial sources, in clinical
use today, are all derived from Actinobacteria. These aerobic Gram-positive bacteria are
highly abundant in soil and marine sediments and constitute one of the largest bacterial
phyla [77]. They have a significantly larger genome size compared to other bacteria and
a high G/C content. They are self-sustainable, making them easy to isolate and cultivate.
Like fungi, they develop a mycelium with spores. During spore formation, the vegetative
mycelium undergoes programmed cell death to reallocate nutrients to the spores. To
prevent other microbes from using these nutrients, they produce secondary metabolites
with antimicrobial activity [7]. Therefore, Actinobacteria and especially the Streptomyces
genus are recognized as specialized producers of secondary metabolites [78,79]. It has
been estimated that members of the genus of Streptomyces alone could produce up to
100,000 molecules with antimicrobial activity [80]. The potential of these bacteria has been
well known for almost a century, resulting in large screening campaigns to exploit the
antimicrobial potential of Actinobacteria. Cubist Pharmaceuticals, for example, screened
over 107 Actinobacteria every year and estimated that a novel antibiotic could be discovered
J. Fungi 2023, 9, 171 7 of 20

at frequencies below 10−7 per random Actinobacteria. Moreover, they estimated that the
global top 10 cm of soil contains 1025 –1026 Actinobacteria, leaving plenty of opportunity for
further screening. Because the burden of fungal infections was often less recognized in the
past, it is unlikely that as many Actinobacteria have been screened for antifungal activity as
for antibiotic properties.
Despite extensive efforts, so far, the only clinically useful antifungals discovered from
Actinobacteria were the polyenes. However, due to nephrotoxicity, their implementation
is limited [81,82]. Over 200 polyene compounds have been described, mainly from Strep-
tomyces [9]. They appear to be the most abundant antifungals produced by Actinobacteria,
outweighing other antifungals by a factor 20 [47]. A screening by Roemer et al. (2011)
confirmed the abundance of polyenes produced by Actinobacteria. Moreover, they also con-
cluded that most antifungals produced by Actinobacteria appear to lack specific targets, with
the majority being ionophores [50]. This resulted in a decreased hit rate for target-specific
antifungals derived from Actinobacteria (9%), compared to fungi (>50%). One strategy to
avoid the rediscovery of polyene antifungals employs the use of a polyene-resistant test
strain. However, this resistance is generally associated with a serious fitness cost [83],
making it hard to use resistant strains in screening efforts to decrease polyene rediscov-
ery. Fortunately, polyenes can be readily identified in extracts due to their distinct light
absorption spectra [84].
Another interesting group of antifungals from actinobacteria are the chitin inhibitors,
nikkomycins and polyoxins. The latter was derived from Streptomyces cacaoi in 1960,
while the former was derived from Streptomyces tendae in 1976 [85,86]. These peptidyl
nucleoside antibiotics are analogs of the substrate UDP-N-acetylglucosamine and, therefore,
act as competitive inhibitors of chitin synthase. Since chitin is a crucial component of
a stable fungal cell wall and is absent in mammalian cells, it is generally considered
a promising drug target [87]. Polyoxin D showed in vitro activity against Coccidioides
immitis, Cryptococcus neoformans, and C. albicans, but failed to remain consistent during
in vivo murine assessments [88–90]. The compound nikkomycin Z showed potent activity
against some infections, such as coccidioidomycosis, but only displayed moderate activity
against Histoplasma capsulatum, C. albicans, and C. neoformans. Furthermore, filamentous
fungi and non-albicans Candida species were practically resistant. It does, however, work
synergistically with glucan synthesis inhibitors and triazoles [91–95]. It underwent clinical
trials in the 1990s, but the bankruptcy of the sponsoring pharmaceutical companies resulted
in the termination of ongoing trials. Stranded as a research topic, the project was continued
by the University of Arizona, which reactivated the clinical studies [93,96–100].
Other notable antifungals that have been discovered more recently from actinobacterial
sources include bafilomycins, neomaclafungins, astolides, caniferolides, and azalomycin
F [101–105]. However, several of these compounds also inhibit the growth of mammalian
cells and bacteria, thereby diminishing their potential for development as medically useful
antifungals.
Streptomyces are, historically, the most successful bacterial genus in terms of antifungal
drug discovery thanks to the polyenes which have become a cornerstone in mycosis
treatment. However, other genera also stood out due to their remarkable antifungal
activity. Bacillus and Pseudomonas species have numerous records in the literature reporting
their antifungal potency. Pseudomonas aeruginosa is a prominent opportunistic pathogen
that displays an antagonistic relationship with fungal pathogens during co-infection. It
secretes an array of metabolites to overcome fungal competitors during infection; as such,
these metabolites are often characterized as essential virulence factors of the pathogen.
These include lactones, alkyl quinolones, rhamnolipids, phenazines, and siderophores
such as pyrrolnitrin. Most act as crucial quorum sensing molecules, iron scavengers, and
overall virulence factors [106–112]. Although they exhibit strong antifungal activity, often
these metabolites suffer from host toxicity, making further drug development challenging.
Bacillus species, especially its most known member bacillus subtilis, have long been known
for their biocontrol properties, tackling diseases caused by fungal phytopathogens. Their
J. Fungi 2023, 9, 171 8 of 20

antifungal activity has been attributed to a multitude of compounds including but not
limited to lipopeptides (surfactins, iturins, fengycins), polyketides (bacillaene, macrolactin),
enzymes, such as chitinases, and volatile compounds, such as pyrazine [113–129]. Although
an increasing number of antifungal agents have been identified and purified from both
bacillus and pseudomonas species, none have been able to make it through drug development
for clinical adaptation.
Microbial dark matter. Most bacteria and, to a lesser extent, fungi cannot be cultivated
in standard laboratory conditions [130]. In natural ecosystems, this so-called “microbial
dark matter” makes up roughly 99% of the microorganisms and comprises a diverse
set of microorganisms. Undoubtedly, unknown natural compounds with antimicrobial
properties stay hidden as this vast potential remains unmined. Soil-derived microorganisms
can roughly be divided into three classes.
The first class, the cultured minority, comprises less than 1% of the total amount of
microorganisms. Almost all bacteria in this group belong to only four phyla, namely Acti-
nobacteria, Proteobacteria, Bacteroidetes, and Firmicutes. All microbial-derived antimicrobials
used today come from this group, but the low-hanging fruits of this group have been
picked [131].
The second class is the in situ cultivable group. These microorganisms cannot be imme-
diately cultivated in a laboratory environment because growth factors, such as siderophores,
are missing [131]. Cultivating these microorganisms requires more advanced methods,
such as, for example, the isolation chip (iChip) developed by Nichols et al. (2010). This
device holds miniaturized microbial growth chambers where single cells are confined
and separated from the environment by a semi-permeable membrane [132]. This protects
slow-growing species from aggressive colonizers that often dominate samples cultivated
in the lab. Additionally, growth factors essential for germination or growth produced by
other microorganisms or present in the soil can permeate through the membranes, making
proliferation possible, which results in pure cultures of potentially novel microorganisms.
Although in situ cultivation can be used to isolate a larger proportion of uncultivable
microorganisms from soil samples, this approach rarely results in the isolation of microor-
ganisms from uncultured phyla. Instead, the in situ cultivated microorganisms are usually
rare or less cultured members of Actinobacteria, Proteobacteria, and Firmicutes [132]. Because
these organisms are closely related to microbes that have already been extensively screened,
a large proportion will likely produce the same or highly similar antimicrobials. Never-
theless, rare isolates can yield novel antimicrobials. This approach has already proven its
success with the discovery of the promising antibiotic teixobactin [133]. Still, it remains to
be seen whether antifungals discovered using this platform find their way to the clinic.
The third and final class are microorganisms that cannot be readily cultured in standard
laboratory conditions, even when in situ cultivation devices, such as the iChip, are used. In
terrestrial habitats, these microorganisms belong to phyla lacking cultured representatives,
such as Acidobacteria, Chloroflexi, and Planctomycete. Cultivation is hard, if not impossible,
for this group. It has been suggested that some of them are intrinsically slow growers
and that cultivation is only possible after growing them for several months in the lab
while retaining the correct conditions [134,135]. It remains unclear why some of these
uncultured microorganisms are so abundant in the soil [130]. Probably, some necessary
factors are still lacking to cultivate these microorganisms in a lab environment. Moreover, it
is unknown whether these microorganisms can produce antimicrobials since they generally
have relatively small genome sizes, ranging from only 0.148 Mb to 2.4 Mb [136]. It has been
estimated that below a genome size of 3 Mb, polyketide synthase (PKS) and non-ribosomal
peptide synthase (NRPS) genes are absent or rare [137]. As per the current literature, since
these genes are critical components of secondary metabolite pathways, it is unlikely that
they are abundant producers of secondary metabolites [79]. Still, it can also not be excluded
that species with a small genome size encode antimicrobial molecules that are not encoded
by NRPS or PKS operons. In contrast, as mentioned before, Streptomyces coelicolor has a
genome size of 7.6 Mb, of which 5–10% of its genomic sequence is dedicated to secondary
J. Fungi 2023, 9, 171 9 of 20

metabolite production [78,138]. Only a limited number of uncultured microorganisms have

been whole-genome sequenced so far. Thus, all we need is the discovery of a novel genus
from the microbial dark matter with a similar coding potential to Streptomyces, enabling us
to unlock another era of highly successful natural product discovery.
Although beneficial, strain cultivation is not necessary for antimicrobial discovery.
A recent study discovered the malacidin class of antibiotics using a culture-independent
approach [139]. A polymerase chain reaction-based approach was used to amplify calcium-
dependent antibiotic gene clusters directly from soil samples. After heterologous expression
in the model host Streptomyces albus, secretion extracts were screened for antibiotic activity
resulting in the isolation and purification of malacidins.

3. Synthetic Compounds
Whereas most antibiotics are of natural origin, the most frequently used antifungals,
the azoles, are of synthetic origin. The reason for this may be the relatively late interest
in antifungal drug discovery. After several decades of steady mortality rates due to
candidiasis, in 1970, mortality rates increased substantially. This rise can be attributed to
the use of immunosuppressive therapies, the increase in immunodeficient patients, such as
those suffering from human immunodeficiency virus (HIV) infections, the increased use of
antibacterial agents with a broad spectrum, and the frequent use of indwelling intravenous
devices. Only in the 1980s were invasive mycoses recognized as a health threat [140].
Consequently, when large-scale screening platforms emerged, the focus resided on bacteria
rather than fungi. So far, only one class of antifungals approved for standalone systemic
use, the azoles, are derived from synthetic compound libraries [141,142].
Synthetic compounds are the result of available techniques and a chemist’s imagina-
tion. Consequently, they occupy a more limited chemical space than natural products [66].
Since the outcomes of these screening efforts are restricted by the envisioned goal and
pharmaceutical and chemical parameters of the included compounds, compound libraries
are generally biased [143,144]. One way to resolve this is by using a synthetic compound
library that is comprised of a diverse set of compounds. For example, the Community
for Open Antimicrobial Drug Discovery (CO-ADD) has composed a library of chemical
compounds from academic sources and is continuously using crowdsourcing to increase
its library size [145]. A proof-of-concept screening resulted in 20–30 times higher hit rates
for bacteria (compared to commercially available libraries) and a hit rate of 0.98% for
fungi [146]. This library will also be used to screen against the fungal targets C. albicans
and C. neoformans.
Imidazole and triazole pharmacophores are relatively abundant in these libraries
and are estimated to constitute around 15% of the hits when screening these libraries
against the opportunistic fungal pathogen Candida albicans [50]. Compounds that enter
these synthetic libraries need to pass through filters to make them a “good drug” later in
the development process. One such rule is the Lipinski rule of five (RO5) which states
that molecules should have a limited size and a lipophilic nature to ensure a good oral
bioavailability [147]. Therefore, most compound libraries are biased toward compounds
that follow these rules but do not necessarily have good antifungal properties. These rules
were defined by comparing the properties of compounds that made it through the first
phase of clinical trials. However, recently it was disproven that molecular weight can be
used to predict oral bioavailability [148]. An additional advantage of these low molecular
weight (<500 Dalton) compounds was their relative ease of synthesis. Therefore, when
RO5 was established in 1997, synthetic compound libraries contained relatively smaller
molecules resulting in a bias towards smaller molecules that were used as drugs. The
molecular weight of approved drugs has been steadily increasing over the past years, and
it has been estimated that good absorption drops sharply above 975 Dalton [149], almost
twice the size originally described in RO5. It is expected that higher molecular weight
molecules will be added to synthetic compound libraries in the future, and this concomitant
increase in complexity could also yield higher hit rates against fungi.
J. Fungi 2023, 9, 171 10 of 20

Synthetic compound libraries are often used for target-based drug discovery, while
natural products are more often screened in whole-cell assays. Unfortunately, target-
based antifungal drug discovery faces identical issues to antibiotic-based drug discovery,
and has so far failed to yield a clinically applied antimicrobial [50,134,150]. One study
exemplifies the difficulties that an in vitro target-based screening can encounter during
translation to in vivo viability screens [51]. During this study, the activator–mediator
interaction responsible for Candida glabrata azole resistance (Pdr1 activation domain and
the Gal11A KIX domain) was targeted. In this screen, small molecules that could inhibit
this interaction would re-sensitize drug-resistant C. glabrata to azole antifungals. A dozen
synthetic compound libraries were screened, totaling over 143,000 compounds. This
resulted in 352 potential inhibitors in an in vitro screening. Due to the presence of the
fungal cell wall, a large proportion of these active compounds lacked the ability to penetrate
the fungal cell wall envelope. Only five compounds showed activity on live cells, with
iKIX1 as the most promising lead, corresponding to less than 2% of all in vitro hits.
Despite the challenges associated with the screens using synthetic compound libraries,
they can still be a very successful approach, as shown by the recent discovery of F901318
(olorofim) [37]. In this study, the F2G company screened 340,000 compounds against the
airborne pathogenic mold Aspergillus fumigatus and discovered a novel chemical series
with potent activity against Aspergillus species, but with no activity against C. albicans. This
might explain why these compounds went unnoticed in previous screening campaigns,
because Candida was typically the target pathogen. This indicates that using a panel of
different fungi as targets can reveal novel compounds with a novel mode of action.

4. Non-Traditional Antifungal Options

Aside from the small-molecule drug treatment for mycosis, other options, so-called
non-traditional antifungals are being assessed by research and, thus, have not been em-
ployed in the clinical context. These so-called “non-traditional antifungals” include pep-
tides, antibodies, vaccines, immunomodulating compounds, mycophages, and virulence
factor inhibitors [33,151,152].
Peptides are promising antifungal alternatives [153]. Antifungal peptides (AFPs)
originate from a natural source or can be synthesized. In December 2022, the antimicrobial
peptide database (APD3) contained 1,277 AFPs [154]. Notable examples of antifungal
peptides include LL-37 and histatin 5 [155,156]. They generally exert their antifungal effect
by disrupting the cell and/or mitochondrial membrane. The AFPs can form an alpha
helix structure, beta-hairpin, a cysteine residue sheet, or a mixture of these conformations
upon interaction with membranes. At present, the majority of AFPs were discovered by
testing their bioactivity during in vitro screening efforts. Bednarska et al. (2016) developed
a platform to target bacterial proteins by cross-aggregation [157]. This platform could
be extended to fungi as well. However, several hurdles need to be overcome before
AFPs can be successfully used as drugs. They are unstable, have a low half-life, and low
bioavailability [158]. Additionally, posttranslational modifications play a crucial role in
their biosynthesis and antifungal properties, and in silico predictions of these modifications
and overall structure-activity relationship (SAR) remain challenging. However, with the
advancements in up-and-coming in silico prediction techniques, the AFP field might see
breakthrough advances within the coming decade.
The host’s innate and adaptive immune response and its effector molecules, antibodies,
control fungal infectious agents by preventing entry, restricting replication, and modulating
the immune response to clear the infection. This combat tool can be exploited by creating
antibodies can mark and combat microbes with high specificity. This promising strategy
has the potential to alleviate the resistance crisis and can be vital for immunocompromised
patients who need immune support [159]. Mycograb is such an example. It is a human
recombinant monoclonal antibody that binds heat shock factor 90 (Hsp90) specifically
at the site that enables the conformational change upon ATP binding, thereby inhibiting
its function [160]. It showed promising synergy with the established antifungal drugs
J. Fungi 2023, 9, 171 11 of 20

fluconazole, caspofungin, and amphotericin B, but was unable to demonstrate standalone

activity in vitro against C. albicans [161,162]. It did, however, demonstrate therapeutic
efficacy in an invasive candidiasis trial [163]. Unfortunately, no approval for clinical use
was obtained from the European Medicines Agency, and this trial was terminated because
of quality concerns due to autoaggregation of the antibody. Despite novel formulations,
therapeutic application remained out of reach for Mycograb, resulting in the discontinuation
of the development of this antibody. Nevertheless, Mycograb provided a vital proof of
concept. Antibodies targeting another part of the cell wall or even receptors, such as GPCRs
(if accessible), could be valuable sources of antifungals in the future.
Vaccine development to prevent fungal infections poses an interesting alternative be-
cause the at-risk patient groups that would benefit from a vaccine are well recognized [164].
Five vaccine categories exist for human fungal diseases, although there is still a long way
to go before commercial application. They are listed as follows with a representative
example [165]: (1) Live-attenuated strains, such as the C. neorformans strain lacking the
sterylglucosidase enzyme [166]; (2) killed fungal strain, such as the vaccine for coccid-
ioidomycosis of formalin-killed spherules [167]; (3) fungal extracts, such as glucan particles
consisting of Cryptococcus alkaline extractions [168]; (4) DNA or RNA vaccines delivering
nucleic acid-encoded antigens, such as the DNA vaccine against Penicillium marneffei using
the cell wall antigen Mp1p [169]; (5) purified fungal associated macromolecules, proteins,
peptides, carbohydrates, and lipids. For example, the previous mentioned NDV-3A vaccine
utilizes a recombinant agglutinin-like sequence 3 protein from C. albicans [168].
Despite the availability of both bacterial and viral vaccines, only a few fungal vaccines
have been enrolled in clinical trials. Even though encouraging results have been obtained,
until now, they have failed to deliver [165,170]. A promising vaccine so far is the NDV-3A
vaccine to treat RVVC. It has shown promising results in a phase 2 trial and has also shown
a protective effect in mice against S. aureus infection [171–173]. So far, vaccine development
to combat C. albicans has received much attention but developing a vaccine for this fungal
pathogen is hard because of three key reasons [170]. First, C. albicans is well known for
its phenotypic plasticity. It can exist in varying phenotypes, including in yeast form, as
pseudohyphae and hyphae, while concomitantly being present as white, grey, or opaque
cells, making the selection of epitopes hard [174]. Second, C. albicans has evolved as an
obligate commensal. Consequently, the immune system naturally tolerates C. albicans when
it is non-pathogenic. Therefore, training the immune system to act against C. albicans
feels contradictory. Third, candidiasis generally occurs in immunocompromised patients
and, thus, mounting an immune response in patients with an out-of-commission immune
system is difficult [170].
Immunomodulatory compounds are another interesting avenue to treat fungal in-
fections. This strategy has been propagated due to advances and successes in immuno-
oncology [33]. One example of such a strategy is the targeting of the JUN terminal kinase
1 (JNK1), a negative regulator of the host’s innate immune response. Inhibitors of JNK
have demonstrated efficacious antifungal resilience in mice [175]. However, similar to
hurdles in vaccine development, patients with mycosis often have an attenuated immune
system; therefore, developing immunomodulatory compounds is challenging. Moreover,
these types of compounds should not aggravate the immunopathology of the patient or
interfere with the effects of other immunomodulatory drugs that are already used to treat
an underlying disease. Finally, due to different immunologic conditions, these compounds
will likely be useful for some, but not for others.
Although not as prevalent as bacteriophages, mycophages might represent a treatment
option for fungal infections [176]. Despite not being studied as much as bacteriophages,
mycophages have been studied predominantly in Aspergillus [177], but have also been
reported in Candida [178]. It has been shown that some mycophages can be transferred from
Aspergillus to Saccharomyces [179], demonstrating that mycophages can have a broad host
range. Infection of Aspergillus with mycophages has been shown to result in a suppression
of mycotoxin production, a decreased growth rate and a reduced spore formation [177].
J. Fungi 2023, 9, 171 12 of 20

However, viral infection of fungi can sometimes also lead to hypervirulent fungal strains. It
is important to note that bacteriophages face tremendous challenges as a treatment option,
and it is likely that mycophages will face similar if not more stringent challenges [180]. For
example, bacteria can develop resistance to phages fast, phages have a limited host-range
(often specific to only a subset of strains), and their large size limits tissue distribution. To
resolve the limited host-range and resistance issues, mixed formulations of different phages
are used, sometimes containing up to 10 different phages, which increases the complexity
and cost during production and storage.
Finally, virulence factor inhibitors have gained attention recently. The major advantage
of virulence factor inhibitors is that they do not directly kill cells, nor do they inhibit the
growth of cells. Instead, as the name implies, they inhibit their virulence capabilities and,
therefore, do not exert as much evolutionary pressure to develop resistance as a traditional
antifungal. However, this has recently been refuted for bacteria, so it is expected that this
could also be the case for fungi [180]. One of the most attractive targets for antifungal
virulence factor inhibitors is the yeast-to-hyphae switch, since it is critical for virulence.
Antisense therapy using antisense oligonucleotides (ASOs), blocking the expressions of
genes necessary for virulence, may be a promising avenue. The ASOs complementarily
bind to their target mRNA by Watson–Crick base pairing. As such, they can restore, reduce,
or inhibit the target protein expression. This approach has been investigated in other
medical fields and has gained FDA approval to treat several afflictions. Unsurprisingly,
antisense therapy is being studied as a novel laboratory tool within the fungal field and
as a potential innovative antifungal strategy for the treatment of mycoses [181–183]. Since
the most common fungal pathogens, Candida, Aspergillus, and Cryptococcus species, have
been studied for decades, their virulence traits are well documented. Antisense therapy
could, therefore, benefit greatly from this fundamental knowledge. Recently, Araùjo
and coworkers (2019, 2022) have developed an ASO targeting the EFG1 mRNA, which
encodes for a central transcriptional regulator of morphogenesis. They reported promising
in vitro results as well as validation in a galleria mellonella assay [184,185]. It should be
noted that currently used antifungals, such as AmB, also block this transition at sub-MIC
concentrations [186,187]. Moreover, during drug development, a novel antifungal should
have at least as good (non-inferiority study) or better (superiority study) activity than the
currently used drugs [188]. Candida infections are still associated with mortality rates of
roughly 40%, indicating a clear need for novel antifungals. The echinocandins and polyenes
are fungicidal and associated with potent anti-biofilm activity. Additionally, their resistance
development frequency is relatively low or absent. Infections in non-immunocompromised
patients are controlled by antimicrobials and the few remaining fungi are cleared by the
immune system. In contrast, systemic fungal infections, such as candidiasis, rarely occur in
patients with healthy immune systems. Consequently, the immune system is inadequate
to clear the infection. Because virulence factor inhibitors by definition cannot clear the
infection, they will probably be useless as curatives in fungal infections. Hence, their
application might be as an adjuvant to antifungal drugs during treatment rather than as
a standalone therapy. However, anti-virulence compounds could be useful in non-lethal
and chronic infections. Examples include RVVC infections and infections of the skin and
nails. A virulence factor inhibitor could demonstrate value in these types of infections as a
prophylactic where the number of episodes can be used as an endpoint.
Most non-traditional antifungals must overcome additional hurdles. For example, a
MIC determination is not always available or even impossible to determine. This requires
another way to determine an efficacious concentration. This is especially problematic
for immunomodulatory compounds that do not directly exert their effect on fungal cells.
Additionally, defining success in vivo during clinical trials will also require novel endpoints
for most drugs [180,188].
J. Fungi 2023, 9, 171 13 of 20

5. Conclusions
The increase in antifungal resistance, the emergence of multidrug-resistant species,
and the grim mortality rates of fungal infections all amount to the continuous need for
novel antifungal compounds. Several sources from natural and synthetic origins were
successfully mined in the past and currently still deliver valuable lead compounds for
drug development. However, novel drug discovery proves challenging due to the frequent
re-discovery of known antifungals and the identification of molecules that already belong
to the scarce antifungal drug classes currently in use. To expand the repertoire of antifungal
agents, one could look at underexplored niches. Since most screening efforts took broad,
rudimentary soil samples, microorganisms that are restricted to specific niches, such as the
rhizosphere or nests of social insects may prove to be a rich source of bioactive compounds
with antifungal properties. Moreover, the creation of large natural product-based libraries
and a transition of synthetic libraries towards a more diversity-oriented composition will
allow the exploitation of a larger fraction of the chemical space, and eventually, expand the
antifungal toolbox. Aside from expanding the chemical vision, tailoring screening goals
to specific pathogenic species, rather than to fungi altogether, will allow the discovery of
family-specific bioactive agents.
Although the field has experienced a standstill of more than two decades since the
introduction of the last distinct antifungal drug class, there is hope on the horizon as
promising candidates from both synthetic and natural origins are currently in the final
phases of development. Like their predecessors, these compounds might become the pro-
genitor of a new class, alleviating the resistance crisis by expanding the therapeutic options.
Furthermore, as in oncological, bacterial, and viral research, non-traditional antifungal
research efforts, such as vaccines, antibodies, anti-virulence factors, immunomodulatory
compounds, and mycophages could revolutionize the field and the way patients are treated.
Finally, there exists extensive fundamental knowledge of fungal pathogens’ essential genes
and their corresponding proteins. Therefore, interesting antifungal drug targets have long
been identified, although identification of compounds that efficiently disrupt said targets
without causing harm to the host is challenging. Anti-sense therapy with tailored ASOs
could overcome this issue and become a new source of man-made antifungal compounds.

Funding: This research was funded by the KU Leuven Industrial Research Fund, grant number
C3/20/008 and G.V. was supported by a grant from The Fund for Scientific Research Flanders (FWO,
grant number 1S43720N).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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