Exploiting Fitness Trade-offs to
Prevent Antifungal Drug Resistance
by
MASSACH
ETT \N TITUTE
OF TECHNOLULGY
Benjamin Matteson Vincent
JUL 0G 2015
B.S., Biological Sciences
Stanford University (2008)
LIBRARIES
Submitted to the Program in Microbiology
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in MICROBIOLOGY
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
February 2015
Massachusetts Institute of Technology 2015. All rights reserved.
Signature redacted
Author _ _...............................................g
,Program in Microbiology
Certified by
Signature redacted
r
ell,
Susan Lindquist
Department of Biology
Thesis Supervisor
Signature
e a t d ....................................
i n t r redacted
Accpteby
by ...... S
Accepted
Michael T. Laub
Department of Biology
Chairman, Microbiology Graduate Committee
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Exploiting Fitness Tradeoffs to Prevent Antifungal Drug Resistance
By
Benjamin Matteson Vincent
Submitted to the Program in Microbiology on January 9 th, 2014 in partial fulfillment of
the requirements for the Degree of Doctor of Philosophy in Microbiology
ABSTRACT
The evolution of drug resistance in pathogenic microorganisms is typically
viewed as an inevitable consequence of the selective pressures imposed by antimicrobial
agents. However, although certain drugs rapidly lose efficacy in the clinic, others remain
refractory to resistance. In my thesis, I tested the hypothesis that antifungal drug
resistance is the outcome of diverse tradeoffs between the fitness benefits of resistance
mutations and their costs upon the adaptive cellular factors that enable the evolution of
new traits. I also pursued the discovery of novel small molecule inhibitors of fungal drug
resistance to further delineate the cellular processes required to support this phenotype.
First, I examined the mechanisms underlying the exceptionally low incidence of
resistance to the polyene antifungal amphotericin B (AmB), a mainstay of antifungal
therapy for over 50 years. Genome sequencing of AmB-resistant clinical isolates and
mutants evolved in the laboratory revealed several loss-of-function mutations in
ergosterol biosynthesis as causes of resistance. However, deeper phenotypic profiling of
these mutants revealed that they all carried massive costs for the ability to tolerate
environmental stressors and deploy virulence factors. I concluded that these costs
sharply limit the emergence of resistance to AmB in the clinic.
AmB is also extremely toxic to patients, a feature seen in many other resistancerefractory drugs. Collaborating with a synthetic chemistry team headed by Martin Burke
(University of Illinois), we demonstrated that new, nontoxic AmB analogs with improved
selectivity for the target (ergosterol) still retain the extremely valuable property of
evading drug resistance. This finding countered the assumption that narrowing the target
specificity of a drug would enable more routes to the emergence of resistance.
Separately, I conducted a high-throughput screen for small molecule inhibitors of
drug resistance in Candida. This screen identified a fungal-selective inhibitor of
cytochrome bcl of the electron transport chain. Collaborating with chemists (JeanBaptiste Langlois and Stephen Buchwald) and structural modelers (Raja Srinivas and
Bruce Tidor), we optimized the potency and fungal selectivity of this compound.
Moreover, these studies provided insight into the role of mitochondrial respiration in
fungal host adaptation, immune evasion, and virulence in an animal model.
Finally, in the appendix I describe work in progress on the discovery and
characterization of a compound that selectively kills Candidaby targeting the
mitochondrial phosphate transporter. Resistance to this agent sensitizes fungi to azoles
and bears severe fitness costs, reminiscent of the case of AmB.
Thesis supervisor: Susan Lindquist
Title: Professor of Biology
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Acknowledgments
Completing the work described in this thesis was only possible through the
tremendous support of many generous people. First, Susan Lindquist has been an
encouraging and supportive advisor and scientific guide to me. She has taught me to
focus my efforts on the most important implications of my research and not get
sidetracked with minor concerns. She has also made it possible for my work to have the
greatest impact it can through enabling some remarkable collaborations for me. I am
extremely lucky as a graduate student to have worked on such important projects with the
full support of many other talented researchers, and she has made all of that happen.
I am also indebted to Sue for creating a wonderful lab environment. The person
most responsible for this is Brooke Bevis, who works tirelessly to make the lab run
smoothly. I can't even begin to thank Luke Whitesell for the amazing technical training,
scientific guidance, and simply good-natured generosity he has provided to me. Ruthie
Shouval also went to great lengths to help me when I needed it. The Lindquist lab has
been a remarkable place for camaraderie and lighthearted fun over these past five and a
half years. As time passes, I will remember the great friendships I've made here as the
best part of my experience. But even more than that, the science that my labmates do on
a daily basis inspires me to do better with my own work.
I would also like to thank other MIT faculty, most importantly my thesis
committee, for offering such constructive and supportive comments during our meetings.
I would also like to thank Alan Grossman for starting the Microbiology Ph.D. program
and keeping such a humble and approachable personality.
Finally, above all, I am most indebted to my parents. As they both earned
doctorates some 40 years ago, they instilled in me that the pursuit of knowledge is the
highest aspiration of mankind. They also know the struggles and pains of surviving
graduate school, and kept me afloat all of these years. Mom and Dad invested in my
education from an early age. They have supported everything I have ever sought to do,
not because they want me to be someone in particular, or want me to be successful, but
only out of love. The greatest joy of my life is to know that my achievements serve to
honor my parents, their parents, and everyone that came before and made all of this
possible.
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Table of Contents
Page
Summary
3
Acknowledgments
5
Table of Contents
7
Chapter One: Introduction
11
Chapter Two: Fitness Trade-offs Restrict the Evolution of Resistance
to Amphotericin B
Abstract
Introduction
Results
Discussion
Methods
References
Figures
36
37
39
41
54
58
68
73
Chapter Three: Non-toxic Antimicrobials that Evade Drug Resistance
Abstract
Introduction
Results
Discussion
Methods
References
Figures
98
99
99
101
108
109
119
124
Chapter Four: A Fungal-Selective Cytochrome bcl Inhibitor
Impairs Virulence and Prevents the Evolution of Drug Resistance
Abstract
Introduction
Results
Discussion
Methods
References
Figures
129
Chapter Five: Conclusions and Future Directions
179
Appendix A: Selective Targeting of the Mitochondrial Phosphate Carrier
Yields an Antifungal Therapeutic Strategy that Evades Resistance
191
Appendix B: Curriculum Vitae
205
7
130
131
134
146
149
168
171
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List of Figures and Tables
Page
Figures
Chapter Two
Figure 2.1: Mechanisms of AmB resistance in Candida
73-74
Figure 2.2: AmB-resistant strains critically depend on high levels
75
of Hsp90 function for survival
Figure 2.3: Constitutive stress response activation in AmB-resistant
76-77
strains
Figure 2.4: AmB-resistant strains are hypersensitive to the stresses
78-79
of the host environment
Figure 2.5: AmB-resistant strains are defective in filamentation
80-81
and adhesion
Figure 2.6: AmB-resistant strains are avirulent in a mammalian model
82-83
of disseminated candidiasis
Figure S2.1 Transposon insertion and heterozygosity analysis in
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AmB-resistant C. albicans
Figure S2.2 : AmB-resistance mutations and heterozygosity analysis
in AmB-resistant C. tropicalis
85-86
Figure S2.3 : Heterozygosity analysis of in-vitro-evolved series
87-88
Figure S2.4 : Minimal inhibitory concentrations of Hsp90 inhibitors
89-90
and cytotoxic compounds
Figure S2.5 : Validation of additional mutant strains
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91-92
Table S2. 1: Strains used in this study
92-93
Table S2.2: Oligonucleotide sequences used in this study
94-97
Chapter Three
Figure 3.1: Synthesis of AmB urea derivatives
124
Figure 3.2: Sterol binding, antifungal, and human cell toxicity
125
of AmB ureas
Figure 3.3: Efficacy and toxicity of AmB ureas in mice
126
Figure 3.4: Characterization of mechanisms and costs of resistance
to AmB ureas
127-128
Chapter Four
Figure 4.1: Discovery of Inz-266 by high-throughput screening
171
Figure 4.2: Inz-266 is a fungal-selective cytochrome B inhibitor
172
Figure 4.3: Structural analysis of Inz-266 binding to cytochrome bc 1
173
Figure 4.4: Optimization of Inz-266 through medicinal chemistry
174
Figure 4.5: JBL- 182 renders fluconazole cidal and reduces the
175
emergence of resistance
Figure 4.6: Inhibition of cytochrome bc 1 by JBL- 182 prevents
176
adaptation to suboptimal carbon sources
Figure 4.7: Loss of cytochrome bc 1 severely reduces fungal virulence
177
and fluconazole tolerance, but increases brain colonization
Figure 4.8: Broad-Spectrum activity of JBL- 182
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178
Chapter One:
Introduction
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FUNGAL PATHOGENS OF HUMANS
Epidemiologv and Pathogenesis of Fungal Infection
Fungi are ubiquitous residents of the human body. In healthy humans, the skin,
oral cavity, gastrointestinal tract and genital mucosa harbor a great diversity of fungal
communities [1]. Fungal outgrowth in these non-sterile sites results in some of the most
frequent infections of otherwise healthy humans. Worldwide, nearly 2 billion people are
afflicted annually with superficial infections of the scalp, nails, and skin (Brown et al,
2012). Over half of women experience vaginal yeast infections at some point in their
reproductive years [2]. Oral yeast infections (thrush) are a common occurrence in both
healthy babies and denture-wearers. Although infections of these bodily surfaces are
unpleasant, they are generally quite minor medical concerns and typically resolve with
brief and facile treatment.
Invasive fungal infections of sterile body sites, on the other hand, are among the
most morbid and severe maladies of mankind. Current estimates suggest that over 1.5
million people worldwide die of invasive fungal infections annually, placing fungi among
the most deadly pathogens on earth [3]. Such invasive fungal infections are most
common in severely immunocompromised individuals, including those with uncontrolled
HIV, patients taking immunosuppressants for solid organ transplants, hematopoietic stem
cell transplant recipients, or chemotherapy patients. However, fungi can also invade the
bloodstream of (relatively) healthy patients undergoing abdominal surgery or central
venous catheter implantation [4]. Depending on the fungal species responsible,
infections can target the lungs, brain, bloodstream, kidneys, or liver, and cause lethality
due to either major organ failure or sepsis. Mortality rates for invasive fungal infections
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range from 30%-90%, depending on the species, body site, and underlying condition of
the patient [5].
This high rate of mortality reflects several problems in the clinical management of
these diseases. Diagnosis of the precise infecting pathogen typically comes too late or
not at all, and clinicians are forced to rely on broad-spectrum empiric therapy [3]. More
importantly, available antifungal drugs suffer from a litany of limitations in their patterns
of systemic distribution, fungicidal efficacy, and patient toxicity [6]. Fungal pathogens
often hide in niches that are poorly penetrated by these agents, deploy adaptive stress
responses to tolerate drug-induced toxicity, and even acquire mutations that desensitize
them to the action of the drug [7-9].
Although a wide variety of fungal species can cause invasive infections, more
than 90% of deaths are caused by members of the Cryptococcus, Aspergillus, and
Candida genera [3]. Cryptococcus neoformans and Cryptococcusgattii are yeast-like
fungi that typically enter the body through inhalation and then invade the central nervous
system in susceptible hosts. These pathogens successfully evade host defenses by hiding
within a thick polysaccharide capsule that is invisible to cells of the immune system.
Aspergillus species are ubiquitous environmental molds inhaled by most humans daily,
but in the severely immunocompromised, they cause a chronic pulmonary disease that is
rarely treatable. Disseminated Aspergillus infections also kill thousands of transplant
recipients annually [10]. Candidaalbicans, however, is the fungal species responsible
for the greatest number of invasive fungal infections, and is the main focus of my work.
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Biology and Virulence of Candida albicans
C. albicans is remarkable among pathogens for the great diversity of surfaces and
organs it readily infects. Unlike most other fungal pathogens, C. albicans has no wellestablished environmental niche, but mainly exists as a commensal of warm-blooded
animals [11]. C. albicans is present in large numbers in the gastrointestinal flora, but can
also be detected in the mouth and vagina of healthy individuals. In these sites, Candida
is typically in equilibrium with host immune factors that limit fungal growth [1].
However, changes in the structure and composition of these environments can lead to
rapid and robust fungal outgrowth. In HIV patients, the decline in CD4+ cell counts tips
the balance in favor of fungi and leads to frequent cases of thrush. Treatment with broadspectrum antibacterial drugs also allows Candidato surpass its competitors and dominate
certain niches, causing secondary infections [12].
Although C. albicans is considered a model fungal pathogen, its genetics and
reproductive cycle of are highly complex, and for many years this complexity limited the
progress of research. C. albicansgenerally grows as an obligate diploid, but haploid
strains of C. albicanshave been described recently [13]. However, all known haploid
strains are slow-growing and avirulent. Meiosis has never been observed in C. albicans.
Instead, a cryptic mating process proceeds through the joining of two different diploid
cell types, white and opaque, and the formation of a tetraploid intermediate [14]. This
tetraploid then loses chromosomes by a poorly understood process, regenerating the
diploid. Although the lack of a haploid stage in its life cycle has hindered the
development of genetic tools for this organism, the creation of auxotrophic markers has
enabled the generation of deletion mutants and heterologous expression sites [15].
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Nevertheless, diploidy prevents facile loss-of-function genetic screens, and partially as a
result, the vast majority of gene products are poorly understood. Further difficulties
include a modified genetic code and the absence of stably replicating plasmids. Despite
these difficulties, the growing importance of C. albicans as a pathogen has fueled the
pace of research.
ANTIFUNGAL DRUGS: MECHANISMS OF ACTION AND RESISTANCE
Azoles
Azoles are by far the most widely used antifungal agents due to their safety and
oral bioavailability, which enables outpatient and prophylactic use. They are generally
split into two groups. The first of these is the imidazoles (including clotrimazole,
ketoconazole, and miconazole), which are only useful for the treatment of skin, hair, and
vaginal infections. The second group comprises the first-generation triazoles, including
fluconazole, which is extremely broadly used in the treatment of mucosal candidiasis.
Newer triazoles (in particular voriconazole) have a broader spectrum of activity, and have
transformed the treatment of life-threatening aspergillosis [16].
Azoles act through inhibition of Erg IIp (lanosterol 14-alpha-demethylase), also
known as Cyp5 1 p [6]. This heme-containing protein catalyzes the removal of a methyl
group from lanosterol in the sterol biosynthetic pathway. Blocking Erg IIp function
causes the accumulation of 14-alpha-methylated sterols, which perturb diverse membrane
functions and prevent cell growth. While newer azoles are fungicidal against Aspergillus,
they are generally only fungistatic drugs against most pathogenic fungi, especially
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Candida. The inability of azoles to kill Candidaallows these infections to persist and
evolve resistance to treatment.
Resistance to Azoles
The broad use of azole antifungals to treat recurrent thrush at the height of the
AIDS epidemic (before the broad introduction of improved antiretroviral therapy) led to
frequent reports of resistance [17]. As Candidais a commensal, rarely spreads between
patients, and appears to lack horizontal gene transfer, resistance typically evolves de novo
[18]. However, the broad use of azoles against agricultural pathogens appears to select
for mutants in Aspergillusfumigatuswith reduced susceptibility to these drugs, causing
grave clinical concern and a dire need to understand mechanisms of resistance [19].
Azole resistance emerges through multiple mechanisms. Alterations in the azole
binding site of Ergi lp, including the R467K and G464S mutations, are frequently
detected in resistant isolates [18]. These mutations (and many others) alter the interaction
between azoles and the heme cofactor of the target enzyme, decreasing its affinity for the
drug [20]. Mutations in the transcription factor Upc2, which controls sterol biosynthesis,
can also contribute to azole resistance [21]. Increased copy number of ERG] 1, which can
occur through formation of a specific isochromosome, is another mechanism frequently
detected in clinical isolates [22]. The most intractable form of azole resistance occurs
through upregulation of multidrug efflux pumps, specifically members of the ATPbinding-cassette (ABC) family and the major facilitator superfamily. Upregulation of
ABC efflux pumps, including Cdrlp and Cdr2p in C. albicans, can occur through gain of
function mutations in the Taclp transcription factor that cause constitutive activation
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[23]. All of these mechanisms appear to have generally neutral effects on the fitness and
virulence of C. albicans, explaining their frequent emergence [24]. Although it has been
studied much less than Candida,Aspergillus evolves resistance to azoles through many
of these same mechanisms.
Resistance to azoles can also occur through loss of function mutations in the
ERG3 gene, encoding sterol delta 5-6 desaturase. These mutations result in the
detoxification of the methylated sterol produced when Ergi ip is inhibited [25].
However, these mutations are rarely observed in the clinic, likely because they reduce the
virulence of C. albicansin most, but not all, strain backgrounds [26,27].
Polyenes
Amphotericin B (AmB) is the only polyene used for invasive fungal infections. It
has the broadest spectrum and the most rapid fungicidal activity of any antifungal agent.
Amphotericin has an unusual mode of action for an antimicrobial: direct small moleculelipid interaction, targeting the fungal sterol ergosterol [28]. Whereas the antibacterials
daptomycin and colistin also directly bind to lipids and can insert themselves in the
membrane, recent work suggests AmB acts differently [29]. It had long been assumed
that AmB kills fungi by forming ion channels within the plasma membrane. However,
chemical synthesis revealed that ion channel formation and cell killing are dissociable
[30]. Further detailed mechanistic studies revealed that AmB does not insert into
membranes, but instead remains on their surface, where it forms large aggregates. These
aggregates have a high affinity for ergosterol and rapidly extract the sterol from fungal
membranes, permeabilizing and killing cells [29]. Key features of this "sterol sponge"
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model, including proof of existence of AmB aggregates in vivo and elucidation of their
structure, remain to be demonstrated. Nevertheless, it presents an attractive explanation
for many of the unexplained phenomena observed with this drug.
An inescapable aspect of the clinical experience with AmB, however, is its
severe, dose-limiting, and occasionally fatal toxicity to patients [31]. AmB is only
partially selective for ergosterol over its human analog, cholesterol [32]. AmB binding to
cholesterol in cell membranes can cause irreversible renal damage, which presents a
challenge as many patients with fungal infections already have limited kidney function.
Newer formulations of AmB in combination with lipids have mitigated, but not
eliminated this issue. In addition, due to its narrow therapeutic index, blood levels of
AmB must be kept very low, which runs counter to the high and repeated dosing
strategies used with most antimicrobials [33].
Resistance to Polyenes
AmB is unique amongst antifungals (and antimicrobials in general) in that resistance to
this agent is exceptionally rare. Repeatedly, large-scale resistance surveillance studies
have identified very few isolates with strong resistance (<1% of all isolates) [34]. The
rarity of resistance is remarkable when it is considered that AmB has been administered
to millions of patients over a span of 50 years, and almost always as monotherapy.
Previous studies of AmB resistance have revealed that disruption of ergosterol
biosynthesis genes (including ERG6 and ERG3/ERG 1I simultaneously) can confer
resistance in vitro [35]. These isolates produced altered membrane sterols, which
presumably have lower affinity for AmB. Several studies have also revealed rare
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Candida mutants recovered in the clinic with amphotericin resistance and altered sterol
profiles. However, the relevance of these mutations to the outcome of therapy in the
patient remains unclear [36,37].
Echinocandins
Echinocandins are natural product derivatives that target the Fks Ip and Fks2p protein
complex that synthesizes beta-1,3 glucan, a major component of the fungal cell wall [38].
These agents lack oral bioavailability and central nervous system penetration, and have a
generally narrower spectrum of activity than other agents. Plasma membrane protein
complexes that synthesize large polysaccharides are notoriously difficult to study.
Hence, little is known about the direct action of echinocandins on their target. However,
their potent fungicidal activity against Candidahas rendered them the drug of choice for
severe invasive candidiasis. One aspect of echinocandin activity that likely improves their
efficacy is their synergistic activity with the host immune system. Inhibition of beta- 1,3
glucan synthesis exposes polysaccharides that are normally buried, rendering the fungi
more immunogenic. This leads to hypersecretion of cytokines when these cells are
encountered by macrophages [39]. This effect likely enhances both innate and adaptive
responses to pathogenic fungi, promoting clearance of the infection.
Resistance to Echinocandins
Although they have only been in clinical use for 10 years, resistance to echinocandins is
becoming a major clinical issue in certain species (particularly Candidaglabrata). The
most commonly observed mechanism of resistance derives from mutations in two "hot-
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spot" regions of the FKS 1 gene encoding the glucan synthase target [40], which decrease
the sensitivity of glucan synthase activity to the drugs. Several of these mutations
decrease the virulence of C. albicanswhen homozygous, which may prevent their
dissemination [41]. Recent efforts have also demonstrated that increased chitin synthesis,
mediated through induction of cell wall stress response pathways, protects fungi from
echinocandins [42,43].
NEW IDEAS TOWARDS THE DISCOVERY OF ANTIFUNGAL AGENTS
Introduction to the problem
Clinical therapy of invasive fungal infections currently relies on agents originally
discovered decades ago. The newest class, the echinocandins, were discovered in the
1970's and spent nearly 30 years in development prior to approval [33]. Azoles were
discovered even earlier, and have been in use since the 1980's. AmB entered the clinic in
1963, before many of the transplant procedures and diseases that predispose to invasive
fungal infection even existed. None of these agents are ideal in terms of patient toxicity,
spectrum of activity, prevention of resistance, ease of dosing, and rapid fungicidal
efficacy against the most obdurate pathogens. But despite the high clinical demand for
improved agents, no new classes of antifungal have even reached phase III trials in the
last 12 years. Economics and regulatory challenges certainly deserve some of the blame
for the paucity of new antifungals. However, even promising preclinical agents are rarely
descried, suggesting that scientific difficulties are the main limitation. New ideas and
methodologies are certainly in order.
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In this section I will briefly outline the difficulties encountered in the pursuit of
novel antifungals (and antimicrobials in general), and the limitations of commonly
employed strategies. I will then shift to a discussion of new approaches in antifungal
discovery including 1) ideas to reinvigorate natural product discovery, 2) targeting
virulence factors, 3) targeting microbial stress responses, and 4) disabling fungal
adaptation to nutrient deprivation by the host. I will then revisit many of these issues in
the conclusion of this thesis to discuss them in light of my findings.
Challenges with traditional approaches to the discovery of antimicrobials
Throughout the golden age of antibiotic and antifungal discovery in the
2 0
th
century, arguably every class of agent was discovered by phenotypic screening for, or
serendipitous observation of, growth-inhibitory effects on whole cells. However, in the
1990's and 2000's, the availability of whole-genome sequences and essential gene lists
ushered in a paradigm-shift in antimicrobial discovery strategies [44]. The new approach
sought to identify gene products that were essential for viability, present in a broad
spectrum of fungi, absent in humans, and amenable to high-throughput screening for
small molecule inhibitors. However, this approach has failed to generate even one novel
class of FDA-approved antibacterial or antifungal [6].
Though few accounts of antifungal drug discovery efforts in this arena are
documented in the literature, substantial detail is available on the failure of targeted
approaches to discovering antibacterials [45]. Strangely, the rate of hit identification in
screens against purified bacterial proteins has been much lower than for purified
mammalian proteins. This unexpected finding may reflect intrinsic differences in
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bacterial protein structure. More interestingly, it might be the result of evolutionary
pressures these proteins have been subjected to in the wild, where they must evade
inhibition by secondary metabolites secreted by competitor organisms. Nevertheless, a
number of effective inhibitors of essential microbial enzymes with excellent potency in
vitro have been discovered, yet never became drugs. A consensus explanation for these
failures is the difficulty posed in engineering the microbial cell permeability upon these
compounds. Known antimicrobial classes have highly discordant physical properties
compared to the scaffolds present in synthetic libraries used in high-throughput screening
[33,46]. The capacity to pass through microbial cell walls and membranes while
avoiding drug efflux pumps is rare in purely synthetic molecules. Indeed, the only purely
synthetic classes of antifungals or antibacterials in broad use are azoles, oxazolidinones,
sulfonamides, and quinolones (with the exception of anti-tuberculosis agents).
Alas, in advancing novel classes of antifungals and antibacterials to approval,
whole-cell phenotypic screens have failed as well [45,47]. Novel synthetic molecules
with useful single-agent antimicrobial activity appear to be lacking in pharmaceutical
screening collections, perhaps due to their limited structural diversity and the physical
properties of these compounds. Certainly, natural products are a much better source of
chemical diversity, and the production of secondary metabolites that kill nearby
competitors is a common survival strategy in the microbial world. Moreover, these
compounds are naturally capable of passing through microbial membranes, and their
biosynthesis may have evolved to prevent resistance mechanisms.
Hence, it is extremely disappointing that natural product-based screening efforts
have also come up short [47]. The complexity of natural product extracts frequently
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complicates the initial screening process. Many hits contain substructures that cause
unfavorable pharmacological properties in man, and their chemical complexity renders
them intractable to synthetic optimization. Most alarmingly, the vast majority of natural
product screening efforts now result in the rediscovery of known classes of
antimicrobials, or new structures active on old targets.
Reinvigorating Natural Product Discovery
Despite the difficulties delineated above, great hope for the future of antimicrobial
natural product discovery lies, ironically, in genomics. Recent advances have enabled
prediction of the structures of secondary metabolites produced by an organism by
sequencing the DNA that encodes its polyketide synthases and other biosynthetic clusters
[48]. Using these methods, the biosynthetic potential of DNA extracted from any
environmental source can be computationally determined, enabling researchers to harness
new sources of chemical diversity from unculturable organisms. The DNA can then be
transformed into tractable host strains for the production and isolation of potentially
useful biosynthetic products, then tested for antimicrobial effects [49]. Though still
unproven in its clinical utility, the potential of this method to expand the universe of
useful antimicrobial chemical matter is immense.
In addition, advances in diagnostics may enable more natural product antifungals
to reach the clinic. The inability to rapidly diagnose the species infecting a patient has
restricted antifungal discovery efforts to the development of broad-spectrum agents. Due
to this requirement, potent natural product antifungals with narrow-spectrum activity
have been discovered and promptly abandoned [47]. These agents could be resurrected if
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rapid, sensitive, and affordable diagnostic procedures to identify the infecting organism
reach the clinic and gain wide use. New diagnostic technologies utilizing sequencing and
RNA probe-based methodologies may enable the realization of this goal [50].
Targeting Virulence Factors
Historically, non-essential targets and compounds that lack significant singleagent growth-inhibitory activity in vitro have been ignored in the development of
antimicrobial agents (with the exception of beta-lactamase inhibitors). However, many
more factors are required for microbial pathogenesis in vivo than are required for mere
growth in vitro, opening up many more targets and potentially much more chemical space
for drug discovery. There are generally two broad types of non-essential targets that
could be exploited: "active" mechanisms that enable damage of host tissue (including
adhesion, invasion, and toxin secretion) and "passive" mechanisms that simply enable
microbial survival within host environments [51]. Inhibitors of active virulence
processes promise a tremendous advantage: they might not select for resistance, since
many virulence strategies are not required for the growth of the individual cells
employing them.
However, targeting active virulence factors in pathogenic fungi may prove
insufficient. Many of the most dangerous fungal pathogens are adapted to life outside of
the host environment. These pathogens cause disease simply by growing and consuming
host tissues; they have not evolved elaborate virulence mechanisms. Moreover, since
invasive fungal infections typically occur in patients with very limited immune function,
antifungal agents need to actively kill their target pathogen to be curative. Nevertheless,
24
a virulence-factor-targeted agent, such as an inhibitor of filamentation in Candida
albicans, could be useful in combination with a more traditional growth-inhibiting
antifungal [52]. Extracellular adhesins such as Als3 that mediate tissue invasion could
also present excellent targets for tractable monoclonal antibody therapies, which could
prevent C. albicans from reaching protected niches such as the brain [53]. Unfortunately,
many of these active virulence processes are not broadly conserved across fungal species.
Given the severe limitations in precise diagnosis of infecting organisms before the
initiation of therapy, identifying broad-spectrum anti-virulence strategies is of the highest
importance.
Targeting Stress Responses
More promising are strategies targeting highly conserved stress response proteins
that enable adaptation to challenges encountered in the host environment. The large scale
identification of genes essential in vivo has recently been enabled by barcode-tagging
methods [54]. More targeted approaches, examining individual stress response pathways,
have also contributed greatly to our understanding of stress adaptation within the host.
One such pathway is the HOG (High Osmolarity Glycerol) MAP kinase pathway [55].
This pathway transduces changes in osmolarity and oxidative stress into a broadly
adaptive transcriptional program, through increased synthesis of osmolytes and other
protective factors [56]. Loss of Hogi renders C. albicanscompletely avirulent in mice,
even though it has no effect on growth under normal conditions in vitro.
Of greatest promise as targets, however, are stress-response proteins that are not
only required for survival in vivo but also for tolerance of (and resistance to) antifungal
25
treatment. Two such proteins that have emerged in the literature are calcineurin and
protein kinase C (PKC) [57-59]. Calcineurin is a Ca2+/Calmodulin activated phosphatase
that responds to membrane perturbation and other stresses by dephosphorylating substrate
proteins, including the transcription factor Crz . In C. albicans, deletion of the catalytic
subunit of calcineurin renders the fungus avirulent, and even prevents survival in serum
[60]. Deletion of calcineurin (or inhibition with FK-506 or cyclosporine) also renders
typically fungistatic ergosterol biosynthesis inhibitors fungicidal [61]. Many of these
same phenotypes are also seen in cells lacking PKC1, a critical regulator of the cell wall
integrity pathway [57].
Critical to the function of PKC, calcineurin, and other signal transduction
pathways is Hsp90 [62]. Hsp90 is a broadly conserved and highly abundant molecular
chaperone that aids in the folding and stabilization of a long list of metastable client
proteins [63]. This list is highly enriched for signal transducers and proteins that undergo
a conformation change in response to stimulation by a ligand or activating partner. In
yeast, Hsp90 is produced in great excess of the amount needed for survival under normal
conditions, and may act as a protein homeostasis buffer that allows for rapid adaptation to
stress [64,65]. The excess buffer of Hsp90 also enables novel mutations to have
immediate phenotypic consequences, including oncogenic mutations in kinases [66].
By responding to the increased demand for signaling from stress response
proteins, Hsp90 enables resistance pathways to multiple antifungals. Inhibition of Hsp90
blocks the emergence of azole resistance in C. albicans, and restores sensitivity to azoles
in mutants that had previously evolved resistance in the clinic [67]. Hsp90 also promotes
resistance to echinocandins in fungi separated by a billion years of evolution [68]. High
26
levels of Hsp90 are also required for fungal virulence in mice (in the absence of
antifungal treatment) in both C. albicans and A. fumigatus [69,70].
Attempts to pharmacologically target these pathways, however, have not yet
proven fruitful. PKC, Calcineurin, and Hsp90 have central roles in cell-mediated
immunity in humans, and their inhibition during a severe infection would likely be
counterproductive. As such, fungal-selective inhibitors are needed, and these have not
yet advanced to the clinic.
Targeting Metabolic Adaptation
Unexpectedly, the work in this thesis uncovered a critical and pharmacologicallytargetable role for central carbon metabolism and mitochondrial respiration in the
pathogenesis and drug resistance of C. albicans. Recent work from many groups has
established that carbon deprivation is an underappreciated form of stress that fungal
pathogens encounter in the course of infection [71,72]. C. albicans is capable of
colonizing such nutritionally disparate body sites as the mouth, vagina, bloodstream,
kidney, brain, skin, nails, and eyes. Moreover, even within a single niche, Candidacan
encounter rapidly fluctuating environmental conditions, including infiltration by immune
cells. As the ability to convert environmental nutrients into biomass and ATP (and thus
new cells) is the fundamental selective pressure on all organisms, a litany of mechanisms
exist to enable fungi to optimally exploit their surroundings. Such mechanisms may
present promising new opportunities for selective therapeutic intervention.
Fermentable sugars, such as glucose, are the preferred general carbon source of
yeasts. However, sugars are rarely abundant in host microenvironments (typically 0.1-
27
0.2%), and in some niches they are completely absent [73]. Though the dominant carbon
source in each niche is poorly defined, they include lactate, acetate, amino acids, and
fatty acids [74]. In the absence of abundant sugars, Candida relies heavily on
gluconeogenesis from these suboptimal carbon sources for many vital processes. One
such process is the biosynthesis of the carbohydrate-rich cell wall. In fact, carbon source
utilization dictates the very structure of the fungal cell wall and thus has critical
consequences for stress tolerance and immunogenicity [75].
Candidaencounters the most severe nutrient deprivation upon engulfment into
phagocytic immune cells (macrophages or neutrophils), where sugars are absent [76]. To
tolerate this environment and escape, C. albicans induces the glyoxylate cycle, enabling
metabolism of non-fermentable 2-carbon compounds including acetate [77]. Indeed, the
role of this pathway is underscored by its deployment by both bacterial and fungal
pathogens, in which it is required for full virulence. A critical requirement for the
successful utilization of all non-fermentable carbon sources is ATP production from the
mitochondrial electron transport chain. In high-glucose conditions, the inhibition of
mitochondrial respiration only partially impairs proliferation in C. albicans [78].
However, when sugars are scarce, mitochondrial function is required to fuel the TCA
cycle, enable gluconeogenesis, and produce ATP through oxidative phosphorylation.
Given the high conservation of most mitochondrial proteins from fungi to
humans, they are rarely mentioned as putative antifungal targets. Previous efforts have
focused on identifying mitochondrial proteins unique to fungi, including the Goal protein
unique to the Candidaclade, or the glyoxylate cycle, which exists across diverse
microbial pathogens but is absent in mammals [77,79-81].
28
However, efforts to unlock
therapeutic value through pharmacological targeting of these metabolic processes have
been limited. Many pathogen-specific mitochondrial proteins are not conserved across a
broad enough spectrum of fungi, or are not druggable by small molecules. Moreover,
given the impressive metabolic flexibility of many pathogenic fungi, inhibiting any one
pathway of metabolic adaptation may be rapidly bypassed by upregulation of others.
Indeed, mutants lacking the glyoxylate are attenuated, but not avirulent [82]. Proteins
with central, conserved, and irreplaceable roles in multiple metabolic pathways, such as
electron transport chain complexes, may present more attractive targets. Unfortunately,
these factors are generally highly conserved from yeast to man. Nevertheless, ample
evidence exists from the history of available antifungals and antibiotics that targeting
processes still present in humans can yield therapeutics with tremendous clinical value.
For example, the target of azoles is well conserved in humans, many classes of antibiotics
target the ribosome, and the new anti-tuberculosis agent bedaquiline targets ATP
synthase. Identifying the atomic-level differences in such central and essential proteins
between their pathogen and human homologs would provide a critical path forward for
the development of new agents.
SUMMARY
Decades of study have provided valuable insights into the mechanisms by which
pathogenic fungi cause disease, the mechanisms by which antimicrobial drugs kill fungi,
and the mechanisms that allow fungi to resist these agents. Yet, our understanding of
why resistance emerges rapidly to some drugs and not others remains limited. Moreover,
feasible strategies for the discovery of new agents active against drug resistant pathogens
29
are lacking. In this thesis, I explore the fitness costs of drug resistance mutations as both
an evolutionary explanation for the clinical rates of resistance and also a powerful tool to
inform the discovery of new agents. I also used unbiased screening to discover new
small molecules that disarm the adaptive cellular responses that enable resistance. With
this work, I seek to provide both a fundamental understanding of the tradeoffs inherent in
the evolution of drug resistance as well as specific targets and strategies for development
of new agents. Examining drug resistance as a form of adaptation to genetic and
environmental change thus provides deep insight into the mechanisms that promote and
restrict the rapid evolution of new traits.
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35
Chapter Two:
Fitness Tradeoffs Restrict the Evolution of
Resistance to Amphotericin B
*This chapter has been published previously:
Vincent BM, Lancaster AK, Scherz-shouval R, Whitesell L, Lindquist S. Fitness tradeoffs restrict the evolution of resistance to amphotericin B. PLoS Biology 2013;11(10).
36
Fitness Trade-offs Restrict the Evolution of Resistance to Amphotericin B
Benjamin Matteson Vincent", Alex Kelvin Lancaster 2', Ruth Scherz-Shouval 2, Luke
Whitesell 2 , Susan Lindquist 2 ,3
1 Microbiology Graduate Program, Massachusetts Institute of
Technology, Cambridge, Massachusetts, United States of America,
2 Whitehead Institute for Biomedical Research, Cambridge,
Massachusetts, United States of America,
3 Howard Hughes Medical Institute, Department of Biology,
Massachusetts Institute of Technology, Cambridge, Massachusetts, United
States of America
E-mail: Lindquist admingwi.mit.edu
Current address: Department of Pathology, Beth Israel Deaconess
Medical Center and Center for Biomedical Informatics, Harvard Medical
School, Boston, Massachusetts, United States of America
*
Abbreviations: AmB, Amphotericin B; CFU, colony-forming-unit; ERG, ergosterol;
FCZ, fluconazole; Hsp90, heat shock protein 90; HUVEC, human umbilical vein
endothelial cell; TNF-a, tumor-necrosis-factor alpha, WT, wild-type
ABSTRACT
The evolution of drug resistance in microbial pathogens provides a paradigm for
investigating evolutionary dynamics with important consequences for human health.
Candida albicans, the leading fungal pathogen of humans, rapidly evolves resistance to
two major antifungal classes, the triazoles and echinocandins. In contrast, resistance to
the third major antifungal used in the clinic, amphotericin B (AmB), remains extremely
rare despite 50 years of use as monotherapy. We sought to understand this long-standing
evolutionary puzzle. We used whole genome sequencing of rare AmB-resistant clinical
isolates as well as laboratory-evolved strains to identify and investigate mutations that
confer AmB resistance in vitro. Resistance to AmB came at a great cost. Mutations that
conferred resistance simultaneously created diverse stresses that required high levels of
37
the molecular chaperone Hsp90 for survival, even in the absence of AmB.
This
requirement stemmed from severe internal stresses caused by the mutations, which
drastically diminished tolerance to external stresses from the host.
AmB-resistant
mutants were hypersensitive to oxidative stress, febrile temperatures, and killing by
neutrophils and also had defects in filamentation and tissue invasion. These strains were
avirulent in a mouse infection model. Thus, the costs of evolving resistance to AmB
limit the emergence of this phenotype in the clinic. Our work provides a vivid example of
the ways in which conflicting selective pressures shape evolutionary trajectories and
illustrates another mechanism by which the Hsp90 buffer potentiates the emergence of
new phenotypes. Developing antibiotics that deliberately create such evolutionary
constraints might offer a strategy for limiting the rapid emergence of drug resistance.
Author Summary
The evolution of drug resistance in human pathogens is considered an inevitable
consequence of the selective pressures imposed by antimicrobial drugs. Yet resistance to
one antifungal drug, amphotericin B (AmB), remains extremely rare despite decades of
widespread use. Here we explore the biological mechanisms underlying this conundrum.
By examining natural and experimental populations of Candidaalbicans, we identify
multiple mutations that confer resistance to AmB in vitro. As with the evolution of
resistance to other antifungals, we find that the chaperone protein Hsp90 is involved in
enabling the evolution of resistance to AmB. We also discover, however, that mutations
that confer AmB resistance impose massive costs on other aspects of fungal
pathogenicity; strains that are resistant to AmB are hypersensitive to attack by the host
immune system and are unable to invade and damage host tissue. Thus, the evolution of
resistance to AmB is restricted by a tradeoff between tolerance of the drug and the ability
to cause disease. We propose that developing new antibiotics for which resistance
presents such dire tradeoffs may be a promising strategy to prevent the evolution of
resistance.
38
Introduction
Understanding how organisms rapidly evolve novel traits is a central problem in both
evolutionary biology and the treatment of infectious diseases. The emergence of drugresistant pathogens not only provides a model for studying the evolution of new phenotypes
but also poses a grave challenge to human health. Antibiotic treatment selects for rare
mutations that alter cellular processes and, thereby, either mitigate the toxicity of the drug
or bypass it altogether. Sometimes resistance mechanisms are completely orthogonal to
the normal biology of the cell, as is the case for the amplification of efflux pumps or the
horizontal acquisition of drug-detoxifying enzymes in bacteria. But often the mutations
that confer resistance alter basic cellular processes in such a way as to create a variety of
new stresses. The latter is especially relevant in eukaryotic pathogens, where the rarity of
genetic exchange within and between populations necessitates the de novo evolution of
resistance [1,2].
Candida albicans is the leading fungal pathogen of humans and the fourth most
common cause of all hospital-acquired infections [3,4]. Normally a harmless commensal,
changes in host immune status allow C. albicans to become pathogenic. Infections range
from superficial thrush to life-threatening systemic disease. Mild to moderate infections
are currently treated with triazoles, which inhibit Ergi1 (lanosterol 14a-demethylase),
preventing ergosterol biosynthesis [5].
Life-threatening systemic infections require
treatment with echinocandin or polyene agents. Echinocandins inhibit the synthesis of
the cell wall polymer (1,3)-p-D-glucan, resulting in loss of cell integrity [5].
The third most commonly employed antifungal is the polyene drug amphotericin
B (AmB), which was the standard of care for -40 years [6]. Its potent fungicidal activity
39
derives from its ability to selectively bind the major sterol of fungal membranes,
ergosterol [7,8]. Among other effects, this binding induces pore formation in the plasma
membrane and results in rapid cell death. While AmB is extremely effective at killing
fungi, its clinical utility is impaired by several liabilities. First, pharmacokinetics and
distribution are poor, allowing some fungi to hide in niches where drug exposure is
limited [9]. Second, AmB induces idiosyncratic systemic reactions involving fever and
tremors. Third, and still more problematic, AmB's cumulative, dose-dependent renal
toxicity limits use in many patients.
Despite these limitations, a remarkable advantage of AmB is that it has been
exceptionally refractory to the evolution of resistance.
After 50 years of use as
monotherapy, the acquisition of AmB resistance in C. albicans remains extremely rare.
For comparison, the antifungal drug 5-flucytosine was introduced several years later than
AmB, but resistance rendered this drug obsolete against Candida in less than 20 years
[10]. In a recent study of 9,252 clinical isolates of C. albicans, 99.8% remained AmBsensitive [11]. Although the less toxic triazoles and echinocandins have recently replaced
AmB as the frontline therapy, AmB still retains frequent use in many settings,
particularly when an infection resists treatment with other drugs [12]. Indeed, resistance
to triazoles emerges frequently and, although echinocandins are relatively new to the
clinic, resistance to echinocandins is also already arising [13-
18].
The best-validated mechanism of resistance to AmB observed in clinical isolates
of C. albicans to date involves a double loss of function in both ERG3 and ERG]] (C-5
sterol
desaturase
and
lanosterol
14a-demethylase,
respectively),
identified
by
biochemical analysis of membrane sterol composition [19- 21]. In other fungal
40
pathogens, sterol analysis of rare AmB-resistant isolates has identified resistant strains
lacking ERG2, encoding C-8 sterol isomerase, and ERG6, encoding C-24 sterol
methyltransferase [22 -24]. However, there has been no systematic analysis of AmB
resistance mutations in Candida using matched isogenic strains. More importantly, the
consequences of these mutations upon the biology and pathogenicity of Candida remain
largely unexplored.
Here we thoroughly explore mutations that can confer AmB resistance in C.
albicans with the goal of understanding why resistance emerges so rarely in the clinic.
Our results establish that the evolutionary constraints imposed by AmB are distinct from
those of other antifungals. They provide insights into the mechanisms by which external
and internal biological stresses restrict evolutionary trajectories.
In addition, our work broadens the role of protein homeostasis regulators as
potentiators for the emergence of new traits.
Finally, our findings suggest a general
strategy for antimicrobial drug development that might be broadly useful in limiting the
emergence of resistance.
RESULTS
Whole Genome Sequencing of AmB-Resistant Clinical Isolates implicates ERG2 and
ERG3/11
As a first step towards understanding the evolution of resistance to AmB in
Candida, we sought to broaden and validate the list of mutations that allow the fungus to
tolerate this drug.
As AmB-sensitive parental strains from which rare AmB-resistant
isolates evolved are not available, the identification of mutations conferring resistance
has proven challenging. Nevertheless, we sequenced the entire genome of two
41
independent clinical isolates that had evolved resistance to AmB, one from C. albicans
and one from the closely related species C. tropicalis. For comparison, we also
resequenced the AmB-sensitive C. albicansreference strain SC5314.
Using paired-end reads, we achieved over 50-fold coverage of these genomes,
which allowed us to detect simple polymorphisms as well as complex genome
rearrangements. As expected, the strains differed from each other and from the reference
strain at more than 20,000 sites. To identify the variants responsible for resistance, we
took advantage of previous work and inspected candidate genes acting in the ergosterol
biosynthesis pathway.
In the C. albicans AmB-resistant isolate, we detected a high density of mispaired
reads at the ERG2 (ORF19.6026) locus (Figure 1A). Further analysis revealed that both
copies of the ERG2 gene in this strain carried an insertion of the TCA2 retrotransposon
(Figure Si A). Whole-genome analysis of polymorphisms indicated that the strain carried
a high level of heterozygosity across its entire genome, with only two small regions of
homozygosity.
Strikingly, one of these included the transposon insertion in ERG2
(Figure SlB).
In the C. tropicalis isolate, the sequence of ERG2 was identical to that of the
AmB-sensitive reference strain, MYA-3404.
However, a mutation was observed in
ERG3 (CTRG_04480), another enzyme involved in sterol synthesis (Figure S2A).
Specifically, phenylalanine replaced serine 257, a residue that is absolutely conserved in
this protein from fungi to mammals (Figure S2B).
In addition, the ERG]]
(CTRG_05283) ORF of this isolate harbored a deletion of 170 nucleotides (Figure S2C).
Again, despite generally high levels of heterozygosity in other regions of the genome, the
42
regions surrounding both ERG3 and ERG]] had become homozygous (Figure S2D).
These results suggest that selective sweeps had operated to fix new mutations to a
homozygous state in both clinical isolates. But, of course, the sequencing of many more
AmB-sensitive and resistant isolates would be necessary to establish this conclusion.
Validation of erg2 and erg3/1J in Laboratory Strains
To validate that either loss of ERG2 or the combined loss of ERG3 and ERG]]
function is sufficient to confer resistance to AmB, we created them anew in a wild-type
background. Because C. albicans is an obligate diploid, we used auxotrophic markers to
sequentially delete the loci, creating homozygous mutations in ERG2 and double
homozygous mutations in ERG3 and ERG1.
To confirm the inactivation of these genes in both lab strains and clinical isolates,
we exploited the unique spectral characteristics of ergosterol. These result from
conjugation of the double bonds C5-C6 and C7-C8, formed and isomerized by Erg3 and
Erg2, respectively. We prepared sterol extracts from both of the clinical isolates and both
of the laboratory mutants along with the isogenic wild-type control. Extracts from the
wild-type laboratory strain exhibited the double-peaked spectrum between 240 and 300
nm characteristic of the conjugated bonds (Figure 1 C). Extracts from both the laboratory
mutants and the clinical isolates did not.
Next, we determined the minimal inhibitory concentration (MIC) of AmB in the
knockout strains and compared it to the wild-type laboratory strain and the clinical
isolates. AmB resistance levels in the two newly created laboratory strains matched those
of the corresponding clinical isolates. For the erg2 mutants this was a 10-fold increase in
MIC (Figure 1 D), and for the erg3 erg]] mutants it was a 20-fold increase. We were
43
unable to obtain further transformants in these mutants for technical reasons, thus
precluding any attempt at complementation of the phenotype. However, we verified our
results with an additional independent mutant in each background (Figure S5). Thus,
laboratory-generated mutants successfully reproduce the resistant phenotypes observed in
clinical isolates.
Laboratory Evolution of AmB Resistance Implicates ERG6 Mutations
To discover other mutations that could confer AmB resistance in Candida, we next
employed in vitro evolution. The drug-sensitive reference strain SC5314 was not suited
to this analysis because we repeatedly found that the resistance that emerged in selections
with this strain was highly unstable. Another strain, C. albicans ATCC- 10231, proved to
be less susceptible to this problem.
To isolate resistant variants, this strain was
inoculated in liquid media containing a low concentration of AmB and serially passaged
seven times into media with a 2-fold higher concentration of AmB at each step.
Surviving cells from each passage were isolated and saved. We then used whole genome
sequencing and alignment of the parental strain with strains from the third, fifth, and
seventh passages to identify the mutations emerging as strains developed resistance.
Alignment
of genome
sequences
and
algorithmic
detection
of novel
polymorphisms emerging within the series revealed a trajectory of mutations in the ERG6
(ORFJ9.1631) gene, encoding A(24)-sterol C-methyltransferase (Figure 1B). Notably,
the parental strain had been heterozygous for a premature stop codon that replaced the
codon for Glu70. A mutation in the other allele of ERG6, AspJ80Gly, appeared at the
second step in the series.
In the third step, the copy number of the Q70Stop allele
increased to a 3:1 ratio relative to the Aspl80Gly allele. Finally, a loss of heterozygosity
44
event, unique to the left arm chromosome 3 where ERG6 resides, resulted in
homozygosity of the nonsense allele (Figure S3A-B).
Several independent selections with this strain-with either a similar gradual
selection process or with selection regimes employing immediate shifts to high drug
concentrations-all involved additional mutations in ERG6 (unpublished data). Finally,
we validated the capacity of ERG6 mutations to create AmB resistance by using
auxotrophic markers to delete the gene in the SC5314 reference strain background. This
resulted in a 12-fold increase in the AmB MIC (Figure 1 D).
Systematic Analysis of AmB Resistance in Late-Stage Ergosterol Biosynthesis
Mutants
To systematically define genes whose inactivation might confer AmB resistance
in vitro, we used homologous site-directed recombination to generate isogenic diploid
deletion mutants for all seven nonessential genes acting in the latter half of the ergosterol
biosynthesis pathway (the steps after cyclization of squalene to lanosterol).
Only the
deletion of ERG2, ERG6, or of ERG3 and ERG]] together conferred more than a 3-fold
increase in the AmB MIC (Figure ID). While other mutations conferring resistance to
AmB may exist, these three are the most critical, as they have all been detected in the
clinic and validated in the laboratory.
Thus, we focused our efforts to explain the
exceptional rarity of clinical AmB resistance on understanding the broader biological
consequences of mutations in ERG2, ERG6, or ERG3 and ERG]].
AmB-Resistant Mutants Have an Unusual and Extreme Dependence on Hsp90
We previously reported that the emergence and maintenance of resistance to
triazole and echinocandin antifungals critically depends on the molecular chaperone
45
Hsp90 (ORF19.6515) [25].
Hsp90 is one of the most abundant proteins in eukaryotic
cells, constituting ~1% of total cellular protein, and acts as a protein homeostasis buffer.
Although Hsp90 is an essential protein, its activity can be reduced up to 10-fold without
impairing normal growth [25- 27]. Previous work has suggested that phylogenetically
diverse organisms use this excess reservoir of protein-folding capacity to promote the
rapid evolution of new traits through a litany of mechanisms [25,27,28].
These include
binding and stabilizing mutant proteins with novel activities, promoting the folding and
maturation of metastable signal transduction proteins that respond to harsh environmental
conditions, and allowing for the release of cryptic genetic variation upon stress.
In
pathogenic fungi, we have shown that mild compromise of Hsp90 function prevents the
de novo emergence of resistance to triazoles and, in fact, reverses the resistance of strains
of which had previously evolved triazole and echinocandin resistance in the clinic
[25,29,30].
Hsp90 promotes antifungal drug resistance by stabilizing calcineurin and
protein kinase C, two signal transducers that promote resistance by mitigating the stress
to the cell wall and membrane that is induced by these drugs [25,30,31].
We asked if Hsp90 also plays a role in the evolution of resistance to AmB, taking
advantage of two structurally unrelated natural products with high specificity for Hsp90
(geldanamycin and radicicol). We examined mutants that had evolved AmB resistance
under drug pressure in the clinic (Figure 2A-B), mutants created deliberately by targeting
genes in the sterol pathway (Figure 2C), and mutants arising at each step in our in vitro
selection (Figure 2D).
We spotted drug-resistant and control isolates on media
containing no drug, fluconazole, or AmB, in either the presence or in the absence of the
Hsp90 inhibitors.
46
As previously described, modest inhibition of Hsp90 completely blocked
fluconazole resistance (Figure 2A, middle panel).
It did not affect growth of the
fluconazole-resistant isolate in the absence of fluconazole (Figure 2A, top panel).
Modest inhibition of Hsp90 also abrogated AmB resistance (Figure 2A, bottom panel).
Surprisingly, however, low concentrations of either of the Hsp90 inhibitors completely
blocked the growth of all of the AmB-resistant strains, even in the absence of AmB
(Figures 2A-D and S4B).
Perturbations in ergosterol biosynthesis can lead to general increases in the
accumulation
of diverse small molecules.
This raised the possibility that the
hypersensitivity to Hsp90 inhibitors might simply be due to an increase in their
intracellular accumulation. To investigate, we determined the MICs of a panel of seven
chemically and mechanistically distinct cytotoxic agents (that do not act through Hsp90)
in all of the resistant strains. These MICs were compared to the MICs of geldanamycin
and radicicol, as well as two synthetic Hsp90 inhibitors from completely different
chemical scaffolds. AmB-resistant mutants were, indeed, generally more sensitive than
wild-type cells to many of the cytotoxic compounds. The decrease from the wild-type in
the MIC of any of these cytotoxic agents ranged from 2- to 8-fold in the erg2 or erg6
mutants and 4- to 16-fold in the erg3 erg]] mutant (Figure S4A-C).
But the decreases
in MIC of the four Hsp90 inhibitors were 18- to 48-fold for the erg2 mutants, 85- to 109fold for the erg6 mutants, and 222- to 480-fold for the erg3 erg]1 mutants (Figure S4BC). Thus, the hypersensitivity of AmB-resistant strains to Hsp90 inhibitors cannot simply
be attributed to a general increase in drug accumulation.
47
Rather, the growth of these
mutants must critically depend on maintaining very high levels of Hsp90 function even in
the absence of AmB.
Was the effect of the Hsp90 inhibitors restricted to growth inhibition, or did they
actually cause cell death? We previously showed that Hsp90 inhibition renders the
typically cytostatic drug fluconazole cytocidal, killing the fungus instead of simply
blocking its growth [29]. Indeed, low concentrations of Hsp90 inhibitors were cytocidal
to the AmB-resistant mutants (Figure 2E). But, once again, in contrast to the fluconazole
resistant strains, Hsp90 inhibition killed AmB-resistant cells even in the absence of AmB.
Thus, the mutations that confer AmB resistance cause a novel and critical dependence on
Hsp90 for the simple maintenance of normal viability.
AmB-Resistant Strains Exhibit Constitutive Activation of Stress Responses
Hsp90 promotes the maturation of a diverse array of metastable signal
transduction proteins, including kinases, phosphatases, and ubiquitin ligases (known as
Hsp90 clients) [32]. These function in many stress response pathways. Thus, the simplest
explanation for the extreme dependence of AmB-resistant strains on Hsp90 is that these
normally nonessential client proteins are required to tolerate the perturbations in cellular
homeostasis caused by mutations in ergosterol biosynthetic enzymes.
To investigate, we first tested our AmB-resistant strains for constitutive
transcriptional activation of a variety of stress response genes. These include targets of
the known HSP90 client calcineurin [30,33], as well as genes involved in the response to
iron starvation or oxidative stress, two stresses tightly linked to membrane sterol
homeostasis. To provide a point of comparison, we exposed wild-type strains to external
stresses known to induce these responses.
48
The AmB-resistant mutants indeed exhibited a constitutive activation of diverse
stress responses (Figure 3A, left panel). Pathways of iron starvation were constitutively
active, most strongly in the erg3/erg]] and erg6 mutants, as evidenced by the high
expression of RBT5 (ORF19.5636), FET34 (ORF19.4215), FTRJ (ORF19.7219), and
FTH1 (ORF19.4802), and SIT] (ORF19.2179). Genes responding to general plasma
membrane and oxidative stressors also showed generally broad elevation [including
CAT]
(ORF19.6229), GPX1 (ORF19.86), CRH]1 (ORF19.2706), and DDR48
(ORF19.4082)].
Induction of calcineurin targets [UTR2 (ORF19.1671), RTA2
(ORF19.24), ECM331 (ORF19.4255)] was observed at varying levels as well, most
strongly in the various lab strains [33,34]. The level of constitutive activation of these
pathways in AmB-resistant strains was in many cases comparable to levels seen in wildtype strains exposed to severe external stresses (Figure 3A, right panel). Intriguingly, the
AmB-sensitive, fluconazole-resistant erg3 mutant did not show dramatic upregulation of
any of the responses tested, but only a weak induction of several iron starvation genes.
These data suggest that the mutations that confer resistance to amphotericin
concomitantly exert an array of stresses to cellular membrane and redox homeostasis.
Nonessential Stress Responses Become Essential in AmB-Resistant Strains
Hsp90-dependent stress-response pathways are not essential for growth of wild-type
strains. In pathogenic fungi, validated Hsp90 clients include calcineurin, the MAP-Kinase
Hogl, and Protein Kinase C (PKC) [30,31,35]. To test whether they become essential in
the resistant mutants, we took advantage of the high conservation of these proteins:
highly selective drugs targeting their human homologs have been developed for diverse
therapeutic purposes, and these are active on the fungal proteins as well. These chemical
49
probes allowed us to selectively reduce the activities of these proteins in the genetically
intractable clinical isolates. In laboratory strains, these compounds allowed us to bypass
the difficulties inherent in maintaining mutations expected to exhibit synthetic lethalities.
To inhibit calcineurin, we used FK-506 and Cyclosporin A, two structurally and
mechanistically distinct inhibitors of the phosphatase.
To inhibit PKC, we used
enzastaurin, a synthetic PKC inhibitor with high selectivity for this kinase, and confirmed
our findings with cercosporamide, a natural product fungal-specific inhibitor of PKC
[31,36]. Treatment with either calcineurin inhibitor inhibited growth of all of the
Amphotericin-resistant strains, with complete growth inhibition of erg2 and erg6 mutants
(Figure 3B). The erg3 erg]] strains were slightly less sensitive to calcineurin inhibitors,
but showed a dramatically increased sensitivity to PKC inhibition.
We also asked if wild-type cells rely on Hsp90-dependent stress responses to
defend themselves from the toxic effects of AmB. To do so, we created genetic
knockouts of these normally nonessential genes in a wild-type background. Indeed, hog]
mutants
were
hypersensitive
to
AmB,
while
strains
lacking
calcineurin
[cnb1(orfl9.4009)] were not (Figure 3C). Mild inhibition of Hsp90, which is sufficient
to impair calcineurin activity, did not change the AmB MIC. However, more extensive
inhibition of Hsp90, which would destabilize Hogl [35], did sensitize cells to the
antifungal. Thus, HogI is required to tolerate the stress imposed by drug treatment, while
the calcineurin and PKC pathways are required to tolerate the stress imposed by
resistance mutations.
Next, we tested the role of Hsp90, Hogl, and calcineurin in the de novo
emergence of AmB resistance. To do so, we generated ERG2/erg2 heterozygotes in a
50
wild-type background and in strains lacking HogI or calcineurin. We then selected for
loss of the remaining allele of ERG2 by plating on media containing AmB. As expected,
ERG2/erg2 heterozygotes that were otherwise wild-type produced resistant colonies at a
rate of ~_1 05
(Figure 3D). Low concentrations of the Hsp90 inhibitor geldanamycin
completely eliminated the emergence of such colonies. Strains lacking calcineurin also
failed to produce resistant colonies, and hog] strains produced only a few small, slowgrowing colonies. We conclude that Hsp90-dependent stress responses are required to
enable the de novo emergence of AmB resistance.
AmB-Resistant Mutants Are Hypersensitive to Stresses Encountered in the Host
Although we have successfully validated the ability of several ergosterol
biosynthesis mutations to confer resistance to AmB in vitro, resistance rarely evolves
during the treatment of infected patients. We wondered if the phenotypic benefit of AmB
resistance might be undermined by fitness costs imposed by the mutations. That is, the
high levels of internal stress that burden the AmB-resistant mutants might make them
unable to withstand the additional external stresses imposed by the host. To investigate,
we tested the ability of the resistant mutants and wild-type control to tolerate a range of
stresses encountered in host environments, including (1) elevated temperatures (fevers
are a universal response to systemic fungal infection and a common side-effect of AmB
treatment); (2) hydrogen peroxide, hypochlorous acid, and nitric oxide (used by
neutrophils to kill Candida); and (3) serum, iron deprivation, and antimicrobial peptides,
as these are other common sources of stress in the host.
While AmB-resistant strains grew similarly to wild-type strains at 37 'C and 39
'C, they grew more poorly at 41 'C (Figure 4A). Resistant mutants were hypersensitive
51
to the presence of peroxide, hypochlorous acid, and the nitric oxide donor DPTANONOate (Figure 4A-C). Resistant mutants also proliferated moderately more slowly
than wild-type cells in the presence of an iron chelator (Figure 4D). Resistant strains
were also sensitive to growth in 100% bovine serum (Figure 4F) at elevated temperature,
but were not more sensitive to the neutrophil-associated
antimicrobial
peptide
Calprotecin/S 1 OOA (Figure 4E).
Next, we tested their susceptibility to attack by neutrophils, the most critical
component of the innate immune system in combating acute fungal infection.
We
isolated human neutrophils from whole blood to 99% purity and activated them by
treatment with recombinant TNF-a. Wild-type or AmB-resistant C. albicans strains were
co-cultured with neutrophils for 6 h, at which point neutrophils were lysed and fungal
growth was measured. The hog] mutant, previously reported to be hypersensitive to
neutrophil attack [37,38], was included as a positive control. AmB-resistant mutants were
indeed significantly hypersensitive to neutrophil attack, exhibiting at least as strong of a
defect as the hog] mutant (Figure 4G).
AmB-Resistant Mutants Are Defective in Filamentation and Tissue Invasion
Another potential fitness cost of AmB-resistance mutations could be a compromise of
pathogenic virulence mechanisms.
C. albicans responds to several stimuli in the host
environment by undergoing dramatic morphological changes, including the adoption of
filamentous hyphal forms. Filamentation enables penetration and invasion of host tissue,
and is a highly validated virulence factor for this pathogen [39,40]. We asked if our
AmB-resistant mutants can filament effectively when exposed to 10% fetal bovine serum
in RPMI culture media. After 4 h of incubation, wild-type strains exhibited long and
52
robust filaments (Figure 5A).
The fluconazole-resistant erg3 mutant exhibited a mild
lay in hyphal protrusion but still formed substantial filaments.
However, the erg2
mutant could only form short and amorphous filaments. The erg6 and erg3 erg]] strains
and clinical isolates were entirely unable to form hyphal extensions, and remained mainly
in the yeast form.
We then asked if this defect in filamentation reduced the capacity of the pathogen
for tissue invasion. Monolayers of primary human endothelial cells were established in
culture, and infected with the C. albicans strains. Lysis of the endothelial cells was
monitored by assaying the release of cytosolic lactate dehydrogenase (LDH).
All
amphotericin-resistant mutants showed dramatic defects in their ability to damage the
monolayer (Figure 5B).
AmB-Resistant Mutants Are Avirulent in Mice
Finally, we compared the virulence of our AmB-resistant laboratory and clinical
strains with that of wild-type and fluconazole-resistant (erg3) strains in a mouse model of
Candida fungemia. To provide a rigorous test, we used a relatively high intravenous
inoculum of 4x 106 fungal cells in young Balb/c mice. At the time of sacrifice, both
kidneys were isolated from each mouse. One was analyzed for fungal burden by
homogenization and plating of CFU; the other was submitted for histological analysis.
The wild-type strain killed all infected mice within 1-2 d (Figure 6A). Necropsy
revealed high viable fungal burden in the kidney, extensive filamentous fungal
morphology, and moderate tissue damage (Figure 6B-C). The erg3 mutant demonstrated
reduced virulence as previously reported [41-43], but still killed all mice in an average of
53
3-4 d. At the time of death, mice infected with this mutant had extremely high kidney
fungal burdens, filamentous fungal morphology, and extensive kidney necrosis.
All AmB-resistant mutants, including the clinical isolate, were completely
avirulent. Some mice showed mild weight loss in the first day after inoculation. But
within a few days all infected mice recovered and appeared healthy.
Kidney fungal
burdens at 12 d postinoculation were at least three orders of magnitude lower than those
of mice infected with wild-type Candidaor the fluconazole-resistant strain (Figure 6B).
Thus, the resistant strains failed to tolerate the host environment or immune attack and
colonize this organ, let alone damage it. Histological analysis demonstrated healthy
kidneys with some signs of resolving acute inflammation, suggesting that innate immune
attack may have contributed to the clearance of these strains (Figure 6C). The lack of
morbidity in mice infected with the clinical isolate suggests that this strain was recovered
from the patient harboring it not because it was virulent but simply because it had
survived AmB treatment.
DISCUSSION
The emergence of drug resistance has diminished the utility of nearly every class
of antimicrobial drug. Yet, 50 years after its introduction, AmB remains as effective as
ever.
The failure of fungi to evolve resistance to AmB presents a considerable
evolutionary puzzle with important consequences for human health. Our work offers a
mechanistic solution. In a comprehensive search for mutations that can produce AmB
resistance, we sequenced rare resistant clinical isolates, evolved resistant strains in the
laboratory, and targeted candidate genes by site-directed recombination. Every mutation
54
that was capable of conferring robust AmB resistance came at great cost to the pathogen.
They all diminished Candida's ability to survive the diverse array of stresses that are
inherent to growth in a mammalian host, crippled a major virulence factor required for
invasive disease (filamentation), and eliminated the capacity to kill mice.
Certainly, AmB therapy often fails, but not because the fungus acquires resistance
to the drug [44,45]. Instead, treatment failure is linked to other factors, including the
inability of the drug to penetrate certain niches of the body, dose-limiting renal toxicity,
or the underlying disease of the patient [6,7,9]. While a small number of AmB-resistant
clinical isolates have been reported in large surveys of clinical strain collections, we
suspect that many (if not all) of these will prove to be avirulent, as was the one we tested.
That is, they may have survived in a superficial niche less exposed to the stresses of the
bloodstream but would not be capable of mounting a virulent systemic infection. Such
strains might persist in patients with extreme immune system deficiencies.
However,
resistance to AmB is rare even in patients receiving myeloablative therapies that
eliminate immune function [6,7]. Even in the absence of stress from the immune system,
the lack of filamentation and hypersensitivity to other aspects of the host environment
(such as fever or iron deprivation) likely restricts the virulence of resistant strains.
Certainly, other explanations have been put forth for the rarity of AmB resistance
and may also be relevant. For example, drugs that target lipids are not susceptible to
resistance caused by substitutions in drug-binding pockets, a common occurrence with
drugs that target proteins. In addition, because AmB acts on the plasma membrane, it is
not susceptible to resistance mediated by increased drug efflux. Nevertheless, our work
indicates that several deletion or loss of function mutations can readily arise in Candida
55
and confer resistance to AmB in vitro. But these mutants do not become prevalent in the
clinic. It might be argued that AmB resistance is rare because it is dosed intravenously
and is not often employed in the types of long-term prophylaxis that breed resistance.
However, the structurally similar polyene nystatin, which has the same ergosterol-binding
mechanism of action as AmB, is widely used as a topical agent in the prevention of
thrush in immunocompromised patients and the treatment of superficial rashes in
neonates.
Although there has been ample opportunity for resistance to emerge and
become a clinical liability, it has not.
Our work also elaborates on the central role played by Hsp90 in potentiating the
evolution of new phenotypes, but here it takes on a novel character. As previously
reported, Hsp90 plays a critical role in the evolution of drug resistance in Candida and
Aspergillus [25]. Hsp90 allows drug-resistant mutants to survive stresses imposed by
triazoles and echinocandins, but is not required to tolerate the mutations conferring
resistance to those drugs alone. Such is not the case for AmB. The alterations in sterol
structure that confer AmB resistance cannot be achieved without causing a high level of
constitutive stress.
High levels of Hsp90, then, become essential to simply support
viability, even in the absence of the drug. Thus, our findings illustrate yet another way
that Hsp90 enables the acquisition of dramatic new phenotypes. Given its conservation,
we suggest its role in supporting stress responses operates very broadly in the evolution
of phenotypic diversity, allowing organisms to acquire mutations that confer novel
phenotypes but simultaneously create stresses that otherwise would not be tolerated.
The clinical experiment of 50 years of AmB use indicates that the emergence of
drug resistance, which widely plagues antimicrobial therapeutics, is not inevitable [46].
56
By elucidating the mechanisms that restrict the evolution of virulent AmB-resistant
Candida, our work suggests a strategy that might be applied more broadly to prolong the
ever-shortening window of efficacy encountered with new antibiotics: the development
of compounds that exploit the high costs of resistance mechanisms. This strategy need
not require the targeting of lipids.
Advances in structural biology and medicinal
chemistry have enabled the design of enzyme inhibitors that are less susceptible to
resistance mediated by point mutations or by drug efflux [47- 50]. Resistance to these
agents may require the microbe to make more complex changes to its physiology and
these, too, may come at a high cost. How might we discover targets that could induce
such constraints upon resistance? One possibility is to focus on essential genes that also
play critical roles in stress responses or virulence processes, for which rewiring of
pathways may fundamentally alter pathogenicity.
In any case, fungal-selective inhibition of Hsp90 presents an attractive mechanism
to prevent the emergence of drug resistance to all three antifungal classes in clinical use.
Rooted in ancient and conserved biological processes, a similar strategy may prove useful
in cancer, where resistance has greatly limited the efficacy of targeted therapeutics.
Indeed, pharmacological inhibition of Hsp90 is now being explored as a strategy to
forestall the emergence of resistance in diverse malignancies [28].
Investigating the
mechanisms that support rapid evolutionary change with an eye to the constant
challenges that cells face in their host environment presents a problem of broad biological
interest with important clinical implications.
57
MATERIALS AND METHODS
Ethics Statement
All animal protocols were conducted in accordance with the Guide for the Care and Use
of Laboratory Animals of the National Institutes of Health. All animals were maintained
according to the guidelines of the MIT Committee on Animal Care (CAC). These studies
were approved by the MIT CAC (protocol #0312-024-15). All efforts were made to
minimize suffering.
Media and Growth Conditions
C. albicans and C. tropicalis strains were routinely maintained at 30 'C in YPD (2%
Bacto peptone, 2% dextrose, 1% yeast extract). Stocks were maintained in 15% glycerol
at -80 'C. For generation of deletion mutants, transformants were selected on synthetic
medium (2% dextrose, 0.67% Difco yeast nitrogen base with ammonium sulfate) with an
amino acid dropout mixture.
RPMI 1640 media (Gibco) was buffered with 165 mM
MOPS, pH 7.0, and supplemented with 2% dextrose.
Whole genome sequencing, alignment, mapping, and variant calling. Using an
Illumina HiSeq platform with paired-end reads, we obtained an average coverage of 50fold. After quality control filtering, reads from each sequenced genome were aligned
against the Candidaalbicans SC5314 reference sequence, unless otherwise specified
(Assembly 21, downloaded from the Candida genome project on June 27, 2011, available
here:
http://www.candidagenome.org/download/sequence/CalbicansSC5314/Assembly21/ar
chive/C albicansSC5314_versionA2 1 -sOl -mO 1 -r03_chromosomes.fasta.gz) using the
BWA aligner [51]. This was followed by variant calling with respect to this Candida
58
albicans reference using the UnifiedGenotyper from version 1.0.5974 (the version we
used throughout these analyses) of the Genome Analysis Toolkit (GATK) [52]. (To
ensure that lower quality SNPs that are present in both a parental and derived strain were
correctly identified as being common, we disabled the maximum deletion fraction in the
-
call to the UnifiedGenotyper module. The specific parameters used were: "-dcov 1000
standemitconf 10.0 -standcallconf 50.0 --maxdeletionfraction 1.0.") For the
Candidatropicalis genome (strain OY5), only a preliminary assembly consisting of 24
scaffolds for the reference example, Candida tropicalis MYA 3404, is currently available.
We downloaded assembly "ASM633vl" from NCBI
(https://www.ncbi.nlm.nih.gov/nuccore?term=GG692395:GG692418[PACC]
on
December 20, 2012). Alignment and SNP calling for the C. tropicalisgenome was
performed as per the C. albicansgenomes. All reads will be available at NCBI under
BioProject accession numbers PRJNA194436
(http://www.ncbi.nlm.nih.gov/bioproject/l94436) for C. albicans and PRJNA1 94439
(http://www.ncbi.nlm.nih.gov/bioproject/194439) for C. tropicalis;the umbrella project
accession number is PRJNA195600 (http://www.ncbi.nlm.nih.gov/bioproject/195600).
Finding Unique Strain-Specific Variants
To identify variants (including SNPs and indels) unique to a strain, we compared the
"parental" strain to individual "derived" strains. We used a combination of custom code
and the GATK's CombineVariants and SelectVariants features to locate, and then rank
by quality, the SNPs and indels detected in open reading frames that were present only in
derived strains. From the previously generated VCF files for each of the parental and
derived strains as described above, the CombineVariants module was used to create a
59
single list of SNPs (specifically we set the module options "-priority" to the name of the
derived strain and the option "-genotypeMergeOptions" to "UNIOUIFY").
With this
output VCF file, we employed the SelectVariants module to detect variants unique to the
derived strain via the option: "-select "set==<derived-strain>".
Additionally, to find
cases where heterozygotes become homozygotes, or vice versa, we again used the
SelectVariants by using the intersection feature: "-select 'set==Intersection". The merged
genotype calls within each common SNP were then further filtered to find high-quality
calls where the zygosity changed. Alignments of the reads for the ranked SNPs and indels
were then visually inspected in the Integrative Genomics Viewer (IGV) for quality
control [53].
In the case of the clinical isolates, we did one pairwise comparison:
between the SC5314
("derived").
wild type and the ATCC 200955 AmB-R clinical isolate
For the in vitro selection experiments, we defined the first isolate (#1) as
parental, and compared the subsequent three serially derived isolates (#2, #3, #4) back to
this parental isolate.
Loss-of-Heterozygosity (LOH) Visualization
To visualize the LOH events in the in vitro selection series (BVO 1 -BV05), we performed
multisample SNP calling using the GATK UnifiedGenotyper module to generate a VCF
file containing all SNPs in all four strains (UnifiedGenotyper options: "-glm SNP -nt 1 -downsample tocoverage 100000"). Following quality filtering of SNPs, from this VCF
file, we created a Python script to generate a list of positions of SNPs (relative to
reference), if present in any of the four strains.
For each SNP position in each of the
strains, we were then able to classify whether it was homozygous for the reference base
(blue), homozygous for the variant base (red), or heterozygous (white). The resulting
60
"heterogram" for all chromosomes was visualized using the quilt.plot function from the R
"fields" package [54]. Regions where a LOH event is likely to have occurred show up as
blocks of blue and red SNPs (regions of high homozygosity) against the backdrop of
white (heterozygous SNPs).
Sliding Window Heterozygosity Analyses
For each SNP in a given strain, we extracted the counts of reads containing the reference
and variant base from the allele depth ("AD") VCF annotation at that position. Using
only SNPs of quality 1,000 or more, we then computed the "base ratio" at each position
by dividing the count of reads for the minor allele base by the total number of reads for
both bases at that SNP position, resulting in values of between 0 (complete
homozygosity) and 0.5 (complete heterozygosity). We then averaged these base ratios
over a 1 kb sliding window for each chromosome. We performed these analyses for the
in vitro evolution series (BVO1-BV04) of C. albicans as well as the single C. tropicalis
isolate (OY5).
Spectrophotometric Analysis of Membrane Ergosterol Content
Sterols were extracted and analyzed as previously described [55]. Equal weights of cell
pellets were used across strains. Absorption patterns were recorded by scanning between
240 and 300 nm at 0.5 nm intervals.
Strain Construction
Strains and primers are listed in Tables Si and S2. Deletion strains were constructed as
described in [56], using HIS1, LEU2, and ARG4 markers (the URA3 marker was not
used, and all strains used were URA3 wild type).
Briefly, PCR products containing
approximately 350 nucleotides of upstream and downstream homology for each gene
61
were generated, and fusion PCR was used with a selectable auxotrophic marker to
generate the knockout construct. Proper insertion of the auxotrophic marker and loss of
the endogenous gene were confirmed by PCR. Two heterozygotes were selected from
each initial knockout transformation for the knockout of the second allele, from which
two knockout strains were tested for each (four total strains). The erg3 erg] mutant was
constructed as previously described [21].
These four strains were compared for
phenotypic concordance in filamentation and stress resistance to minimize the effect of
secondary mutations; additional data on a second mutant strain for key mutants is
presented in Figure S5.
Minimum Inhibitory Concentration and Growth Assays
Antifungal susceptibility was determined in flat bottom, 96-well microtiter plates
(Costar) using a broth microdilution protocol as described [25]. Overnight cultures were
grown at 30 'C in YPD for at least 16 h, and cell density was measured by OD600 before
seeding approximately 103 cells per well in YPD or RPML media, at 30 'C or 37 'C, as
indicated in figure legends. Growth was measured at 24 or 48 h postincubation by alamar
blue (Invitrogen) fluorescence with excitation at 550 nm and emission at 590 nm, and in
certain
cases
confirmed
by measurement
of OD600
after
agitation using
a
spectrophotometer (Tecan). MIC80 was defined as the concentration of drug reducing
growth by 80% relative to the wells containing no drug. For susceptibility to AmB and
stress response inhibitors (Figures ID, 3B-C, and S5A), growth scores were determined
by normalization of the values for each sample to the values obtained for the wild-type
strain in the absence of AmB (or in the DMSO negative control for Figure 3B).
For
these assays (Figures 1D, 3B-C, and S5A), each condition was tested in duplicate and
62
Relative growth data were quantitatively
repeated on at least two different days.
displayed
in color using Java TreeView
1.1.3
(http://itreeview.sourceforge.net).
Sensitivity to stressors (Figure 4B-E) was similarly determined by microplate dilution
and reading of alamar blue dye fluorescence; here, growth scores were calculated by
dividing the growth value of the mutant by the growth value of the wild type in each
particular stress condition. For stress assays (Figures 4B-E and S5B-C), data represent
the mean of six wells, pooled from experiments performed on two separate days.
Statistical significance was determined in Graphpad Prism 5.0 using the Student's t test
function. Error bars represent the SEM for each group. Serum sensitivity was performed
as previously described [57].
Cidal and static effects of Hsp90 inhibition were tested generally as previously described
[29]. Cells were grown overnight in YPD and diluted to a concentration of 104 cells/mL
at 30 'C in YPD containing Hsp90 inhibitors at the indicated concentrations. After 24 h,
cells were plated onto YPD at two dilutions and colonies counted.
Drugs used in growth assays included AmB (Fungizone, Invitrogen), fluconazole (TCI
chemicals), radicicol (A.G. Scientific), geldanamycin (A.G. Scientific), Cyclosporin A
(CalBiochem),
FK-506
Enzastaurin (LC Labs).
(A.G.
Scientific),
Cercosporamide
(Sigma-Aldrich),
and
All drugs were dissolved in DMSO, with the exception of
fluconazole (H 20) and AmB, which was obtained as an aqueous suspension with sodium
deoxycholate.
Agar Plate Growth Assays
Spotting assays were performed by growing overnight cultures of strains in YPD at 30
'C, washing in PBS, and resuspending in PBS at a concentration of 5x 106 cells/mL. Four
63
5-fold serial dilutions were performed before spotting. All RPMI-agar plates were used
within 6 h of pouring due to the potential instability of AmB and peroxides; it is
recommended to test a range of AmB concentrations in agar plates due to potential
chemical instability.
Selection of AmB-resistant colonies was performed using
ERG2/erg2i heterozygotes in wild-type, cnbAz, or hogli/
backgrounds on RPMI-
agar plates containing 0.4 gg/mL AmB; the wild-type was also selected on media
containing AmB and 2.5pM geldanamycin. Strains were grown overnight in YPD at 30
'C, washed in PBS, and plated at a density of 8x 106 cells per plate, and incubated for 2 d
at 37 'C before photographs were taken.
In Vitro Gradual Selection of AmB Resistance
ATCC 10231 was grown overnight at 30 'C in YPD and 2x 108 cells were inoculated into
one liter of YPD containing 0.25 [tg/mL AmB at 30 'C. Cultures were grown shaking for
24-48 h until turbidity was observed; cells were then removed, washed in PBS, and split
for freezing glycerol stocks or reinoculation into media with a 2-fold higher concentration
of AmB at the same cell density. The process was repeated until the concentration of
AmB reached 32 pg/mL.
The process was repeated in three independent selections.
Strains were then thawed from glycerol stocks and struck to single colonies for future
MIC assays.
Neutrophil Killing Assay
Neutrophils were prepared fresh from the blood of a healthy human donor following
standard protocols, using Histopaque 1077 density gradient centrifugation and hypotonic
erythrocyte lysis [58].
After isolation, neutrophils were activated by treating with
recombinant TNF-a (10 ng/mL). Killing assays were performed essentially as described
64
in [38,59]. Briefly, neutrophils were co-cultured with log-phase C. albicans strains at a
1:1 ratio, with both cell types at a concentration of 10 4/mL.
Control wells were
inoculated in the identical conditions but without neutrophils added.
Plates were
incubated at 37 'C in a humidified incubator for 6 h, at which point neutrophils were
lysed by adding one volume of water containing 0.1% Tween-20 and a 1:40 dilution of
Alamar blue; wells were vigorously pipetted up and down. Alamar blue fluorescence
was measured after 90 min of incubation at 37 'C.
Relative growth was measured by
dividing values obtained in the presence of neutrophils by those obtained in their absence
for each strain.
Control wells lacking C. albicans were included to verify that this
treatment does not quantify growth of the neutrophil cells. Results from three separate
plates are shown; growth of each mutant strain is presented as a fraction of the wild-type
growth from the same plate. Statistical analysis was performed using paired Student's t
test in Microsoft Excel.
Filamentation Assay
Hyphal induction was performed by growing C. albicans overnight at 30 *C in YPD,
washing in PBS, and diluting 1:100 into RPMI + 10% fetal bovine serum at 37 'C
(Sigma-Aldrich). After 2 or 4 h, cultures were briefly concentrated by centrifugation and
visualized by DIC microscopy.
Endothelial Cytotoxicity Assay
Endothelial cell invasion assays were performed with human umbilical vein endothelial
cells (HUVEC's, Lonza) as previously described [60].
Monolayers were infected with
C. albicans strains at a 1:1 HUVEC:fungus ratio and assayed for cytotoxicity with the
CytoTox-96 Lactate dehydrogenase assay (Promega) after 6 h of co-incubation.
65
Cytotoxicity was quantitated as the fraction of LDH release relative to a 100% value of
wells treated with 1% Triton X-100 and a baseline value of HUVEC cells not infected
with Candida. Error bars are indicative of the standard error of the mean for each group.
Error bars indicate SEM.
Results pooled from two experiments are displayed (six
replicate wells per experiment). Statistical analysis was performed using unpaired
Student's t test in Microsoft Excel.
Quantitative RT-PCR Expression Analysis
For measurement of the expression of stress response genes, strains were grown
overnight in YPD 30 'C and diluted to OD600 of 0.15 in YPD, then grown for 5 h to
mid-log phase and either centrifuged without treatment or subjected to different stresses.
For stressed wild-type cells (SN250 strain), the conditions were as follows: AmB
treatment with 1 ptg/mL AmB for 15 min, Osmotic shock with 0.3M NaCl for 10 min,
Nitrosative stress with 2 mM DPTA-NO (Cayman Chemical) for 15 min, calcium shock
with 150 mM calcium chloride for 10 min, oxidative stress with 10 mM tert-butyl
peroxide for 10 min, and iron chelation with 500 iM bathophenanthroline sulfonate for 4
h. Cultures were centrifuged at 1,500 g for 5 min and quickly flash frozen in liquid
nitrogen. Total RNA was isolated with an RNeasy column kit (Qiagen), normalized to
equal amounts of total RNA across samples, and reverse transcribed for 120 min with the
high capacity reverse transcription kit (Applied Biosystems). qPCR was performed with
SYBR green mastermix (Applied Biosystems) on an Applied biosystems AB17900
thermal cycler, using oligonucleotides described in Table S2.
Each measurement was
obtained from an average of four wells, including two biological replicates and two
technical replicates.
Expression analysis was performed by the comparative ACt
66
quantitation method, comparing mutant or stress-treated strains to wild-type untreated
strains, using nomalization to four internal control genes: TDH3, ACTi, TEF3, and
RPP2B; the mean value obtained from the four normalizations was used. For
representation by heatmap (Treeview), relative expression levels were determined by
dividing each value by the maximum expression level for that gene in any tested
condition, with the untreated wild-type samples set as the baseline (as certain genes were
induced over 50-fold and others were not induced greater than 4-fold in any condition).
Murine Model of Systemic Infection
We utilized 7-9-wk-old female Balb/c mice from Charles River laboratory (n = 8 mice
for WT, 10-14 mice for mutant strains).
Each strain was tested in two independent
experiments (performed at different times), and data were pooled. Strains to be injected
were grown overnight in YPD, diluted, and grown for 5 h into mid-log phase at 30 'C,
then washed twice in phosphate buffered saline (PBS), counted by hemocytometer and
plating of dilutions, and resuspended in PBS at a concentration of 4xl07 cfu/mL. We
used 100 [tL of each suspension to inject mice by lateral tail-vein injection. Mice were
weighed daily and monitored for signs of morbidity and sacrificed when body weight
decreased by more than 20%. Kidneys were removed and either homogenized in PBS
and plated for viable colony units (in duplicate) or submitted for fixation and staining
with Periodic-acid Schiff stain. A veterinary pathologist was consulted for histological
analysis. All experimental procedures were carried out according to NIH guidelines and
MIT protocols for the ethical treatment of animals.
67
Acknowledgments
The authors express gratitude to M. Brophy and L. Nolan for purified Calprotectin, J_
Love and T. Volkert for genome sequencing, J. Kohler and C. McLellan for useful
discussions, V. Vyas for strains, and J. Funt for custom Python code used in the
generation of heterozygosity plots from VCF files.
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72
J
-
~AL ~
A
SC5314
Wild-Type
ATCC 200955
AmB-R
Clinical isolate
Chr2:
153368
Chr2:
157825
ERG2
B
i
#1
in vitro
selection
WTIQ7OStop
DIBOG/QO7Stop
#2
for AmB
resistance
fI
#3
D180G/Q70Stop (1:3)
#4
Q7QStop/Q7OStop
ERG6
Chr3:
Chr3:
466245
465115
[AmB]
D
Total Sterol Extract
C
C. albicans Wild-Type
C. albicans Clinical isolate
erg2
1.00.8.0
35 0.1 0.2
(pgfmL)
0.4 0.8 1.6
erg3
0.6-
erg4
0.4.
erg5
0.2'
erg3.ergll
erg6
erg24
0
0240
280
260
300
Wavelength
-
WT
-
erg2 -
C. tropicalis
Clinical
C. tropicalis Wild-Type
C. tropicalis Clinical Isolate
erg3;ergll
C. albicans
Clinical
0.0 0.25 0.50 0.75 1.0
Relative Growth
73
Fi2ure 1. Mechanisms of AmB resistance in Candida. (A) Alignment of reads from
whole-genome sequencing of C. albicans wild-type strain SC5314 and AmB-resistant
clinical isolate ATCC 200955 demonstrates transposon insertion in ERG2 in the clinical
isolate. The ERG2 locus is shown. Colored reads are indicative of mate-pairs that do not
both map to the same chromosome, but instead one end to ERG2 and the other end to the
TCA2 locus (elaborated in Figure SlA). Reads were visualized with the integrative
genomics viewer (IGV) [53]. (B) Alignment of selected strains from whole-genome
sequencing of in vitro-evolved AmB-resistant series identifies causal mutations.
Mutations in ERG6 ORF are highlighted, and the corresponding amino acid changes are
indicated. Strain #1, 0 generations (founder); Strain #2, 60 generations; Strain #3, 120
generations; Strain #4, 240 generations. Two segments of IGV visualization for ERG6
were joined to allow visualization of both mutations in one image; point of joining
indicated by "::". (C) Spectrophotometric analysis of sterols reveals lack of C5-C6:C7-
C8 conjugation in AmB-resistant clinical isolates as well as laboratory-generated erg2
and erg3 erg]] mutants. Sterols were isolated by saponification and heptane extraction
and analyzed spectrophotometrically between 240 and 300 nm, following established
methods [55]. (D) AmB susceptibility of clinical isolates and laboratory-generated
mutants in every nonessential gene in the latter half of the ergosterol biosynthesis
pathway (after cyclization of squalene to lanosterol). Mutants were generated in SN152
strain background using HIS 1, LEU2, and ARG4 markers [56]. AmB susceptibility was
determined by microplate dilution in RPMI at 37 'C for 24 h, repeated in duplicate;
growth was normalized to wild-type in the absence of AmB.
74
A
B
A0.5pM
Geldanamycin
C. tropicalis WT
C. tropicalis AmB-R
Geldanamycin
-
Wild-Type
FCZ-Resistant
AmB-Resistant
0.5pM
-
C. tropicalis WT
C. tropicafis AmB-R
Wild-Type
FCZ-Resistant
AmB-Resistant
8 pg/mL
1 pg/mL
AmB
L
C
-
FCZ
L
Wild-Type
1 pglmL
AmB
FCZ-Resistant
AmB-Resistant
Hsp9O
inhibitor
MIC (AM)
E
Radicicol MIC
Geldanamycn MIC
-0- Amphotein MIC
D
Wild-Type
erg3AA
arg2A/A
erg6A/
erg3A/A erg11AlM
Clinica
-A-
-4-
-15o Amphotericin
r 1 28 MC (sgImL)
11.00
25.
-0.75
4.
3.
2.
*0.50
4
;
5
Sequential Isolate Number
Geldanamycin
erg2MA
erg6NA
erg3/I1IA
Clinhal
104.
102m
Fold
Population
Change
7
101K
10.
j1
10-2.
-0.28
3
0.25 pM
AmB
Wild-Type
3-A
2
0.4 pgimL
105.
40.
351
1
No Drug
(RPMI, 37-C)
10-4
10-8
;
N
Geldanamycin
E Radicicol
Figure 2. AmB-resistant strains critically depend on high levels of Hsp90 function
for survival. (A) Mild inhibition of Hsp90 not only reverses AmB resistance but
selectively kills AmB-resistant isolates. Spot assays (5-fold serial dilutions) of wild-type
(SC5314), fluconazole-resistant (Isolate #2, [61]), and AmB-resistant (ATCC 200955) C.
albicans on RPMI media containing antifungals and/or Hsp90 inhibitors at the indicated
concentrations. (B) Increased dependence on Hsp90 for AmB resistance is conserved in
a C. tropicalis clinical isolate. Spot assays of C. tropicalis reference strain MYA-3404
(AmB-sensitive) and ATCC 200956 (AmB-resistant) on media containing AmB,
geldanamycin, or both compounds. (C) Isogenic, laboratory-generated AmB-resistant
mutants also show an increased dependence on Hsp90. Spot assay of laboratorygenerated mutants on AmB and geldanamycin. Fluconazole-resistant erg3 mutant and
AmB-resistant clinical isolate are provided for reference. Full MIC data in liquid culture
for all strains are provided in Figure S4. (D) In vitro selection for AmB resistance leads
to hypersensitivity to Hsp90 inhibition. 24-hour MIC80 (drug concentration reducing
growth by 80 percent) of geldanamycin, radicicol, and AmB for each isolate from in vitro
evolution series, tested in YPD at 30 'C. Note the discontinuity in the left y-axis due to
the dramatic decrease in Hsp90 inhibitor MIC. (E) Hsp90 inhibition is cidal to AmBresistant strains. Viability assays of wild-type and AmB-resistant C. albicans strains in
the presence of the Hsp90 inhibitors geldanamycin or radicicol. Strains were incubated
in liquid culture for 24 h with the drugs and then plated for surviving colony-forming
units. Drugs were used at 5 ptM in all strains. Error bars indicate SEM; each
measurement was performed in duplicate.
75
A
oo
fe.
of
Wild-Type
AmB
erg3AIA
NaCl
erg3, 1A/A
Fe Chelator
erg2A/A
DPTA-NO
Clinical
Ca
erg6AIA
Peroxide
U_
Is
L
+
2
-1.00 -0.83 -0.6 -0.50 -0.33 -0.16 0.00 0.16 0.33 0.50 0.86 0.83 1.00
Relative Expression
DMSO G dA RAD
B
Hsp90
FK506 CsA
CRD ENZ
Calcineurin
PKC
AmB (pg/mL)
C
0
C.a. Wild-Type
Wild-Type
C.a. Clinical
cnbIA/A
C.a. erg2
0.05 0.10 0.20 0.40
hog1A/ A
C.a. erg6
1pM GdA
C.a.
erg3/11
SpM
C.t. Clinical
GdA
C.t. Wild-Type
0.00 0.25 0.50 0.75 1.00
Relative Growth
0.00 0.25 0.50 0.75 1.00
Relative Growth
Wild-Type
hogIA/A
cnb1IA
76
Wild-Type;
2.5 pM Geldanamycin
Figure 3. Constitutive stress response activation in AmB-resistant strains. (A) AmB
resistant mutants constitutively express diverse stress response genes at high levels in the
absence of any external stressors. qRT-PCR profiling of stress response genes in wildtype, fluconazole-resistant (erg3), and AmB-resistant mutants of C. albicans grown in
rich (YPD) media with no added stressors (left panel), compared with wild-type strains
treated with a variety of acute stressors known to activate these stress response pathways
(right panel). Strains were grown to mid-log phase in YPD, and RNA was isolated by
established methods. For stress treatment of wild-type strains (right panel), the indicated
stressors are: AmB, 1 pg/mL AmB; NaCl, 0.3 M NaCl; Fe Chelator, 500 pM
bapthophenanthroline disulfonate; DPTA-NO, 2 mM DPTA-NONOate; Ca , 150 mM
CaCl 2 ; Peroxide, 10 mM tert-butyl peroxide. Expression levels of diverse stress response
genes were quantified and normalized to four internal control genes: TDH3, TEF3,
ACT1, and RPP2B; the mean value obtained from these four normalizations was used.
To generate quantitative comparisons for color visualization, the relative expression level
of each gene in each sample was divided by its maximal expression level observed (in
any of the deletion strains or stress conditions; see Materials and Methods for further
description). (B) Calcineurin and PKC pathways are differentially required for survival of
AmB-resistant strains. Growth of AmB-resistant laboratory mutants and clinical isolates,
as well as wild-type controls, in the presence of small molecule stress response inhibitors
at the following concentrations: Geldanamycin and Radicicol, 2 pM; FK-506, 5 tg/mL;
Cyclosporin A, 5 pg/mL; Enzastaurin and Cercosporamide, 5 pg/mL. Growth was
assayed after 24 h; values indicate means of duplicate measurements, normalized to wildtype in DMSO. (C) Hogl, but not calcineurin or PKC, is required for wild-type levels of
AmB tolerance in the absence of ergosterol biosynthesis mutations. Wild-type, cnbi, and
hog] strains, as well as wild-type with 1 or 5 pM geldanamycin, were tested for AmB
MIC by microplate dilution. Growth was assayed after 48 h to highlight differences in
drug tolerance between strains; values indicate means of duplicate measurements,
normalized to the wild type in the absence of AmB. (D) Hogl, Cnbl, and high levels of
Hsp90 are required for the de novo emergence of AmB-resistant colonies. ERG2/erg2
heterozygotes from wild-type, cnb], or hog] backgrounds were plated at a density of
8x 106 cells per plate on media containing 0.6 ptg/mL AmB. The wild-type was also
plated on media containing AmB and 2.5 pM geldanamycin. Plates were photographed
after 3 d.
77
A
-
-
Wild-Type CM
erg3A/A
erg2A/A
-
erg6A/A
erg3, 11A/A
Clinical
37'C
41'C
39'C
Hypochlorous Acid
B
1.00-
37'C
39'C
0.5mM
0.25mM
Peroxide
Peroxide
DPTA-NONOATE (NO donor)
C
1
1.00T
Relative
Growth 0.75-
0.75
0.50-.
Relative
Growth 0.50.
0.25-
0.25-
0.00
1
I
0.081
Tf
441
E
Calprotectin/S1OOA9 Peptide
BPS (Iron Starvation)
1.25-
1.00Relative
Growth
0.75.
Relative 1.00Growth
0.75-
0.50-
0.50-
0.25]
0.25-
T
0.00
G
F
Neutrophil:Candida
Co-Culture
Serum Proliferation, 39.5*C
1.00-
+0 WT
CFUImL
+
erg2AIA
1
erg6A/A
Relative 0.75-
erg3A/Aerg11A/A
+ Clinical
+
Growth
0.50-
102
0.25-
0
4"M
Ulp
0.00
48
Tim (hr)
~&I" / 4
78
Figure 4. AmB-resistant strains are hypersensitive to the stresses of the host
environment. (A) AmB-resistant strains are sensitive to very high febrile temperatures
and extremely sensitive to oxidative stress, especially at elevated temperature. Wild-type
and AmB-resistant strains were spotted by serial dilution on RPMI media with or without
tert-butyl peroxide at the concentrations indicated. (B-E) AmB-resistant strains are
hypersensitive to stresses encountered in the host environment. Wild-type and AmBresistant strains were grown in RPMI media containing 2 mM of hypochlorous acid (B)
or 4 mM of the nitric oxide donor DPTA-NONOATE (C), two neutrophil-secreted
products that are the critical final effectors of anti-Candidaimmunity. Sensitivity to iron
deprivation was tested by growth in RPMI + 500 iM of the iron chelator
bathophenanthrolinedisulfonic acid (D). No increase in sensitivity to the antimicrobial
peptide Calprotectin (10 pg/mL) (E) was observed. Growth values were obtained by
normalization to wild-type growth in the same condition. All mutant strains were
significantly more sensitive than wild-type to the tested concentrations of hypochlorous
acid, DPTA-NONOate, and BPS at 37 'C (**p < 0.01, two-tailed Student's t test), but not
to calprotectin. Values indicate the mean of two independent experiments of three
replicates each; error bars indicate SEM. (F) AmB-resistant strains proliferate more
slowly than wild type in serum at elevated temperature. Wild-type and AmB-resistant
strains were inoculated in 100% fetal bovine serum at 39.5 'C, and viable colony-forming
units were determined by plating dilutions on YPD after 24 and 48 h. Error bars indicate
SEM. (G) AmB-resistant strains are hypersensitive to killing by neutrophils. Human
neutrophils were isolated from whole blood (see Materials and Methods) and activated by
treatment with recombinant TNF-a. Candida were added to wells containing neutrophils
at a 1:1 effector:target ratio and incubated at 37 'C for 6 h, at which point neutrophils
were lysed and Candidagrowth was quantitated with Alamar blue. Percent growth for
each strain was calculated as the fraction of growth in the presence of neutrophils to
growth in the absence of neutrophils; mutants were normalized to growth of the WT on
each plate. The experiment was performed with a total of three biological replicates from
two separate days; error bars indicate mean and SEM.
79
YPD, 30 0C
Serum/37 0 C
Serum/37 0C
2 hr
4 hr
A
WildType
erg3AA
erg2lA
erg6AA
erg3AIA
erglI A
AmB-R
Clinical
Isolate
40-
-TPercent
30Cytotoxicity
(LDH Release)
*
B
F,,i1
2010-
40
0
80
Figure 5. AmB-resistant strains are defective in filamentation and tissue invasion.
(A) AmB-resistant mutants fail to properly induce filamentous growth upon stimulation.
Wild-type strains and resistant mutants were grown in YPD and then inoculated into
RPMI media containing 10% fetal bovine serum at 37 'C. Strains were analyzed by DIC
microscopy after 2 and 4 h. (B) AmB-resistant strains cause much less damage to
endothelial monolayers than wild type. Monolayers of HUVECs (human umbilical vein
endothelial cells) were established and infected with C. albicans. After 6 h, endothelial
cell cytotoxicity was assayed by quantifying LDH release, using uninfected cells as a
negative control and cells lysed with 1% Triton X-100 as 100% lysis control. Data were
pooled from two independent experiments with six replicate wells each; error bars
indicate SEM. All mutant strains were significantly less cytotoxic than wild type (*p <
0.05, **p < 0.01, two-tailed paired Student's t test).
81
Mouse Tall Vein Infection
100.
-
-
A
-u- WT
+ erg3AIA
Percent 75
Survival
erg2AI4
erg6AIA
erg3A/Aerg1 IAIA
+ Clinical
50
-+-
250.
-
1
0 1
B
.
2
3
4
7
.
.
5 5 7 3 9
Time (days)
.
10 11 12
Kidney Colony Forming Units at Sacrifice
107Total CFU
104105*
103102101.
own
-7*..
AA
vY0
AA
I>'
*44~~4
C
C
Wild-Type
erg3AA
erg2AA
erg6AA
82
erg3AA;ergllAA
Clinical Isolate
AmB-resistant strains are avirulent in a mammalian model of
Figure 6.
disseminated candidiasis. (A) AmB-resistant mutants do not cause morbidity even when
injected into mice at a high inoculum. Wild-type, erg3, and AmB-resistant mutants were
grown into log phase for 5 h in YPD, counted, and 4x 106 cells of each strain were
injected into the tail vein of 7-9-wk-old Balb/c mice (n = 8-14 mice per strain). Mice
were monitored for weight loss and sacrificed after a >20% drop in body weight or
appearance of morbidity. All surviving mice were sacrificed after 12 d. (B) AmBresistant strains are unable to colonize the mouse kidney. Colonies were counted after
homogenization and plating in duplicate of viable candida from one kidney of each
infected mouse at the time of sacrifice. As wild-type, erg3, and other mutants survived
for different periods of time before sacrifice, direct comparisons cannot be made between
these groups based on CFU values. However, the extremely low CFU values from AmBresistant strains are typically indicative of sterilization. (C) AmB-resistant strains do not
damage the mouse kidney. Kidneys from each infected mouse were fixed and stained
with periodic acid-Schiff stain to visualize Candida and kidney pathology. Wild-type
and erg3 strains demonstrated filamentous growth and severe kidney pathology, which
was even greater in the erg3 strain. Examples of sites where fungi are observed are
highlighted with a black arrow; fungi appear as long filaments with a purple color. No
viable Candida were seen in sections from mice infected with AmB-resistant strains.
Scale bar, 50 gm.
83
Insertion region
A
WildType
I
*,
i~'
AmphB-
Transposon
Resistant
Mate pairs
-.-
~
------
t~1
TCA2 Transposon Endogenous Locus
(Chromosome 7)
ERG2
(Chromosome 1)
B
Chri
Chr2
Chr3
Chr4
Chr5
Chr6
Chr7
ChrR
0.4
Mean
Heterozygosity
0.1
ERG2 (Chrl:155,291 to 154,638)
Supporting Information
Figure S1. Trasposon insertion and heterozygosity analysis in AmB-resistant C.
albicans. (A) Insertion of TCA2 retrotransposon into ERG2 locus. Mapping of reads
from wild-type (SC5314) and AmB-resistant (ATCC 200955) C. albicans. Left, ERG2
locus on chromosome 1; right; TCA2 locus on chromosome 7. Mate-pairs of reads in the
AmB-resistant strain in which one mate maps to ERG2 and the other to TCA2 (identified
by long insert sizes) are depicted in the lower panel. All screenshots were generated by
the Integrative Genomics Viewer (IGV) [53]. (B) Whole-genome heterozygosity analysis
of C. albicans ATCC 200955. Single-nucleotide-polymorphisms (SNPs) were analyzed
for base ratio at each variant site to determine an allelic ratio; the mean heterozygosity
value for total heterozygosity is 0.5, and for total homozygosity the value is 0.0. Base
ratios were averaged over a 1 kb sliding window for each chromosome.
84
S258F
ERG3
B
C.
A.
H.
LHKPHHKWIVCTPFASHAFHPVDGFFQSLPYH
LHKPHHKWIMPSPFASHAFHPLDGWSQSVPYH
LHKPHHIWKIPTPFASHAFHPIDGFLQSLPYH
albicans
fumigatus
sapiens
C
ERG11
C. tropicaliscontigs (unassembled)
D
a
aa
a
z
zz
z
S
00
528
0 0 03
1
a
a
a
a
5,
2,
Z
Z
Z
Z
5,
8,
1
06
05
07
08
a
2,
09
0.4-
Mean
Heterozygosity
0.1
ERG3 ERG11
85
a
a
z
z
2
10
1
Figure S2
AmB-resistance mutations and heterozygosity analysis in AmB-resistant C.
tropicalis. (A) Mutation in ERG3 changing conserved serine 275 residue to phenylalanine
in C. tropicalis ATCC 200956. (B) S275 is universally conserved in Erg3 homologs.
Alignment of protein sequence surrounding S275 from Erg3 homologs of C. albicans, A.
fumigatus (XP_747563), and H. sapiens (BAA33729). (C) 170-nucleotide deletion from
ERG]] of C. Tropicalis ATCC 200956, detected as the complete absence of reads
mapping to this region of the gene. Unnecessary lines generated by IGV (not
representative of data) were removed. (D) Heterozygosity analysis of ATCC 200956; the
incomplete assembly of the C. tropicalis genome requires the use of smaller sequence
contigs.
86
.. .
....
..
..
.....
...
...........
....................
A
cd
cd
chri
ews
chrS
d"r
B
chtR
-
U
chi7
Ca21chr3_C albicansSC5314
I
0
Isolates
CW)
6
#1
#2
0
#3
6
#4
E-
2e+05
6e+05
4e+05
position
87
8e+05
Figure S3
Heterozygosity analysis of in vitro-evolved series. (A) Whole-genome analysis of loss
of heterozygosity events across four sequenced isolates from in vitro-evolved series
(from Figure 1B). A list of all SNP positions in the four strains was compiled and then
analyzed for heterozygosity by base ratio (see Materials and Methods).
Sites
homozygous for the reference base are depicted in blue, homozygous for the variant base
in red, and heterozygous in white. The loss of heterozygosity is focused on the left arm
of chromosome 3, which contains ERG6. (B) Heterozygosity analysis of chromosome 3
demonstrates loss of heterozygosity in isolate #4. Sliding-window analysis of base ratio
along chromosome 3 depicts loss of heterozygosity in the left arm of chromosome 3.
88
A
Wild-type
CE1 efg2-1Ml Clinical isolate
M erg6-1-
&
MIC,
PM
-
643216.
-il
2-
erg3erg 11-
U.5.
0.250.125
0.0625
0.03125
19\1
B
N Wild-type
32
MIC,
PM
pM
32
-
4
2
11
0.5
0.25
0.1250.0625-
19N
C
U
F ~]
U*
U*
U*
1024-
4?
<1.
Fold Sensitivity Ratios (WT MIC/Mutant MIC)
Compound/Strain
Anisomycin
Benomyl
Nocodazole
MNNG
4NQO
Mycophenolic Acid
Nourseothricin
Geldanamycin
Radicicol
NVP-AUY922
SNX-2112
erg2-/4.00
2.00
2.00
2.00
4.00
1.67
8.00
32.14
24.00
27.31
21.33
ATCC200955
4.00
2.00
2.00
3.00
2.00
5.00
8.00
28.80
18.00
27.31
21.33
89
erg6-/- erg3, 11-/8.00
5.33
4.00
2.00
8.00
4.00
6.00
3.00
8.00
4.00
10.00
2.50
16.00
8.00
480.00
96.00
384.00
96.00
222.61
109.40
256.00
85.33
erg2-/clinical isolate
erg6-/erg3ergll--
Figure S4
Minimal inhibitory concentrations of Hsp90 inhibitors and cytotoxic compounds.
(A) Sensitivity of AmB-resistant mutants to diverse cytotoxic compounds. MIC80 of
each strain against each compound was determined by microplate dilution assay in YPD
at 30 'C. (B) MIC80 of strains to four Hsp90 inhibitors in YPD at 30 'C. (C) Fold
sensitization, relative to wild-type, of mutant strains to cytotoxic agents and Hsp90
inhibitors (Hsp90 inhibitors highlighted in red for clarity). Table indicates the value
obtained when the MIC80 for each compound in the wild-type strain is divided by the
MIC80 for that compound in that mutant strain.
90
0
A
0.05 0.1
0.2 0.4 0.8 1.6
[AmB] (pg/mL)
Wild-type
erg2A/A #2
erg6A/A #2
0.00 0.25 0.50 0.75 1.00
Relative Growth
erg3/ergllA/A #2
c
B
DPTA-NONOATE (NO donor)
Hypochlorous Acid
In
1.00-.
1.00-
Relative
Growth
Relative
0.75.
Growth
0.50-
0.60-
0.25n nn
0.75-
0.25-
II
,_
I
e
**
.*--
~r~i
*r
499
At-
A
D
YPD, 30"C
RPMl+FBS, 37-C, 4hr
Wild-Type
erg2A/A
#2
erg6AIA
#2
erg3AIA
erg1IA/A
#2
91
Figure S5
Validation of additional mutant strains. (A) AmB susceptibility of a second,
independently generated laboratory deletion mutant in ERG2, ERG6, or ERG3 and
ERG]], performed as described in Figure 1D. (B) Sensitivity of each additional mutant
to hypochlorous acid (B) and DPTA NONOate (C), performed and analyzed as described
in Figure 4B-C (**p < 0.01, Student's t test). (D) Filamentation of each additional
mutant in response to stimulation by fetal bovine serum at 37 'C in RPMI media,
performed as described in Figure 5A.
Strain Name/Name in
Genotype
Source
Prototrophic (naturally erg2/erg2)
ATCC
Prototrophic (naturally erg3/erg3,
erg]I/erg1)
Prototophic
ATCC
Prototrophic
As ATCC 10231, erg6 D1 80G
As ATCC 10231, erg6 D1 80G (3:1)
As ATCC 10231, erg6 Q70stop/Q70stop
Prototrophic
ATCC
This study
This study
This study
White TC, 1997
LEU2/leu2A HIS1/his1A arg4A/arg4A
URA3/ura3A::imm 434
IRO1/iroIA: :imm 434
leu2A/leu2A his1A/hislA arg4A/arg4A
URA3/ura3A: :imm 434
Noble et al, 2005
Text
ATCC 200955 (AmB-R
Clinical C. albicans)
ATCC 200956 (AmB-R
Clinical C. tropicalis)
MYA-3404 (C.
tropicalis WT reference
strain)
ATCC 10231/in vitro #1
AmB-R#2/in vitro #2
AmB-R#3/in vitro #3
AmB-R#4/in vitro #4
Fluconazole-R #2
(CaCi-2)
SN250/Wild-Type
SN152
ATCC
Noble et al, 2005
IRO1/irolA::imm434
BV06/erg2A/z
BV07/ erg3J/
BV08/ erg4A/A
BV09/ erg5AI/A
As SN 152,
erg2A::C.d.HIS]/erg2A:C.m.LEU2
As SN 152,
erg3A::C.d.HIS/erg3A:C.m.LEU2
As SN152,
erg4A::C.d.HIS/erg4A:C.m.LEU2
As SN152,
92
This study
This study
This study
This study
BV10/ erg6l/
BV1 1/ erg3A/A
erg] zA/A
erg5A::C.d.HISJ/erg5A:C. m.LEU2
As SN 152,
erg6A::C.d.HISJ/erg6A:C.m.LEU2
As SN152,
erg3A::C.d.HIS/erg3A:C.m.LEU2
This study
This study
erg]JA:C.d.ARG4/ergl lA/C. d.ARG4
BV12 /erg24/z
BV13/ cnbi/zI
BV14/ hogzI/z
BV16/ WT,
ERG2/erg2A
BV17/ cnb z/zA,
ERG2/erg2A
As SN152,
erg24A.C. dHISJ/erg24A:C.m.LEU2
As SN152,
cnblA::C.d.HIS/cnbIA:C.m.LEU2
As SN152,
hogIA::C.d.HIS/hogIA:Cm.LEU2
As SN250,
ERG2erg2A::C.d.ARG4
As SN152,
cnbi A::Cd. HISJ/cnbi A:C.m.LEU2;
This study
This study
Noble et al, 2010
This study
This study
ERG2/erg2A::C.dARG4
BV18/ hogl/A,
ERG2/erg2A
As SN152,
hogIA::C.dHIS/hogIlA:Cm.LEU2;
ERG2/erg2A::C.dARG4
Table S1
Strains used in this study. List of strains used in this study.
93
This study
Name
Use
Sequence
SN-2
bridging primer for all
deletions
ccgctgctaggcgcgccgtgACCAGTGTGATGGATATCTGC
ERG2-del-upF
bridging primer for all
deletions
Upstream deletion
fragment
ERG2-del-upR
Upstream deletion
fragment
SN-5
ERG3-del-upF
Downstream deletion
fragment
Downstream deletion
fragment
Check insertion of
deletion
checking loss of
endogenous gene
checking loss of
endogenous gene
Upstream deletion
fragment
ERG3-del-upR
Upstream deletion
fragment
ERG2-del-dnF
ERG2-del-dnR
ERG2-delupcheck
ERG2intcheck-F
ERG2intcheck-R
ERG4-del-upF
Downstream deletion
fragment
Downstream deletion
fragment
Check insertion of
deletion
checking loss of
endogenous gene
checking loss of
endogenous gene
Upstream deletion
fragment
ERG4-del-upR
Upstream deletion
fragment
ERG3-del-dnF
ERG3-del-dnR
ERG3-delupcheck
ERG3intcheck-F
ERG3intcheck-R
gcagggatgcggccgctgacAGCTCGGATCCACTAGTAACG
AGTAATGGTTGCTCCGGTTG
CACGGCGCGCCTAGCAGCGGTGATACGTTTCGGTTCTCTGTC
GTCAGCGGCCGCATCCCTGCTAATAGAGGGGGCACGGATT
ATCCATGGCAACAAATGAAA
AATCGTATCCCTTCGTCGTC
CCACGGCCATTATGATTGAT
GATTGATCCAGTACCAGCATGA
CCTTTGCATTTGCTAAAATCTG
CACGGCGCGCCTAGCAGCGGAAAATTGGCTAAACCGAATCC
GTCAGCGGCCGCATCCCTGCCATCTTTGTTTTGGACCATTGA
CAAAGCCAACCAGATCATCA
TGTGCAGGAATGCAATTGTT
GATTTCCCAAGTCTCCCAAA
GGAAGAACCCATCAACTGGA
AAT GAC GTC TCC CCC TCT TT
CAC GGC GCG CCT AGC AGC GGT GTT GAT GGT TGT AAA ATG AAA AA
94
ERG5-del-upF
Downstream deletion
fragment
Downstream deletion
fragment
Check insertion of
deletion
checking loss of
endogenous gene
checking loss of
endogenous gene
Upstream deletion
fragment
ERG5-del-upR
Upstream deletion
fragment
ERG4-del-dnF
ERG4-del-dnR
ERG4-delupcheck
ERG4intcheck-F
ERG4intcheck-R
ERG6-del-upF
Downstream deletion
fragment
Downstream deletion
fragment
Check insertion of
deletion
checking loss of
endogenous gene
checking loss of
endogenous gene
Upstream deletion
fragment
ERG6-del-upR
Upstream deletion
fragment
ERG5-del-dnF
ERG5-del-dnR
ERG5-delupcheck
ERG5intcheck-F
ERG5intcheck-R
ERG6-del-dnR
ERG6-delupcheck
ERG6intcheck-F
ERG6intcheck-R
ERG 1-delupF
Downstream deletion
fragment
Downstream deletion
fragment
Check insertion of
deletion
checking loss of
endogenous gene
checking loss of
endogenous gene
Upstream deletion
fragment
ERGL 1-delupR
Upstream deletion
fragment
ERG 1-deldnF
ERGI1-deldnR
ERGL 1-delupcheck
ERGI 1intcheck-F
ERGI1intcheck-R
ERG24-delupF
Downstream deletion
fragment
Downstream deletion
fragment
Check insertion of
deletion
checking loss of
endogenous gene
checking loss of
endogenous gene
Upstream deletion
fragment
ERG24-delupR
Upstream deletion
fragment
ERG24-deldnF
ERG24-deldnR
Downstream deletion
fragment
Downstream deletion
fragment
ERG6-del-dnF
GTC AGC GGC CGC ATC CCT GCT TGC TAA GCT ACA TAA TAT CAA AAT
CA
CCA CTG GTC CAA AAC AAC AA
CCA CTG GTC CAA AAC AAC AA
GCT GGG GTT CCA TTC AGT TA
AAT GGA GAT CCA AAC CCA CA
GCAACGCGTAACAACACACT
CACGGCGCGCCTAGCAGCGGAAAGTGAATGTTTTGTGGTTTTTG
GTCAGCGGCCGCATCCCTGCGAGGGAAATAAGTAGGTCAAAACAA
CGAGGTTGCTCCATCAATTT
GTTCCGGTTGGATCGAATTA
TGCTTCATCTCGTGATTTGG
AATGATCGGGAAATTGACCA
CGATGGCCGTAGTTCTTACA
CACGGCGCGCCTAGCAGCGGGAAAATGAAAGGTTCTTTAACTTGA
GTCAGCGGCCGCATCCCTGCGGGGCTTGACAAACAACAAG
GCTAAGCCCATTTTCAGTGG
CCGAAATATCATTGGGAACG
GTGGTGTAGGTGGTCCTGGT
GTTGTTTGTCAAGCCCCATT
TTTGAGAACAGCCACACGAC
CACGGCGCGCCTAGCAGCGGTGAGTTATGATCTTCTTGAAAAGAAAC
GTCACCGGCCGCATCCCTGCCGGCAACTTTCTTTCGATTC
AGCAGATGATGCTGGAACCT
GCCGGGAATAACTGAGAAAA
TTTGACCGTTCATTTGCTCA
TAGCTTTGGCAGCAGCAGTA
TTGAACCCCATCCAAATTGT
CACGGCGCGCCTAGCAGCGGAACTTTGGTTAGTTATTTTGTGGAAA
GTCAGCGGCCGCATCCCTGCAAAACAAACAAGGAAAGCGAAT
AAATGATGCCGAAATCGAAC
95
ERG24-delupcheck
ERG24intcheck-F
ERG24intcheck-R
CNBl-del-upF
CNB1-delupR
Check insertion of
deletion
checking loss of
endogenous gene
checking loss of
endogenous gene
Upstream deletion
fragment
Upstream deletion
fragment
CNBl-del-dnF
Downstream deletion
fragment
CNBl-deldnR
CNB1-delupcheck
CNB1intcheck-F
CNB1intcheck-R
Downstream deletion
fragment
Check insertion of
deletion
checking loss of
endogenous gene
checking loss of
endogenous gene
YHB1-RT-F
RTPCR
AAGTTGCTCCTCCTGCTGGTAAT
YHB1-RT-R
RTPCR
TCCTTGTCTGTAGCTGGGTCATAGA
RBT5-RT-F
RTPCR
GTTCTGTTGAATCTTTGGCCAC
RBT5-RT-R
RTPCR
AGCCTTAGCCAAAGCGTCA
FET34-RT-F
RTPCR
ATGAGGAGGAGGAAGAACAAAGC
FET34-RT-R
RTPCR
GGGCTAGAAGAGGAACCAGTTG
FTRT-RT-F
RTPCR
TATCATTGCCACGGTCTTGA
FTR1-RT-R
RTPCR
GGACCAGAACCGTTTTCAGA
FTH1-RT-F
RTPCR
CCCAACTGAATCTGATGACCTTAC
FTHI-RT-R
RTPCR
GCCAACACAGCAGCATTTAC
SITI-RT-F
RTPCR
CTGCCAGTCAACCCGTCTATG
SIT1-RT-R
RTPCR
AACTCCAAACGACCAAGGACAT
CRHI-RT-F
RTPCR
CCAGTTCTTCATCCAGCTCA
CRH II-RT-R
RTPCR
CCAATCAATGCAACAAAGCC
DDR48-RT-F
RTPCR
TTCGGTAAAGACGACGACAAAGA
DDR48-RT-R
RTPCR
GCCAAATGAAGAGGATCCATAAGA
STP4-RT-F
RTPCR
TCCTTTCAAGAACATCGATTCA
STP4-RT-R
RTPCR
TTATGCATCCAATCATCGACA
CAT1-RT-F
RTPCR
TTG GTC AAC ACG GTC CAT T
CAT1-RT-R
RTPCR
CCA TAA GCA CCG GAA CCT T
GPX1-RT-F
RTPCR
ATG GCA AGA ACC AGG CAC TA
GPX1-RT-R
RTPCR
CTG GAT CTG CTT GTT CAC CA
UTR2-RT-F
RTPCR
CCATGCTGTTCTCAATTTGGTA
UTR2-RT-R
RTPCR
TCTAGGCATAGGCATACAAGCA
RTA2-RT-F
RTPCR
AAG AGC CAC ACA AGC GAT TT
RTA2-RT-R
ECM331-RTF
ECM331 -RTR
RTPCR
TCC CGT GAA TAA CCA CCA AT
RTPCR
GAGTATTCTCAACTCCCCATCG
RTPCR
AAATCGCAAAGGAAGGTAATGA
TEF3-RT-F
RTPCR
CCA CTG AAG TCA AGT CCG TTG A
TEF3-RT-R
RTPCR
CAC CTT CAG CCA ATT GTT CGT
AAGGTTTCCAGACATTCCACA
TGGCTGGCCTATTTAGCTTG
ACGACCAATGAACCAATCGT
TTCTGGTGGCTCATTCTTTG
GGGAAGAGAATTGAACAGGTTG
CACGGCGCGCCTAGCAGCGGTTGTCAGTAGTTGTTGAAGTCGAA
GTCAGCGGCCGCATCCCTGCCAACAGTTTTGGTTTAGTTCATGT
TTGTTGAGCAGGTTGCTGTC
TAAAGATGGGTCAGGGCAAA
CGTCCAAATCAGCTTCCATT
96
ACT1-RT-F
RTPCR
GCT TTT GGT GTT TGA CGA GTT TCT
ACT1-RT-R
RTPCR
GTG AGC CGG GAA ATC TGT ATA GTC
TDH3-RT-F
RTPCR
ATC CCA CAA GGA CTG GAG A
TDH3-RT-R
RTPCR
GCA GAA GCT TTA GCA ACG TG
RPP2B-RT-F
RTPCR
ACT TAG CTG CTT ACT TAT TGT TAG
Table S2
Oligonucleotide sequences used in this study. Oligonucleotide primer sequences used in
the generation of mutants and RT-PCR experiments.
97
Chapter Three:
Non-Toxic Antimicrobials that Evade
Resistance
*This chapter has been submitted for publication and is under peer review
**My contribution included designing and performing the experiments included in Figure
4, as well as writing the text describing these experiments and portions of the abstract and
discussion
98
Non-toxic antimicrobials that evade drug resistance
Stephen A. Davis', Benjamin M. Vincent 2,3, Matthew M. Endo', Luke Whitesell 3 , Karen
Marchillo, David R. Andes5 , Susan Lindquist 3,4 *, and Martin D. BurkeI*
1Howard Hughes Medical Institute, Roger Adam Laboratory, Department of Chemistry,
University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States.
2Microbiology Graduate Program, Massachusetts Institute of Technology,
Cambridge,
Massachusetts, United States of America 3 Whitehead Institute for Biomedical Research,
Cambridge, Massachusetts, United States of America, 4 Howards Hughes Medical
Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts, United States of America 5Departments of Medicine and Medical
Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin, United
States. *To whom correspondence should be addressed
ABSTRACT
Drugs that act more promiscuously provide fewer routes for the emergence of resistant
mutants. But this benefit often comes at the cost of serious off-target and dose-limiting
toxicities. The classic example is the antifungal amphotericin B (AmB), which has
evaded resistance for more than half a century. We report dramatically less toxic
amphotericins that nevertheless evade resistance. They are scalably accessed in just three
steps from the natural product, and bind their target (the fungal sterol, ergosterol) with far
greater selectivity than AmB. Hence, they are less toxic and far more effective in a mouse
model of systemic candidiasis. Surprisingly, exhaustive efforts to select for mutants
resistant to these more selective compounds revealed that they are just as impervious to
resistance as AmB. Thus, highly selective cytocidal action and the evasion of resistance
are not mutually exclusive, suggesting practical routes to the discovery of less toxic,
resistance-evasive therapies.
Introduction
Less selective pharmacological action is generally associated with decreased
vulnerability to resistance, but also with increased toxicity" 2 . The classic example is
amphotericin B (AmB), an exceptionally resistance-evasive but also highly toxic
antifungal agent that has remained the last line of defense in treating invasive fungal
infections for over half a century 3 . An excess of 1.5 million people die from such
99
I
infections each year 4, in large part because the extreme toxicity of AmB is dose-limiting'.
Extensive efforts to develop a clinically viable less toxic amphotericin have been made,
but without success5 . Moreover, it has remained unclear whether such a decrease in
toxicity would come at the cost of an increase in vulnerability to pathogen resistance.
For decades the pursuit of a less toxic amphotericin was guided by the widely
accepted model in which AmB (Fig. la) kills cells via ion channel-mediated membrane
permeabilization5 ' 6 . This model suggests that improving the therapeutic index of this drug
requires the selective self-assembly of oligomeric ion channels in yeast vs. human cells, a
problem that has been very challenging to approach rationally. Contrary to this model, it
was recently shown that AmB primarily exists as a large extramembranous aggregate
which kills yeast by simply binding 7 and extracting8 ergosterol, and likely kills human
cells by similarly binding cholesterol 9 . Ergosterol is critical for many different aspects of
yeast physiology 0-14 , and mutations that alter sterol biosynthesis in a manner that confers
resistance abrogate fungal virulence15 , explaining the failure of fungi to evolve AmB
resistance in the clinic. This new sterol sponge model enabled efforts to improve the
therapeutic index of AmB to focus on the simpler problem of selectively binding sterols,
and this yielded the recent discovery of a new derivative, C2'deoxyAmB (C2'deOAmB)
(Fig. la) that binds ergosterol but not cholesterol and is toxic to yeast but not human
cells9 . Limited synthetic access to this derivative, however, has hindered its further
development and the determination of whether this improvement in therapeutic index is
coupled to a decreased capacity to evade resistance.
To rationalize the greater ergosterol-selective binding observed with C2'deOAmB we
took into consideration several new structural insights regarding these prototypical small
100
molecule-small molecule interactions. First, the mycosamine appendage is critical for
binding both ergosterol and cholesterol1 6 . Recent solid-state NMR evidence also confirms
direct contact between the A and B rings of ergosterol and the AmB polyene motif in the
sterol sponge complex8 . Furthermore, a recent crystal structure of an AmB derivative1 7
(Fig. Ic) reveals a water-bridged hydrogen bond between the C2' and the C13 hydroxyl
groups. We propose that such intramolecular hydrogen bonding fixes the relative
positions of the mycosamine appendage and the polyene motif enabling AmB to bind
both ergosterol and cholesterol, and that deletion of the C2' hydroxyl group favors a shift
to an alternate conformer that selectively binds ergosterol. Alternatively stated, this
model predicts that a protein-like' 8 -22 ligand-selective allosteric effect underlies these
small molecule-small molecule interactions. Guided by this model, and further
encouraged by previous reports of modest but promising improvements in therapeutic
index 5, we pursued more synthetically accessible disruptions of a putative similarly
ground state-stabilizing intramolecular salt bridge between the C41 carboxylate and C3'
ammonium ions (Fig. la).
RESULTS
Three-step synthesis of AmB ureas
All previously reported derivatives of AmB have maintained a C I6-C41 carboncarbon bond5 . Enabling us to explore a new chemotype, we discovered that treatment of a
minimally protected variant of AmB (1) with diphenyl phosphoryl azide (DPPA) cleanly
promotes a stereospecific Curtius rearrangement in which the C16-C41 bond is cleaved
and the resulting isocyanate is intramolecularly trapped by the neighboring C15 alcohol23
101
to form an oxazolidinone 2 (Fig. ld). This particular oxazolidinone, in turn, is
surprisingly reactive to ring-opening with primary amines under mild conditions to yield
a new class of urea containing amphotericins (3) having a C16-nitrogen bond.
Interestingly, the parent heterocycle, 2-oxazolidinone, is unreactive under the same
conditions.
We further found that 1 can be directly converted to 3 in a scalable one-pot operation
involving serial addition of DPPA, an amine, and aqueous acid. Starting with 1 g of
fermented AmB and using methyl amine as the nucleophile, this overall three-step
sequence yields 264 mg of AmB methyl urea (AmBMU) (Fig. le). Employing ethylene
diamine produces 236 mg of AmB amino urea (AmBAU), and in a four step-variant,
reaction with P-alanine allylester followed by deallylation yields 124 mg AmB
carboxylatoethyl urea (AmBCU). This chemistry thus provides rapid, efficient, and
scalable access to these new derivatives starting with the natural product that is already
fermented on the metric ton scale.
AmB ureas are selectively toxic to yeast
With this new series of AmB derivatives in hand, we first determined their sterol
binding properties via isothermal titration calorimetry (ITC) (Fig. 2a)9 . AmB binds both
ergosterol and cholesterol, but the aglycone, amphoteronolide B (AmdeB), binds neither
sterol 16 . Like C2'deOAmB 9, all of the new C16 urea-containing derivatives retain the
capacity to bind ergosterol but, within the limits of detection of this experiment, show no
binding to cholesterol.
This sterol binding selectivity translated into a major improvement in therapeutic
index in vitro (Fig. 2b). Specifically, we determined the minimum inhibitory
102
concentration (MIC) against Saccharomyces cerevisiae and the minimum hemolytic
concentration (MHC) against human red blood cells for AmB, a series of previously
reported C41 and/or C3'-modified derivatives5 , and the new AmB ureas. AmB is a potent
antifungal (MIC 0.5 gM), but is also highly toxic to human red blood cells (MHC 8.6
RM) (entry 1). AmdeB is non-toxic to both (entry 2)9,24. Previously reported
modifications including methyl esterification (AmBME)25,26, carboxylic acid reduction
(C41MeAmB) 24 , methyl amidation (AmBMA) 27, and double modification to form a
triazoloethyl
amide
bis-aminopropyl
derivative
(AmBTABA) 28 produce modest
improvements in therapeutic index (entries 3-6), with the best results obtained with
AmBTABA (MIC 0.25 pM, MHC 49 pM) (entry 6). In contrast, all of the AmB urea
derivatives retained antifungal activity but show dramatically reduced toxicity to human
red blood cells. The MHC of AmBMU and AmBAU exceeds the limits of solubility in
this assay (500 pM) (entries 7,8). Remarkably, the only structural difference between
AmBMA (entry 5) and AmBMU (entry 7) is the insertion of a protonated nitrogen atom
between the C16 and C41 carbons (Fig. la,c).
The activities of the AmB urea derivatives were further tested against a series of
pathogenic Candida strains (Fig. 2c) 29 . Both AmBMU and AmBAU demonstrate potent
antifungal activity against all strains tested. AmBCU retained activity, but was in general
somewhat less potent. The compounds were also tested for toxicity against human renal
proximal tubule epithelial cells (RPTEC), the critical site of toxicity for AmB in patients.
They all showed little or no toxicity to hTERT1 RPTEC 30 , and substantially reduced
.
toxicity to the more sensitive primary RPTEC 31
103
AmB ureas are more efficacious and less toxic in mice
Based on all of these results, AmBMU and AmBAU were judged to be especially
promising and thus further evaluated for efficacy and toxicity in vivo (Fig. 3a-d)32 . In a
mouse model of disseminated candidiasis both AmBMU and AmBAU were substantially
more effective than AmB at reducing fungal burden in the kidneys at all three tested
doses (1, 4 and 16 mg/kg, intraperitoneal injection). The differences in efficacy were
most pronounced at the 16 mg/kg dose at 24 hours post inoculation. Relative to AmB,
AmBMU reduced the fungal burden by 1.2 log units (p < 0.0001), and AmBAU reduced
the fungal burden by nearly 3 log units (p < 0.0001). We speculate that an improved
pharmacological profile, potentially due to a >20 fold increase in water solubility relative
to AmB, may contribute to this unexpected dramatic improvement in antifungal activity
for the new compounds in vivo.
Acute mouse toxicity was determined by single intravenous administration of AmB
or its derivatives to healthy, uninfected mice and monitoring for lethality (Fig. 3d). All
mice in the 4 mg/kg AmB dosage group died within seconds. AmBAU was drastically
less toxic with >50% lethality only observed at the 64 mg/kg dosage group. Strikingly, all
mice dosed with even 64 mg/kg AmBMU survived with no observable toxicity.
AmB ureas still evade resistance
We next investigated the impacts of increased ergosterol selectivity on the
development of resistance to these analogs. Due to its unique mode of action7, AmB is
not susceptible to most of the common mechanisms of resistance to other antimicrobials.
Its lipid target is not as readily mutable as proteins or RNA; it is unaffected by efflux
pumps; and its polyene macrolide structure is not a substrate for secretion via drug-
104
detoxifying enzymes. Moreover, ergosterol plays a central role in many aspects of yeast
physiology' 0 ~4 . Mutations in genes involved in ergosterol biosynthesis can change sterol
structures in ways that confer AmB resistance in vitro34, however, these mutations have
enormous fitness costs in vivo, crippling fungal virulence". Consequently, resistance
rarely, if ever appears in the clinic35 . We asked if the improved sterol selectivity of
AmBMU and AmBAU rendered them more vulnerable to the evolution of resistance.
We first determined the MIC of AmB, AmBMU and AmBAU against a panel of labgenerated strains carrying mutations in all seven of the non-essential C. albicans latestage ergosterol biosynthesis genes (Fig. 4a)'. Surprisingly, AmBMU and AmBAU
displayed an in vitro resistance profile that was very similar to AmB (Fig. 4a). As for
AmB, only erg2, erg6 or erg3/ergll mutants showed substantial resistance to AmBMU
and AmBAU, and all of these mutants are known to be avirulent15 . Thus, known
ergosterol biosynthesis mutations do not appear to be a threat to the efficacy of AmBAU
and AmBMU.
We next used exhaustive unbiased selections for survival in the presence of the
compounds to ask if any other mutations could confer resistance to AmBMU or AmBAU.
One-step selections on plates containing 8X the MIC of AmB, AmBMU or AmBAU
yielded no colonies with stable resistance to any of the drugs, even after ethyl
methanesulfonate mutagenesis. We then utilized a gradual resistance-selection protocol in
liquid culture, with serial two-fold increases in drug concentration. Most serial selections
ended in extinction of the lineage. However, we did recover 5-8 mutants for each drug
that exhibited a ;>4-fold increase in MIC. Importantly, all of these substantially resistant
105
mutants were cross-resistant to all three compounds, suggesting no new mechanisms of
resistance unique to AmBMU or AmBAU (Supplementary table I).
To identify the mutations responsible for resistance in these selections, we used
whole genome sequencing of the WT strain as well as all 11 of the in vitro-evolved
resistant mutants, including both strongly and weakly-resistant isolates (Supplementary
table 1). Most mutants with strong resistance to AmB or the derivatives contained
mutations in the ERG2 or ERG6 locus that subsequently underwent a loss of
heterozygosity (Fig. 4b)'. Unexpectedly, we also identified 3 independent mutants with
low-level (~2-fold)
AmB resistance mediated through a recurrent substitution in
ORF19.7285 (D216Y), an uncharacterized WD40 repeat protein conserved across fungi.
However, these mutants were no more resistant to AmBMU and AmBAU than to AmB
(Supplementary table I).
We then asked if any of the mutants with substantial resistance to AmBMU or
AmBAU (>4-fold MIC increase) could elude the dramatic fitness defects previously
demonstrated for AmB-resistance. As previously reported 5 , all AmB-resistant mutants
are extremely sensitive to oxidative stressors, which are continually encountered during
the course of infection. In addition, they become highly dependent on the molecular
chaperone Hsp90, which supports diverse fungal stress responses' . All of the mutants
resistant to AmBMU or AmBAU were likewise severely sensitized to the oxidative
stressor tert-butyl hydrogen peroxide and the Hsp90 inhibitor geldanamycin (Fig. 4c,d).
Previous work has
also
demonstrated
that
AmB-resistance
mutations
disable
filamentation, a key driver of virulence in Candida15 . Again, in response to stimulation
106
with fetal bovine serum at 370 C, all mutants with strong resistance to AmBMU or
AmBAU were unable to form the filaments observed in the wild-type (Fig. 4e).
We next asked if resistance to AmBMU or AmBAU reduces competitive fitness in
vivo. To do this, we infected mice with a pool of strains, consisting of the wild-type
parent (AmBMU and AmBAU-sensitive) and 15 AmBMU or AmBAU-resistant mutants
(with each strain comprising 1/ 1 6 th of the total population). After allowing the infection
to proceed for four days (in the absence of drug treatment), we euthanized the mice and
tested the drug sensitivity of fungal colonies isolated from in the kidneys to determine the
AmBMU and AmBAU-resistant fraction of the final population. Indeed, even over this
short period of infection, the percentage of the surviving population resistant to AmBMU
or AmBAU dropped dramatically and the drug-sensitive parent rapidly overtook the
population (Fig. 4f).
Finally, we tested if any of our AmBMU or AmBAU-resistant mutants retained the
capacity to cause lethal infection. To do so, we inoculated mice with pools of resistant
mutants and compared their survival to mice infected with wild-type strains. At a low
inoculum (to match that of an individual resistant strain) the wild-type strain killed all
infected mice (Fig. 4g). Wild-type strains subjected to the same mutagenesis and in vitro
passaging used to generate the resistant strains also killed all of the mice. In stark contrast,
all mice infected with pools of mutants selected for resistance to AmB, AmBMU or
AmBAU survived the infection (Fig. 4g). Thus, AmBMU and AmBAU are no more
vulnerable to resistance than AmB, which has evaded resistance despite widespread
clinical utilization for over half a century.
DISCUSSION
107
Our findings collectively reveal that selective antimicrobial action and evasion of
resistance are not mutually exclusive. Here, compounds that bind with high selectivity to
a pathogen-specific lipid evade the emergence of virulent resistant strains, suggesting
major costs in fitness are caused by even small changes in the structure of this lipid. This
is likely because ergosterol is a central molecular node 36 in yeast physiology, being
critical for the function of membrane proteins10, endocytosis", vacuole fusion12
membrane compartmentalization", and pheromone signaling
4
Our results further suggest that AmBMU and AmBAU are exceptionally promising
candidates for replacing AmB as a less toxic treatment for invasive fungal infections.
These new derivatives bind ergosterol but not cholesterol, are substantially more effective
and less toxic than AmB in vivo, yet still evade resistance. Moreover, these compounds
are accessed in just three steps from AmB, which is already fermented and commercially
available on the metric ton scale. All of the reagents used in their synthesis are already
employed on the process scale to prepare other pharmaceuticals, including diphenyl
.
phosphoryl azide 37 39
Our results also support a novel ligand-selective allosteric-effect model for guiding
the rational development of other non-toxic amphotericins. This model predicts that
disruption of intramolecular polar interactions between functional groups on the
macrolide core and the mycosamine appendage cause a conformational shift in the
molecule, from one that binds both ergosterol and cholesterol to one that selectively binds
ergosterol. Strikingly, the portion of AmB that contains all of these functional groups,
i.e., the module comprised of C13-C23, is 100% conserved in every member of the
mycosamine-bearing polyene macrolide family of natural products40 . This includes a
108
collection "aromatic polyenes" that are reported to be orders of magnitude more potent
.
than AmB 4 1 and effective against even very challenging to treat Aspergillus infections4 2
Thus, synthesizing the analogous urea derivatives of other polyene macrolide natural
products could lead to ultrapotent and/or broader spectrum yet still minimally toxic new
antifungal agents.
More broadly, our results show that targeting pathogen specific and polyfunctional
lipids represents a promising blueprint for achieving the highly sought combination of
low toxicity and evasion of resistance. Intriguingly, nisin, which binds a bacterial
polyfunctional lipid, lipid II, has similarly evaded resistance despite half a century of use
as a food preservative . It was recently discovered that binding of the same lipid
underlies the action of defensin peptides 44, critical components of innate immunity that
have retained antibiotic activity over more than two billion years of evolution. Other
recent studies increasingly show that specific lipid-transmembrane protein interactions
are critical for diverse cellular functions4 5 . Thus, as new microbe-specific and
polyfunctional lipids are discovered4 6 , they present outstanding targets for the rational
development of other non-toxic yet still resistance-evasive antibiotics.
Methods
General Reaction Conditions
Due to the light and air sensitivity of amphotericin B (AmB), all reactions were
performed in oven or flame dried glassware under an atmosphere of argon under low
light conditions. Compounds were stored under an anaerobic atmosphere. All solvents
were dispensed from a solvent purification as described by Pangborn and coworkers 47
109
(THF, Et2 0: dry neutral alumina; DMSO, DMF, CH 30H : activated molecular sieves).
Triethylamine
and pyridine were freshly
distilled under nitrogen
from CaH2.
Camphorsulfonic acid was recrystallized from ethanol. Water was doubly distilled or
obtained from a Millipore (Billerica, MA) MilliQ water purification system. Reactions
were monitored by RP-HPLC using an Agilent 1200 Series HPLC system equipped with
an Agilent Zorbax Eclipse C 1 8 3.5-pm, 4.6 x 75 mm column with UV detection at 383 nm
at 1.2 mL/min, or an Agilent 6230 ESI TOF LC/MS system equipped with an Agilent
Zorbax Eclipse C18 1.8-pm, 2.1 x 50 mm column with UV detection at 383 nm at 0.4
mL/min. Full experimental details and characterization for new compounds appear in the
Supplementary Information.
Extinction Coefficient Determination
Extinction coefficients (L mol~1 cm') were determined as previously reported
117,000), C41MeAmB
121,000), AmBMU
(6406 =
(6406 =
(6406
164,000), AmdeB
102,000), AmBMA
= 87,000), AmBAU
(6406
(6406
(6406 =
=
102,000), AmBME
114,000), AmBTABA
= 87,000), AmBCU
(6406
(6406
-
and were as follows: AmB
(6406 =
= 58,000).
Isothermal Titration Calorimetry
ITC was performed as previously reported.
Growth Conditions for S. cerevisiae.
S. cerevisiae was maintained with yeast peptone dextrose (YPD) growth media
consisting of 10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, and 20 g/L agar for
110
solid media. The media was sterilized by autoclaving at 250'F for 30 min. Dextrose was
subsequently added as a sterile 40% w/v solution in water (dextrose solutions were filter
sterilized). Solid media was prepared by pouring sterile media containing agar (20 g/L)
onto Coming (Coming, NY) 100 x 20 mm polystyrene plates. Liquid cultures were
incubated at 30'C on a rotary shaker and solid cultures were maintained at 30'C in an
incubator.
Growth Conditions and MIC Assay for C. albicans, C. tropicalis, C. parapsilosis, and
C. glabarata.
The organisms were maintained,
grown,
subcultured, and quantified on
Sabouraud dextrose agar (SDA; Difco Laboratories, Detroit, MI). Twenty-four hours
prior to the study, the organisms were subcultured at 35'C.
Minimum Inhibitory Concentration (MIC) Determination
MIC determinations were performed in duplicate on at least two occasions using
.
the Clinical and Laboratory Standards Institute M27-A3 microbroth methodology 48
Hemolysis Assays
Hemolysis experiments were performed following known procedures9 . Whole
human blood (sodium heparin) was purchased from BioreclamationlVT (Westbury, NY)
and stored at 4'C and used within two days of receipt.
WST-8 Cell Proliferation Assays
111
Primary Renal Proximal Tubule Epithelial Cells Preparation.
Primary human renal proximal tubule epithelial cells (RPTECs) were purchased
from ATCC (PCS-400-010, Manassas, VA) and immediately cultured upon receipt.
Complete growth media was prepared using renal epithelial cell basal medium (ATCC,
PCS-400-030), renal epithelial cell growth kit (ATCC, PCS-400-040), and penicillinstreptomycin (10 units/mL and 10 ug/mL). Complete media was stored at 4'C in the
dark and used within 28 days. Primary RPTECs were grown in CO 2 incubator at 37 'C
.
with an atmosphere of 95% air/5% CO2
TERT1 Renal Proximal Tubule Epithelial Cells Preparation.
TERTI human renal proximal tubule epithelial cells (RPTECs) were purchased
from ATCC (CRL-4031, Manassas, VA) and immediately cultured upon receipt.
Complete growth media was prepared using DMEM:F12 media (ATCC, 30-2006),
triiodo-L-thyronine (Sigma, T6397), recombinant human EGF (Life Technologies,
PHG03 11), ascorbic acid (Sigma, A4403), human transferrin (Sigma, T8158), insulin
(Sigma 19278), prostaglandin El (Sigma, P7527), hydrocortisone (Sigma, H0888),
sodium selenite (Sigma, S5261), and G418 (Sigma, A1720). Complete media was stored
at 4C in the dark and used within 28 days.
TERTI RPTECs were grown in CO 2
.
incubator at 37'C with an atmosphere of 95% air/5% CO 2
WST-8 Reagent Preparation.
WST-8 cell proliferation assay kit (10010199) was purchased from Cayman
Chemical Company (Ann Arbor, MI) and stored at -20'C and used within 6 months of
112
receipt. WST-8 reagent and electron mediator solution were thawed and mixed to prepare
the WST-8 reagent solution. The solution was stored at -20'C and used within one week.
WST-8 Assay.
A suspension of primary or TERTI RPTECs in complete growth media was
brought to a concentration of 1 x 105 cells/mL. A 96-well plate was seeded with 99 pL of
the cell suspension and incubated at 37'C with an atmosphere of 95% air/5% CO 2 for 3
hours. Positive and negative controls were prepared by seeding with 100 [tL of the cell
suspension or 100 pL of the complete media. Compounds were prepared as 5 mM (AmB)
and 8 mM (AmBAU, AmBMU, AmBCU, and AmdeB) stock solutions in DMSO and
serially diluted to the following concentrations with DMSO: 8000, 6000, 4000, 3000,
2000, 1500, 1000, 800, 600, 400, 300, 200, 100, 50, 25, 10, 5, 2.5, 1, 0.5, 0.25, and 0.1
pM. 1 pL aliquots of each solution were added to the 96-well plate in triplicate, with each
column representing a different concentration of the test compound. The 96-well plate
was incubated at 37*C with an atmosphere of 95% air/5% CO 2 for 24 hours. After
incubation, the media was aspirated and 100 pL of serum-free media was added and 10
pL of the WST-8 reagent solution was added to each well. The 96-well plate was mixed
in a shaking incubator at 200 rpm for 1 minute and incubated at 37'C with an atmosphere
of 95% air/5% CO 2 for 2 hours. Following incubation, the 96-well plate was mixed in a
shaking incubator at 200 rpm for 1 minute and absorbances were read at 450 nm using a
Biotek HI Synergy Hybrid Reader (Wanooski, VT). Experiments were performed in
triplicate and the reported cytotoxicity represents an average of three experiments.
113
Data Analysis.
Percent hemolysis was determined according to the following equation:
% cell viability =
Abs
ap-
Abs-ng.
neg. X
xsample
100%
Abs.pos.- Abs-neg.
Concentration vs. percent hemolysis was plotted and fitted to 4-parameter logistic
(4PL) 49 dose response fit using OriginPro 8.6.
The MTC was defined as the
concentration to cause 90% loss of cell viability.
Ethics Statement
All animal procedures were approved by the Institutional Animal Care and Use
Committee at the University of Wisconsin according to the guidelines of the Animal
Welfare Act, The Institute of Laboratory Animal Resources Guide for the Care and Use
of Laboratory Animals, and Public Health Service Policy.
In Vivo Murine Efficacy Study
All studies were approved by the Animal Research Committee of the William S.
Middleton Memorial VA Hospital (Madison, WI). Efficacy was assessed by CFU count
in the kidneys of neutropenic mice with a disseminated fungal infection as described
previously by Andes et a,''0 5 1 . A clinical isolate of Candida albicans (K-1) was grown
and quantified on SDA. For 24 hours prior to infection, the organism was subcultured at
35'C on SDA slants. A 106 CFU/mL inoculum (CFU, colony forming units) was
prepared by placing six fungal colonies into 5 mL of sterile, depyrogenated normal
(0.9%) saline warmed to 35'C. Six-week-old ICR/Swiss specific-pathogen-free female
mice were obtained from Harlan Sprague Dawley (Madison, WI). The mice were
114
weighed (23-27 g) and given intraperitoneal injections of cyclophosphamide to render
neutropenia (defined as <100 polymorphonuclear leukocytes/mm 3). Each mouse was
dosed with 150 mg/kg of cyclophosphamide 4 days prior to infection and 100 mg/kg 1
day before infection. Disseminated candidiasis was induced via tail vein injection of 100
tL of inoculum. AmB, AmBAU, or AmBMU were reconstituted with 1.0 mL of 5%
dextrose. Each animal in the treatment group was given a single 200 pL intraperitoneal
(ip) injection of reconstituted AmB, AmBAU, or AmBMU 2 hours post-infection. Doses
were calculated in terms of mg of compound/kg of body weight. At each time point (6,
12, and 24 hours post-infection), three animals per experimental condition were
sacrificed by CO 2 asphyxiation. The kidneys from each animal were removed and
homogenized. The homogenate was diluted serially 10-fold with 9% saline and plated on
SDA. The plates were then incubated for 24 hours at 35'C and inspected for CFU viable
counts. The lower limit of detection for this technique is 100 CFU/mL. All results are
expressed as the mean logio CFU per kidney for three animals.
In Vivo Murine Toxicity Study
All studies were approved by the Animal Research Committee of the William S.
Middleton Memorial VA Hospital (Madison, WI). Uninfected Swiss ICR mice were used
for assessment of infusion toxicity. Groups of five mice were treated with single
intravenous doses of AmB, AmBAU, AmBMU (reconstituted with 1.0 mL of 5%
dextrose), or sterile pyrogen-free 0.85% NaCl administered via the lateral tail vein over
30 seconds. Dose levels studies included 0.5, 1, 2, 4, 8, 16, 32, and 64 mg/kg. Following
administration mice were observed continuously for one hour and then every 6 hours up
115
to 24 hours for signs of distress or death.
Resistance Studies
Minimum Inhibitory Concentration and Growth Assays
Susceptibility of wild-type and resistant strains to AmB, AmBAU, AmBMU, tertbutyl peroxide (Sigma-Aldrich), geldanamycin and radicicol (A.G. Scientific) was
determined in flat bottom, 96-well microtiter plates (Costar) using a broth microdilution
protocol adapted from CLSI M27-A3. Overnight cultures (14-20hr) were grown at 30'C
in YPD, and approximately 5x10 3 cells were seeded per well. For AmB, AmBAU, and
AmBMU, MIC assays were performed at 37'C in RPMI buffered with MOPS (0.165M)
with 10% fetal bovine
serum (Sigma-Aldrich)
added; for tert-butyl peroxide,
geldanamycin, and radicicol, MIC's were determined in YPD at 30'C. MIC's were
determined after 24h incubation as the concentration of compound resulting in no visible
growth in wells.
For quantitative display of growth at drug dilutions, OD 60 0 was
measured in a spectrophotometer (Tecan) and displayed as heat maps using Java
TreeView 1.1.3 (http://jtreeview.sourceforge.net).
Media and Growth Conditions
C. albicanswas generally grown and maintained as described previously". Stocks
were stored in 15% glycerol at -80*C; strains were generally grown in YPD media at
30'C. Drugs were added directly to media from DMSO stocks.
116
In Vitro Gradual Selection of AmB, AmBAU, or AmBMU Resistance
Selection of resistance to AmB, AmBAU, and AmBMU was performed as
follows. 1 mL overnight (14-20hr) cultures of SC5314 (WT) were washed in PBS, then
treated with 3% EMS (Ethyl Methanesulfonate, Sigma-Aldrich) for 45 min. Cells were
then washed 4x in YPD and resuspended in YPD and allowed to recover for 3h. Cells
were then inoculated to an OD600 of approximately 0.025-0.05 in 10OmL YPD
containing 0.25ptM AmB or AmB-AU, or 0.375pM AmB-MU. After 24-72 hours, a lmL
aliquot was removed from any culture that had grown to saturation and subjected to
another round of mutagenesis in the same manner as described above. After recovery,
cells were then inoculated into a new YPD flask containing 2x higher concentration of
the same drug. Cultures that grew were subjected to one more round of EMS mutagenesis
before inoculating into a 2-fold higher drug concentration (total of 3 rounds of EMS
mutagenesis) and then passaged at 2-fold higher increments of drug concentration until
reaching 2pM AmB or AmB-AU, or 3gM AmB-MU. Cultures were passaged once more
at 2pM AmB or AmBAU or 3pM AmBMU, then plated onto YPD media and frozen in
glycerol stocks before further evaluation.
Filamentation Assay
Hyphal induction was performed by growing C. albicansovernight at 30'C in
YPD, washing in PBS, and diluting 1:100 into RPMI+10% fetal bovine serum (SigmaAldrich) at 37'C in a culture tube on a rotating wheel. After 3h, cultures were washed in
PBS and resuspended in 250gg/mL Calcofluor white in a microcentrifuge tube, and
shaken at 30'C for 10 min. Cells were then washed twice in PBS, concentrated 10-fold,
117
briefly sonicated in a water bath, and mounted on slides for visualization under a DAPI
filter set at 60X magnification.
Murine Model of Systemic Infection
All animal protocols were performed in accordance with the Guide for the Care
and Use of Laboratory Animals of the National Institutes of Health. Animals were
maintained according to the guidelines of the MIT Committee on Animal Care (CAC).
These studies were approved by the MIT CAC (protocol #0312-024-15). We used 7-12week-old female Balb/c mice ordered from Taconic farms for all mouse virulence
studies. All strains were prepared for inoculation by diluting overnight cultures (14-20h)
1:100 into YPD and growing into log phase for 4-5 hours, then washing 3x in PBS
before. Strains were injected into the lateral tail vein in a volume of 100 pl. For mouse
survival experiments, strains were grouped as follows: The wild-type Mutagenized pool
consisted of 5 SC5314 colonies subjected in parallel to mutagenesis and passaging (as
described above) without drug exposure, injected as of 1.6x10^5 cfu per strain (8x10^5
cfu total inoculum per mouse); the Wild-type low inoculum was the SC5314 parental
strain injected at 1.6x10A5 cfu.
AmB-Resistant, AmBAU-Resistant, and AmBMU-
resistant pools were comprised of strains isolated from each selection in the presence of
the indicated drug, using strains that exhibited >4-fold MIC increase for the drug used.
Individual resistant strains were present in the pools at 1.6x1 0 A5 cfu per mouse (8x 1 0 A5
total inoculum per mouse when pooled). Each strain or pool of strains was tested in at
least two independent experiments, and data were pooled. Mice were weighed daily and
monitored for signs of morbidity and sacrificed when body weight decreased by 20%, or
118
when signs of extreme distress were apparent.
For the competitive infection with
quantification of kidney burden, a pool comprised of 16 strains at equal fraction of the
population, one SC5314 wild-type and 5 strains each from selections for resistance to
AmB, AmBAU, and AmBMU was used, with 3x1OA4 cells of each strain inoculated per
mouse (4.8x10A5 total inoculum). Three mice were used per experiment, in a total of two
experiments.
4d after infection, mice were sacrificed and kidneys were removed
aseptically, homogenized, and plated onto YPD plates.
Pools of the inoculum
immediately before injection were also plated. 184 colonies were randomly selected from
the pre-infection and 184 from the post-infection plates and tested for growth in 96-well
plates in the presence of 1 pM AmBAU or 1.5 gM AmBMU, and the fraction of wells
from the pre and post-infection pools exhibiting growth in either drug was determined.
Whole Genome Sequencing, Alignment, Mapping, and Variant Calling
.
Whole genome sequencing and analysis was performed as previously described 5
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Acknowledgements Portions of this work were supported by the National Institutes of
Health (R01GM080436, R01GM080436-S), Howard Hughes Medical Institute (HHMI),
and The Mathers Foundation. M.D.B is an HHMI Early Career Scientist and S.L. is an
HHMI Investigator.
Author Contributions S.A.D., B.M.V., L.W., D.R.A., S.L., and M.D.B. conceived the
study and designed the experiments. S.A.D., B.M.V., M.M.E., and K.M. performed the
experiments. S.A.D., B.M.V., S.L., and M.D.B. wrote the manuscript.
Author
Information
www.nature.com/reprints.
Reprints
and
permissions
Correspondence
information
and requests
is
available
at
for materials should be
addressed to M.D.B. (mdburkegillinois.edu) or S.L. (lindquist@wi.mit.edu).
Competing Financial Interest The University of Illinois has filed patents on
amphotericin ureas.
123
a
OH
/
13
OH OH
"Me
e
'cIntramolecular
OH OH 0,,,
0
1
Me,
Me
41 C0 2
putative
j
R
salt bridge
NH 3
2'
0
R= OH amphotericin B (AmB)
H C2'deoxy AmB (C2deOAmB)
HO
HH
N
.OH
ergosterol
Me,.
Me
mycosamine
131A
Me
d
OH
O-
O,,
_
H
cholesterol
OH
N>2
OH
OHOH
O,
,OH
0
N.Me
NN'
H
H
1. FmocONSuccinimide
0
O
AmBMU
OH
'~--Me
OH
NH 2
OH
264 mg
64% average yield per step
2. CSA, MeOH
3. DPPA, Et3N;
H2NCH 2CH 2 NH2 ;
HCO 2H, H 20
OH
OH OH 0,.N
o-
OH
,OH
~O
N
H
H
-- Me
OH NH2
AmBAU
OH
236 mg
61% average yield per step
_NH2
H
OHNH
OH
1. FmocONSuccinimide
2. CSA, MeOH
3. DPPA, Et3N;
P-Alanine Allylester;
HCO 2 H, H 20
4. Thlosallcyllc Acid,
Pd(PPh3 )4
ig
OH
N' R
H
N
3
AmB
1. FmocONSuccinlm
2. CSA, MeOH
3. DPPA, Et3N;
H2NMe;
HCO 2H, H 20
.1OH
0,
H20OH
NH
2R
H AH
H', H 20
\5ONHFmoc
I
OH
2
OH
Me
Me
OMe
MSOH
,OH
_,,H
H
HO
OMe
Me
Me
Me
r,
H
me
/
polyene
Me
,
/ //
.,OH
HO:
M
HO
b
HO
0
Me,,
OH OH 0,,
,OH
OO
0
N
H
0
N
H
OH
OOH
AmBCU
NH 2
Kr
124 mgOH
58% average yield per step
Figure 1: Synthesis of AmB urea derivatives a. Chemical structures of AmB and
C2'deOAmB. b. Chemical structures of ergosterol and cholesterol c. X-ray crystal
structure of N-iodoacylAmB showing an intramolecular water bridged hydrogen bond
between the C2' and C-13 hydroxyl groups. d. General scheme for synthesis of AmB
ureas. e. Synthesis of AmBMU, AmBAU and AmBCU from AmB in only 3 or 4 steps.
124
a
b
Ergosterol Bindng
-10
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i
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AmBMU
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7
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OH
R
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mycosamine
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mycosamine
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0.25
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NH2
nycosamine
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mycosamine
OH
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R'
OH
1
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M
AMBMEA
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sterol-f roe LUVs
E cholest eroI-containing LUVs
-25
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C
3
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C.
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S. creWsiae red blood cells
.
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Figure 2: Sterol binding, antifungal, and human cell toxicity of AmB ureas a. Total
net isotherms from ITC showing AmB or derivatives binding to ergosterol (top) or
SD. *,
cholesterol (bottom). Values represent the mean of at least three experiments
(MIC)
concentration
inhibitory
Minimum
b.
significant.
not
p:0.05, **, p<0.001; NS,
90%
causing
(MHC)
against S. cerevisiae and minimum hemolytic concentration
hemolysis against human red blood cells. c. MIC against clinically relevant yeast
pathogens (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis) and minimum toxic
concentration (MTC) causing 90% toxicity against human hTERT 1 renal proximal tubule
epithelial cells (RPTEC) and human primary RPTECs.
125
C
b
a
I mgfkg
I)
L
16 ntkg
4 mrg
+Contmi
7.0-
--
6.5-
+AmBMU
6.0-
-- AmBAU
AmB
5.55.04.5.
4.03.5
I
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12
18
6
24 0
Tism (houn)
d
U
12
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24 0
6
12
Tis (hours)
18
24
Drug Lettialy by Does
100
so-
i6
a
-e- AmB
so-
-J
a-
AmBAU
-
C
+
40-
AmBMU
20-
u~w
l'6
32
4
64
Dose (mg")
Figure 3: Efficacy and toxicity of AmB ureas in mice. Quantification of the fungal
burden in the kidneys of C. albicansinfected neutropenic mice 2, 6, 12 and 24 hours after
a single IP injection of AmB, AmBMU or AmBAU at dosages of a. 1 mg/kg b. 4 mg/kg
or c. 16 mg/kg. P-values are relative to AmB at each indicated time point, *, p<.05, **,
p<0.001, ***, p<0.0001, d. Dose response toxicity assessed via determination of lethality
upon single IV injection of AmB, AmBMU and AmBAU to healthy mice at doses of
ranging from 0.5 to 64 mg/kg (5 mice per dosage). Mice were monitored for survival up
to one day.
126
a
AmBAU
AmBMU
AmB
0.00
WT
0.17
-
erg2A
erg3AA
0.33
erg4AA
0.50
0.67
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rg6IA
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00
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1
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0
12
8
4
dAMBMU MIC (pM)
AmBAU-R#1
16
Geldanamycin Sensitivity
9
I
AmBAU-R#3
AmBAU-R#7
30
20
0
0
0
AmBAU-R#10
Competitive Fitness
CL
C
16
1A00m
-
AmBMU and
AmBAU Resistant
0.8
12
Mbuse Overall Survival: Pooled Infection
g
AmBMU and
AmBAU Sensitive
1.0
8
AmBMU MIC (IM)
ERG2
e
4
-
75-
-
-
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-
50
AmB-Resistant pool
AmBMU-Resistant pool
AmBAU-Resistant pool
WT-Low Inoculum
WT-Mutagenized Pool
0
0.4
CL
25-
0.20*
0.0Injected Pool
In Kidney after 4d
0
2
127
4
8
6
Time (days)
10
12
14
Fig 4: Characterization of mechanisms and costs of resistance to AmB ureas. a.
Activity of AmB, AmBMU and AmBAU against C. albicans ergosterol biosynthesis
mutants. Growth (as judged by ODonn at 24 h) is shown relative to WT with no compound
added. Scale bar indicates relative growth ranging from bright green (equal to WT
growth, 1.00) to black (zero growth, 0.00). b. Complete genome sequencing of parental
wild-type (SC5314) and mutants selected for resistance to AmB, AmBMU or AmBAU.
ERG2 ORF is shown; color coded mutations represent blue=C, red=T, brown=G,
green=A in the mutant strain. c,d. MIC of tert-butyl peroxide and geldanamycin
compared to AmBMU for all resistant isolates selected; each point indicates one or more
isolates. e. Filamentation in response to serum at 37'C. Representative images of wildtype and mutants selected in AmB, AmBMU or AmBAU, (all cross-resistant), stained
with Calcofluor White. Scale bar = 10 Em f. Competitive infection of mice with 16
different strains (1 WT and 15 that are resistanct to AmBMU and AmBAU). 3x10 4 cells
of each strain injected per mouse (4.8x 105 total inoculum). Fraction of pool sensitive or
resistant to AmBMU and AmBAU determined before tail-vein injection and 4d later after
isolation from kidneys. g. Mouse overall survival after tail-vein injection. Mice were
injected with a pool of 5 passaged and mutagenized WT strains each at 1.6x10 5 (8x10 5
total inoculum), 1.6x10 5 cells of parental WT (WT-Low), or pools of 5 resistant mutants
with 1.6x1 05 cells of each mutant.
128
Chapter Four:
A Fungal-Selective Cytochrome be 1
Inhibitor Impairs Virulence and Prevents the
Evolution of Drug Resistance
*This chapter is currently in preparation for submission
129
A Fungal-Selective Cytochrome bcl Inhibitor Impairs Virulence and Prevents the
Evolution of Drug Resistance
Benjamin M. Vincent, Jean-Baptiste Langlois, Raja Srinivas, Alex K. Lancaster, Ruth
Scherz-Shouval, Luke Whitesell, Bruce Tidor, Stephen L. Buchwald, Susan Lindquist
Affiliations
1 Microbiology Graduate Program, Massachusetts Institute of Technology, 77
Massachusetts Avenue, Cambridge, Massachusetts, 02139, United States of America,
2 Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge,
Massachusetts, 02142, United States of America,
3 Department of Chemistry, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, United States
4 Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts, 02139, United States of America
ABSTRACT
To cause disease, a pathogen must adapt to the challenges of its host environment. The
leading fungal pathogen Candidaalbicans readily colonizes nutrient-poor bodily niches,
withstands attack from the immune system, and can tolerate treatment with azole
antifungals, often evolving resistance. To discover agents that block these adaptive
strategies, we screened 300,000 compounds for inhibition of azole tolerance in a drugresistant Candida isolate. We identified a novel indazole-based compound that converts
azoles from fungistatic to fungicidal drugs by selective inhibition of mitochondrial
)
cytochrome bcl. We synthesized >100 derivatives to optimize potency (to 0.4 pM IC 50
and fungal selectivity (>25-fold over human). In addition to reducing azole resistance,
targeting cytochrome be 1 prevented C. albicans from adapting to the nutrient-deprived
macrophage phagosome, and greatly curtailed its virulence in mice. Inhibiting
mitochondrial respiration and restricting metabolic flexibility with this synthetically
tractable chemotype provides an attractive therapeutic strategy to limit both fungal
virulence and drug resistance.
130
INTRODUCTION
Many pathogens have evolved to optimally exploit very particular niches within
the host. But rigid specialization comes with several costs. Specialist pathogens struggle
to adapt to the dramatic differences in nutrient composition, immunological surveillance,
and drug exposure that characterize different host niches.
Because of this, hyper-
specialized pathogens fail to disseminate and colonize new niches, which limits their
reproductive fitness. Furthermore, when perturbations of host physiology cause dramatic
changes in local environmental conditions, pathogen populations that cannot adapt go
extinct. In such circumstances, the adaptive plasticity of generalist pathogens provides
strong evolutionary benefits.
Candida albicans provides a paradigm for the study of a flexible and resilient
pathogen. It is the most common cause of fungal infection in humans worldwide.
In
healthy humans, C. albicans thrives as a harmless commensal of the oral cavity and
gastrointestinal tract [1]. Immunocompetent patients can suffer from recurrent infections
(e.g. diaper rash and vaginal infections), but they typically resolve quickly. Mild to
moderate immunosuppression often leads to more significant C. albicansinfections of the
oropharynx, esophagus, and other superficial sites. In severely immunocompromised
patients, Candida and other fungal pathogens can invade the bloodstream and internal
organs, causing sepsis, multi-organ system failure, and death in up to 40% of patients [2].
Invasive fungal infections are now responsible for over 1.5 million deaths per
year worldwide [3]. The incidence of such infections is increasing due to climate change
and the expansion of human populations into new environments, which exposes them to
131
new pathogenic fungi [4].
Exacerbating
the problem, the increasing use of
immunosuppressive therapies and the rapidly rising rate of diabetes and other
immunosuppressive systemic illnesses renders human populations ever more susceptible
to fungal infection.
Compounding the problem yet further, the relatively recent
evolutionary divergence of humans from fungi (relative to their divergence from bacteria)
reduces the number of targets available for selective therapeutic intervention.
Indeed,
only three classes of antifungal drug are broadly used in the clinic-and none is ideal [5].
Azoles are the most widely used antifungals in the clinic due to their safety and
good oral bioavailability. They block fungal growth by disrupting ergosterol biosynthesis
(through inhibition of the Erg 11, lanosterol 14-alpha demethylase enzyme) [6]. Other
synthetic compounds with single-agent, broad-spectrum antifungal activity have not been
approved for systemic or mucosal infections, despite extensive efforts to discover and
develop them [5]. Though safe and highly effective in certain settings, azoles do have
several drawbacks. First, their activity is fungistatic, not fungicidal, against Candida and
most other fungal pathogens. This limited activity allows pathogens to persist, creating
ideal conditions for the emergence of resistant strains. Indeed, azole resistance is
increasing in prevalence, and is typically driven by pump-mediated drug efflux or by
mutations in ERG 1, the drug target [7].
A second major liability of azoles is their inability to clear most types of
disseminated fungal infection in patients with limited immune function. These patients
require intravenous treatment with either polyenes or echinocandins, large natural
product-derived molecules with fungicidal activity. However, these agents are limited by
poor therapeutic index (in the case of the main polyene, amphotericin B) or limited brain
132
penetration and species spectrum (in the case of echinocandins). Even with optimal
treatment, the mortality rate from invasive fungal infections remains extremely high (4090% depending on the species) [3]. Clearly, new therapeutic strategies are sorely needed.
In the treatment of bacterial infections, combination therapy has proven highly
effective and slowed the emergence of resistance. This has not been possible with the
agents approved for the treatment of fungal infection, due to in large part to antagonistic
antimicrobial activites. Given the safety and efficacy of azoles, agents that render them
fungicidal (in combination) and limit the emergence of resistance would have tremendous
clinical utility. Promising targets for such intervention include two signal transduction
proteins that enable survival of azole-induced membrane stress, calcineurin and protein
kinase c (PKC) [8-10]. These metastable proteins require high levels of the molecular
chaperone Hsp90 for function [11,12]. Inhibition of Hsp90, calcineurin, or PKC all
render azole treatment fungicidal and dramatically reduce the emergence of resistant
strains. However, the human homologs of these highly conserved proteins play critical
roles in host stress tolerance and immune responses, necessitating fungal-selectivity if
they are to be used in the context of an active infection. Selectivity has been difficult to
realize. Other potential strategies for reducing azole resistance [13] have not been
pursued in drug development or successful in the clinic.
Here we took another approach: unbiased high-throughput phenotypic screening
for agents that bestow fungicidal activity upon azoles. Out of more than 300,000
compounds screened, we discovered a novel, fungal-selective inhibitor of cytochrome
bcl (complex III) of the electron transport chain that had particularly promising
properties. Exploring the effect of inhibiting cytochrome bcl revealed a critical role for
133
mitochondrial respiration in fungal virulence. The chemical tractability of the indazole
scaffold allowed us to improve its potency, fungal selectivity, and stability. Our findings
suggest that the selective targeting of fungal respiration represents a promising approach
for restricting the emergence of drug resistance and sensitizing fungi to attack by the
immune system.
RESULTS
Identification of novel small molecules that reverse fluconazole resistance in C.
albicans
To find new agents that might be useful in antifungal combination therapy, we
screened for compounds that are fungicidal in combination with fluconazole and prevent
the emergence of fluconazole resistance. We employed a previously reported isolate of
C. albicans(referred to as CaCi-2) that had evolved resistance to fluconazole during the
treatment of an HIV-infected patient [11,14]. We screened this isolate at 8 pg/ml
fluconazole, which slows, but does not stop, its growth (Figure IA). Of 302,509
compounds screened, 1654 compounds (0.5%) inhibited growth >75% in combination
with fluconazole (Figure IB). Hits were filtered for potency (IC50 < 1 pLM), lack of
toxicity to mouse 3T3 fibroblasts (IC50 > 26pM), and lack of single-agent activity
without fluconazole (IC50 > 26pM).
Next we filtered the screen hits for compounds with activity against a clinical
isolate that had emerged later in the treatment of the same patient and had higher levels of
resistance (CaCi-8; [14]). We then eliminated chemically reactive compounds,
compounds with metal-chelating motifs, and promiscuous compounds that hit multiple
134
other assays run using the same chemical library. 30 candidates were advanced and
retested from dry powders, and 3 were chosen for structure-activity relationship studies.
Detailed information on the primary screen is freely available at the PubChem database
(Pubchem AID 2007). An indazole derivative (Inz-266) was chosen for target
identification and further development due to its chemical tractability.
Inz-266 Blocks Mitochondrial Respiration by Targeting Cytochrome bcl
To better understand the effects of Inz-266 on fungal cells, we generated a profile
of compound-induced phenotypes under a variety of growth conditions and in the
presence of various stressors. These conditions included high salt, oxidative stress,
nutrient deprivation, and exposure to diverse toxic agents. We then compared the profile
of phenotypes induced by Inz-266 to that of other small molecules that sensitize C.
albicans to azoles, including inhibitors of Hsp90, Calcineurin, PKC, histone deacetylases,
PKA, and ARF cycling [8,10,13]. Inz-266 produced a unique phenotypic profile (Figure
IC). Most intriguingly, Inz-266 potently inhibited growth of both C. albicans and the
model yeast S. cerevisiae in media containing glycerol as the sole carbon source, while
only mildly slowing growth in glucose (Figure 1 D). While fungi can ferment glucose
and other sugars, utilizing glycerol as the sole carbon source requires cellular respiration.
This result suggested that Inz-266 might interfere with mitochondrial function.
As a way to identify the target of Inz-266, we exploited its potent, single-agent
inhibition of respiratory growth to select resistant mutants for whole-genome sequencing.
A wild-type S. cerevisiae strain (BY4741) was plated on media containing glycerol as
carbon source and 1 OpM Inz-266. After 7 days of incubation, two colonies with robust
135
growth were isolated. The resistance of these isolates was confirmed and their genomes
were sequenced together with that of the parental BY4741 strain. Alignment of mutant
and parental genomes followed by polymorphism discovery (Materials and Methods)
revealed that both resistant mutants contained an identical mutation in the cytochrome b
(COB) gene of the mitochondrial chromosome (Figure 2A). This mutation replaced
phenylalanine 90 with tyrosine (F90Y) in the mitochondrial cytochrome B protein, which
is the core enzymatic subunit of cytochrome bc 1 (Complex III) of the election transport
chain.
Cytochrome bc 1 is an 11-protein complex embedded in the mitochondrial inner
membrane that couples the oxidation of ubiquinol to the reduction of cytochrome C [15].
Cytochrome B, which bears the mutation that conferred resistance to Inz-266, forms the
core catalytic component of the bel complex. The enzymatic activity of the complex is
to convert ubiquinol and oxidized ferricytochrome C into ubiquinone and reduced
ferricytochrome c, which simultaneously transporting two protons into the mitochondrial
intermembrane space. This enzymatic activity is critical to oxidative phosphorylation in
two ways. First, it establishes and maintains the proton gradient along the mitochondrial
inner membrane, thus enabling ATP synthase activity through chemiosmotic coupling.
Second, by producing a reduced form of cytochrome C, it enables complex IV
(cytochrome C oxidase) to utilize molecular oxygen as the terminal electron acceptor in
mitochondrial respiration. Taken together with the specific inhibition of respiratory
growth by Inz-266, the resistance conferred by the cytB F90Y mutation suggested that
cytochrome bc 1 might be the direct target of Inz-266.
136
Inz-266 Inhibits Cytochrome bcl Activity in Isolated Yeast Mitochondria
To determine if Inz-266 inhibits cytochrome bc 1, we used a well-established in
vitro assay of its enzymatic activity using purified yeast mitochondria, permeabilized
with Triton X- 100 [16]. This assay exploits a spectral shift in cytochrome C upon its
reduction by cytochrome bcl and ubiquinol. Indeed, Inz-266 exhibited dose-dependent
inhibition of cytochrome bcl from both S. cerevisiae and C. albicans IC50's of 2.5 and
8.0 ptM, respectively (Figure 2B-E). To provide yet stronger proof that cytochrome B is
the relevant target of Inz-266, we performed the same assay with mitochondria purified
from the indazole-resistant cytochrome b F90Y mutant and asked if the enzymatic
activity was resistant to Inz-266. Indeed, a four-fold higher concentration of Inz-266 was
required to achieve 50% inhibition of the F90Y mutant enzyme than for WT (Figure 2B).
This 4-fold increased resistance to Inz-266 in the enzymatic activity of the F90Y mutant
mitochondria closely parallels the 4-fold increased resistance to growth inhibition by Inz266 in this mutant (Figure 2C).
To confirm that loss of cytochrome B function confers azole sensitivity in C.
albicans, we used genetic tools to disable the complex. Methods to selectively mutate the
mitochondrial genome in C. albicans are unavailable. Instead, we deleted a nuclearencoded gene, RIP], which encodes a conserved member of the complex that is required
for cytochrome bc 1 function [17]. We knocked out both copies of the RIP] gene in the
CaCi-2 background, and reconstituted the gene to control for potentially extraneous
effects of the deletion. riplA/A mutants from both backgrounds were indeed
hypersensitive to azole treatment, mimicking the effects of Inz-266 (Figure 2D).
137
Inz-266 is selective for yeast over human cytochrome bcl
The cytochrome bel complex is highly conserved from fungi to humans. To
determine the relative fungal selectivity of Inz-266, we tested it on mitochondria purified
from HEK293 human embryonic kidney cells. Inz-266 inhibited human cytochrome bcl
activity with an IC50 of 45 pM, 8.3-fold higher than the IC50 for the fungal homolog
(Figure 2E).
We then interrogated the toxicity of Inz-266 to respiring mammalian cells. In
tissue culture, most mammalian cell lines do not require mitochondrial respiration for
proliferation. To force respiration, we cultured cell lines in media containing galactose
instead of glucose [18]. We used a luminescent assay of cellular ATP content as a
stringent test for inhibition of mitochondrial respiration (the dominant source of ATP
production in these conditions) in HepG2 hepatocellular carcinoma cells and NIH-3T3
mouse fibroblasts. Inz-266 inhibited proliferation in these cell lines under forced
respiration conditions only at 32 pM (the solubility limit), and only mildly at that (Figure
2F). These results suggested that the inhibition of respiration by Inz-266 is at least
moderately selective for fungal over human cells.
Mutational and Structural Analysis Suggests Inz-266 Binds the Cytochrome B Qo
Site
Cytochrome b is a large protein with multiple deep binding pockets. To
investigate regions critical for Inz-266 activity, we isolated additional Inz-266-resistant
mutants and sequenced their mitochondrial cytochrome B genes. From these selections,
we identified P270L and L275S mutations to confer resistance to Inz-266 (Figure 3A).
Along with F90Y, P270L and L275S map to a small region of the previously solved
138
crystal structure of the protein, known as the Quinol-oxidizing (Qo) pocket (Figure 3B-C,
Hunte et al, 2000). This site is the target of the antimalarial drug atovaquone, which,
unlike Inz-266, is a ubiquinol substrate analog (Trumpower et al, 2000).
An alignment of the structures of the Qo site from the yeast and mammalian
(bovine) homologs revealed several key differences that could be exploited for selective
inhibition (Figure 3C). Most prominently, L275 in the fungal enzyme is replaced in the
mammalian enzyme with a bulkier phenylalanine residue, which protrudes into of the
central cavity of the Qo site. To determine if this residue is involved in the fungal
selectivity of Inz-266, we tested a previously-described yeast L275F mutant [19]. This
mutation conferred ~4-fold resistance to Inz-266. We also noted that the previously
described G143A mutant conferred strong resistance to Inz-266 (Figure 3A). We
conclude that the Qo pocket is the site for Inz-266 binding. Moreover, differential
interactions with fungal L275 and human F275 residues in this site are at least partially
responsible for the fungal selectivity observed (Figure 3A).
To better define the binding mode of Inz-266, we used computational docking of
the compound onto the structure of cytochrome b. Inz-266 was docked into a 16A grid
surrounding the Qo site, using a previously solved structure of the cytochrome bc 1
complex (PDB: 1EZV). After sampling possible conformations of Inz-266 and their
position within the active site, a lowest energy structure was computed (Figure 3D).
Notably, in this conformation, Inz-266 made close contacts with Gly143, Pro270, and
Leu275, all of which confer resistance to the compound upon mutation (Figure 3C).
Taken together, these data very strongly suggest that the Qo pocket is the binding
site for Inz-266. While this site is the known target of several agricultural fungicides,
139
none have been pursued for the treatment of human fungal infections due to their lack of
selectivity for fungi, poor pharmacological properties. or inactivity against relevant
human pathogens [20]. The low molecular weight and chemical tractability of Inz-266,
combined with its potency and fungal selectivity, made it an attractive lead for further
development through medicinal chemistry efforts.
Optimization of Inz-266 Through Medicinal Chemistry
To improve the potency and selectivity of Inz-266, we synthesized and tested a
total of 125 analogs (Selected compounds described in Figure 4). We noted that
substitution of the phenyl group of Inz-266 with fluorine and trifluoromethyl groups
conferred 37-fold enhanced potency against the fungal enzyme, but only 9-fold enhanced
potency against the human enzyme (JBL-202, JBL-22, and JBL-84, Figure 4).
Substituting a methyl group or fluorine atom at the 7 position of the indazole ring also
improved potency 5-fold (JBL-94). Combining these modifications produced JBL-140,
which demonstrated good potency and selectivity (IC50 0.026 jiM, 40-fold fungal
selective).
Unfortunately, all of these compounds contain a highly labile ester group, which
is susceptible to hydrolysis in the presence of serum esterases. However, we determined
that a more stable methyl-tetrazole (JBL- 114) or pyrazine (JBL-218) moiety at this
position retained moderate antifungal activity. Combining this substitution with JBL- 140
yielded JBL-182, which displayed strong antifungal activity (IC50 0.38 gM vs CaCi-2
with fluconazole), and almost 30-fold fungal selectivity (IC50 10.59 JIM vs respiring
HepG2 cells). While the fungal selectivity of JBL-182 may require further optimization,
140
we note that the clinically useful antimalarial drug atovaquone (which targets cytochrome
bcI) was to ic to respiring HepG2 cells with an IC50 of 12.2 pM, a concentration that it
typically achieves in patients [21]. This indicates that partial inhibition of the Qo site of
cytochrome bcl can be tolerated in humans.
Inhibition of Cytochrome bel Prevents the Emergence of Fluconazole Resistance
and Renders Fluconazole Fungicidal
This potent, selective, and more stable compound provides a valuable tool that
allowed us to investigate the role of mitochondrial respiration in diverse aspects of fungal
pathogenesis. We first interrogated the effect of cytochrome B inhibition on the
emergence of resistance to fluconazole. We plated C. albicans on media containing
fluconazole, JBL-182, or both drugs (Figure 5A). Resistant colonies emerged at a high
rate (-0. 1 %) on the plates containing fluconazole alone. Plates containing JBL- 182 alone
exhibited reduced colony size, but no reduction in the total number of colonies. However,
the combination of JBL- 182 with fluconazole completely abrogated the emergence of
resistant colonies (Figure 5A).
Up to this point, it was unclear whether JBL- 182 merely enhances the fungistatic
activity of fluconazole, or if the combination renders cells inviable. To distinguish
between these possibilities, we treated liquid cultures of C. albicanswith fluconazole,
JBL- 182, or both compounds, and measured viable colony forming units recoverable
from the culture over time. As expected, fluconazole alone slowed growth of the wildtype strain but did not cause a decrease in viability (Figure 5B). JBL-1 82 also partially
141
slowed growth alone. Importantly, combination of fluconazole with JBL-182 reduced the
number of viable cells by 97%. The fungicidal effect of fluconazole was even stronger
against the riplA/A mutant (Figure 5B). Thus, JBL-182 both prevents the emergence of
new resistance to fluconazole and transforms fluconazole from a fungistatic compound to
a fungicidal one.
Inhibiting Cytochrome B with JBL-182 Restricts Carbon Source Utilization
Having established the effects of JBL- 182 in combination with fluconazole, we
explored the consequences of cytochrome bc 1 inhibition alone on the physiology and
virulence of C. albicans. A key function of mitochondrial respiration across eukaryotes
is to support ATP production and biomass expansion in the absence of sugars. In the
laboratory, fungal pathogens are typically studied in high glucose conditions (2%), in
which ATP can be readily produced from glycolytic fermentation without respiration.
However, in host environments, sugars are much scarcer. In the bloodstream, readily
fermentable sugars are present at 0.1-0.2%, and in other niches they are present at lower
concentrations or completely absent [22]. Candidathus relies on utilization of
suboptimal carbon sources, including lactate, acetate, fatty acids, and amino acids [23].
Might JBL- 182 restrict the metabolic flexibility of C. albicans by preventing utilization
of these carbon sources?
Under conditions of high glucose (and thus fermentation), JBL- 182 caused very
little growth inhibition in a disc diffusion on agar plates (Figure 6A). In plates containing
only fetal bovine serum as the source of nutrients (-0. 1-0.2% glucose), JBL- 182 caused
slight growth inhibition. And in media containing the non-optimal sugar galactose,
142
which is abundant in the GI tract [24], JBL- 182 also caused moderate growth inhibition.
But when non-fermentable carbon sources were tested, the iiihibitory effect of JBL-182
was dramatically stronger. On media containing amino acids, lactate, or acetate as the
carbon source, JBL- 182 exhibited potent and profound growth inhibition (Figure 6A,
bottom panel).
Inhibition of Cytochrome bcl Sensitizes C. albicans to Macrophages
Perhaps the most severe nutrient limitation that C. albicansencounters is upon
engulfment into the macrophage phagosome [25]. Upon phagocytosis, C. albicans
induces the glyoxylate cycle of 2-carbon metabolism and induces a morphogenetic
program of filamentation, to enable escape. To determine if inhibition of fungal
cytochrome B might impair this process, we co-cultured C. albicans engineered to
express blue fluorescent protein (BFP) with mouse bone-marrow-derived macrophages,
with or without JBL- 182. As expected, the macrophages phagocytosed the fungi within
30 minutes, with or without the compound (Figure 6B). The fungi showed filamentous
growth and escaped the macrophages within 2 hours in the absence of JBL- 182. In
contrast, with JBL- 182 C. albicans remained trapped in the phagosome (Figure 6B-C).
Similar effects were observed when macrophages were infected with the rip]A/A mutant,
confirming that loss of cytochrome B function is responsible for this phenotype (Figure
6B-C).
We also asked if JBL- 182 would sensitize C. albicansto growth inhibition by
macrophages. In a 14 hour co-culture, untreated wild-type C. albicans evaded
macrophage attack and grew robustly (Figure 6D). Inhibition of cytochrome bc 1 by JBL-
143
182 (or deletion of RIP 1) sharply curtailed fungal growth in the presence of
macrophages. These results suggest that inhibition of cytochrome bc 1 strongly
synergizes with macrophages in blocking the growth of C. albicans.
Cytochrome bcl Is Required for Fungal Virulence and Drug Resistance in Mice
Given the effects of cytochrome bc 1 inhibition on properties key to fungal
pathogenesis, we asked if its function is required for fungal virulence in a whole animal
model. Pilot experiments determined that JBL- 182 will require further optimization of
pharmacological parameters for to achieve sufficient target engagement for use in mice.
As a surrogate for the effects of the compound, we examined our riplA/A mutant
generated in the CaCi-2 fluconazole-resistant background. In the absence of drug
treatment, CaCi-2 (and the riplA/A:RIP1 re-integrant strain) killed mice within several
days, as expected (Figure 7A). In stark contrast, the rip]A/A mutant did not cause severe
disease in the mice for over two weeks.
However, after 17-20 days, mice infected with the rip]A/A strain exhibited
agitated behavior and a head-tilt, suggestive of a possible central nervous system (CNS)
infection, and were promptly euthanized. Indeed, the brains of these mice carried high
fungal burden (data not shown). To quantitatively test if cytochrome bc 1 compromise
increased colonization of the CNS in mice, we infected animals with a low inoculum of
WT, rip]A/A, and the RIPl reconstituted strain. We allowed this infection to proceed for
four days, then sacrificed the completely asymptomatic mice and quantified fungal
144
burden in the brain and kidney. While the rip]A/A mutant was almost completely
defective in kidney colonization, it exhibited approximately 5-fold higher fungal burden
in the brain than the wild-type or re-integrant strain (Figure 7B). Thus, while the riplA/A
mutant does not kill mice through the typical means of kidney colonization, this mutant
has an increased capacity to colonize the brain, where it can cause lethality. This might
limit the utility of targeting cytochrome be 1 as monotherapy.
Treatment with azoles is generally insufficient to cure disseminated candidiasis,
as their fungistatic activity is insufficient to fully eradicate the fungus. We tested
whether the fungicidal effects of disabling cytochrome bc 1 in the presence of azoles
would enable a lasting cure of the infection. We infected mice with a high inoculum of
fungus and treated them for 3 days with a standard dose of fluconazole that controls but
does not eradicate the infection. We then stopped treatment and monitored for recurrence
of the infection over the next four weeks. In mice infected with CaCi-2, severe infection
recurred within 1-2 weeks of stopping treatment. Only 10% of these mice survived
(Figure 7C). In mice infected with the riplA/A mutant, 90% survived, with only one
mouse succumbing to neurological symptoms after more than 3 weeks.
Given the increase in brain colonization in mice infected with strains lacking
cytochrome function, we tested if this concern was eliminated by fluconazole treatment.
We infected mice with a low inoculum of CaCi-2 or rip]A/A, allowed infection to
proceed for four days, treated for 3 days with fluconazole, and then waited four more
days before sacrificing mice. Quantification of fungal burden in both the brain and
kidney established that fluconazole treatment eliminated brain colonization in these mice
(Figure 7D). Thus, even if cells lacking cytochrome bc 1 function were to succeed in
145
colonizing the brain, they should remain highly susceptible to fluconazole. Thus, these
two antifungal strategies strongly potentiate each other.
JBL-182 Inhibits Fungal Pathogens Separated by Over a Billion Years of Evolution
While we have focused on C. albicans, many other fungal pathogens that cause
life-threatening invasive infections respond poorly to azoles and other available agents
[3]. The Qo binding site for JBL- 182 is strictly conserved across these evolutionarily
distant species, including the critical L275 residue, which dictates the fungal selectivity
of this scaffold (Figure 8A). To determine if JBL-182 might have efficacy against a
broad range of other fungal pathogens, we tested its effects on Scedosporium prolificans,
Aspgerillus terreus, and Rhizopus oryzae. As a single agent, JBL-182 exhibited
promising growth inhibition of all three fungi. When grown on media requiring
respration, this activity was markedly enhanced (Figure 8B). Notably, these species are
also closely related to agricultural pathogens that cause devastating crop failures. Hence,
the fungal specificity afforded by the binding pocket of JBL- 182 may provide avenues
for the pursuit of antifungals across diverse settings.
Discussion
The evolution of mechanisms that permit flexibility and resilience in the face of
environmental challenges empowers many fungi to be extremely formidable pathogens.
Currently, the chemical arsenal we have to combat them is severely limited, creating a
dire need for new approaches. Here we demonstrate that mitochondrial respiration
presents an attractive target for intervention because it is required for these adaptive
146
strategies. Surprisingly, despite its conservation from fungi to humans, our work
demonstrates that it is possible to pharmacologically disable respiration across separated
by a billion years of evolution without impairing respiration in human cells.
We discovered and optimized a potent and fungal selective indazole-based
inhibitor of cytochrome bc 1 that abrogates the ability of C. albicansto tolerate treatment
with azole drugs and impairs their ability to evolve azole resistance. Disabling
cytochrome bc 1 also prevents fungal adaptation to nutrient starvation, sensitizes C.
albicansto macrophages, and curtails virulence in mice. Restricting the adaptive
plasticity of fungal pathogens by inhibiting respiration may present a broadly useful
approach to the development of sorely-needed combination therapies. Moreover,
targeting cytochrome bc 1 may provide an avenue for the treatment of fungi that are
refractory to treatment with azoles and other available agents.
Considerable progress has been made to identify metabolic pathways that are
absent in humans and might therefore present attractive therapeutic targets. One such
example is the glyoxylate cycle, which enables diverse pathogens to survive carbon
starvation [22,26,27]. However, given the formidable metabolic flexibility of C.
albicans,the pharmacological targeting of any one metabolic pathway alone may prove
insufficient as a therapeutic strategy. In support of this notion, mutants lacking a
functional glyoxylate cycle exhibit only partially reduced virulence [27]. In contrast, the
mitochondrial electron transport chain is a central molecular node in metabolic networks,
and is required for many pathways of metabolic adaptation. It is thus unlikely that fungi
can readily tolerate or bypass its inhibition and retain virulence [28]. We anticipate that
mitochondria will prove essential to many other fungal virulence processes, as others too
147
have begun to reveal [29,30]. Although targeting such highly conserved proteins might
initially seem unwise, there are certainly many examples of effective antimicrobials that
exert their effects through proteins shared with man, such as azoles or ribosome-targeting
antibiotics.
Though promising, inhibiting fungal cytochrome bc 1 must overcome several
challenges to be useful as a therapeutic strategy. One challenge is to design compounds
that can target this hydrophobic pocket in the inner mitochondrial membrane, crossing
both the plasma membrane and the outer membrane to do so, and yet retain sufficient
water solubility for effective delivery. Recent advances in drug formulation may mitigate
this concern [31]. In addition, a higher degree of fungal selectivity than we have achieved
to date (40-fold) might be necessary to guarantee safety in patients. The successful use of
the cytochrome bc I inhibitor atovaquone in antimalarial therapy suggests that such
challenges can be overcome. Moreover, recent advances in the crystallization of fungal
cytochrome bc I could aid in the structure-based design of agents with improved potency
and selectivity [32].
The increased brain colonization of C. albicans with disabled cytochrome B (the
rip]A/A mutant) could also present a challenge. However, the clinical relevance of this
phenomenon is unclear [33], and combination therapy with azoles, which eliminate this
colonization, would appear to greatly mitigate this concern. Another challenge will be to
determine if mitochondrial inhibitors can be effective against Candidaglabrata, in which
loss of mitochondrial respiration appears to increase azole resistance through induction of
drug efflux pumps [34]. But even if targeting of mitochondrial respiration is ineffective
148
against C. glabrata, it may be effective in the treatment not only of C. albicans but also of
diverse fungal pathogens that are refractory to treatment with current agents [3].
Most (arguably all) classes of antibiotics and antifungals of the 201h century were
identified by phenotypic screening of natural product extracts and synthetic libraries,
assaying simply for single-agent microbial growth inhibition (both intentionally and
serendipitously). Yet, novel classes of natural product antimicrobials are increasingly
hard to discover, and their development into drugs poses substantial challenges [35]. In
the past two decades, efforts have swung toward target-based screening of purified
proteins against synthetic chemical libraries. This approach has failed to deliver any
novel classes of antifungals or antibacterials in clinical practice [6,36]. In part, this
failure is due to the fact that synthetic compounds with good activity in cell-free assays
fail to traverse microbial cell walls and membranes. Recent efforts have returned to
phenotypic screening, using synthetic chemical libraries to search for single agents
capable of inhibiting microbial growth. However, results suggest that the few chemical
classes with useful activity as single agents have already been discovered [36]. As an
alternative approach, we propose that unbiased phenotypic screening for growth
inhibition in combination with older antimicrobials might uncover vast regions of
chemical space with previously unknown therapeutic potential [37,38]. To this end, we
hope that the discovery and optimization of JBL- 182 will provide a broadly applicable
blueprint for similar efforts against other pathogens.
Materials and Methods
149
High I hroughput Smaji Moiecule Screen
Methods used in the high-throughput screen are described in depth in [39].
Fungal Growth and Strain Manipulation
C. albicansand S. cerevisiae were maintained under standard conditions as described
previously [28]. Assays of fungal growth were generally performed as described in [28],
with the following specifications. Phenotypic profiling experiments were performed in
96-well flat-bottom plates by diluting overnight YPD cultures to a starting OD of 0.0003,
then assaying growth in drug and stress conditions after 24 or 48 hours (as indicated) by
reading OD600. In these experiments, drugs and concentrations included Conditions
included 0.9M NaCl, 0.05 jg/mL aureobasidin, 80 piM bathophenanthroline disulfonic
acid, 0.5pg/mL cerulenin, 2mM tert-butyl peroxide, 40 mg/L calcofluor white, 2 pg/mL
tunicamycin, 0.1 pg/mL caspofungin, 25 gM simvastatin, and 0.008% SDS. Small
molecule inhibitor odrugs were used at either 2.5 ptM (Brefeldin A), 5 ptM
(Geldanamycin, FK-506, cercosporamide), 10 ptM (Trichostatin A, Inz-266), 15 pM
MDL-12,330A), or 20 pM (rotenone). Synthetic media used a base of Difco YeastNitrogen-Base (YNB) with complete amino acid supplement, and addition of 2% glucose
or 2% glycerol. Growth results were quantitatively displayed in heatmaps using
Treeview as described in [28]. Minimal inhibitory concentration (MIC) assays were
performed at 2 fold concentration increments in 96-well plates over 24-72 hours as
described in figure legends. The MIC was defined as the lowest concentration that
results in no visible fungal growth.
150
Alignment of Yeast and Bovine Cytochrome bcl
Cytochrome bcl from Saccharomyces cerevisiae
(PDB: 1P84) was aligned to
cytochrome bel from Bos taurus (PDB: 1SQV), using the "align structures" feature in
Pymol.
Structural Docking (Raja Srinivas and Bruce Tidor)
Ligands were docked into the active site using the GLIDE package (Schrodinger suite).
The initial unbound ligand conformation was generated using a SMILES
sequences,which was converted to a 3D structure using Ligprep (Schrodinger Suite). For
docking, the native ligand, ubiquinol, was removed from the active site of the crystal
structure (PDB Code: 1EZV). A grid of 16A was generated centered on the active site.
To generate an improved bound conformation, we used an inverse design approach,
which has been described in detail before. (Citations here-see below) In short, an
ensemble of scaffold conformations is generated in the active site, with appropriate
attachment points for the functional groups. The top docked conformation of Inz-266 was
used to generate an initial conformation for the scaffold. We thoroughly sampled in both
translation and rotation throughout the active site to generate an ensemble of possible
scaffold position. We further sampled the bond between the Indazole and the benzene
every degree (Bond shown in green). We rotamerized the functional group by
enumerating all dihedrals every 30 degrees and employed a Dead-end Elimination/A*
approach, which guarantees the global minimum energy conformation. Finally, we
151
rescored the top conformations using an energy model with increased accuracy as
described before.
Selection of resistant mutants and whole genome sequencing
Selection of mutants resistant to Inz-266 was performed by plating 107 cells per plate on
YNB-glycerol-agar plates containing Inz-266. Plates were incubated for 7 days before
picking colonies and retesting their resistance in liquid media. Isolates were retested for
resistance to cycloheximide to eliminate nonspecific efflux mutants. DNA extraction,
whole genome sequencing and polymorphism detection was performed as described in
[28].
Cytochrome B enzyme assay
The cytochrome B enzyme assay was adapted from [16].
Briefly, compounds were
diluted in a 2x reaction buffer containing 100mM Tris-Cl pH 7.4, 10mM sodium azide,
0.02% BSA, and 0.1% Tween-20. Mitochondria (-5 pg protein) and cytochrome C (final
concentraion 50tM) were then added, and the reaction was initiated by the addition of
ubiquinol (50pM), reduced from ubiquinone with dithionite.
Reaction plates were
incubated at room temperature for 10 minutes before reading absorbance at 550nm.
IC50's were calculated in Graphpad Prism 6.0. Antimycin A (200nM) was included on
every plate as a control for 100% inhibition.
Mammalian Cell Respiration Toxicity
Compounds were tested for toxicity to respiring mammalian cells as described in [18].
Cells were initially passaged in High-Glucose DMEM, then washed and resuspended in
glucose-free DMEM with 10mM galactose and added to 96-well white tissue culture
152
plates at 10,000 cells per well. After 72 hours, ATP production was used as a surrogate
for growth and assayed using Cell-titer-glo (Promega) at 1:5 dilution.
Bone-Marrow Derived Macrophage Co-Culture
Macrophages were isolated from GFP-tubulin Balb/c mice using standard methods [40].
After 7-9 days in culture, macrophages were added to 12-well plates containing glass
cover slips at 3x10 5 cells/well, and fungi were added at an MOI of 4. Cells were fixed
with 3% formalin before visualization by fluorescence microscopy. Fungal growth over
14hr in the presence of macrophages was assayed by Alamar Blue dye reduction (control
experiments demonstrated very low background macrophage dye signal).
Mouse infection experiments
All mouse experiments were performed under MIT protocol 0312-024-15 and in
accordance with NIH standard for the ethical treatment of animals. Mouse experiments
were performed as described in [28] with the specific inoculum used noted in the figure
legend. 7-12 week-old female Balb/C mice were used in all experiments. Fluconazole
was prepared at 1 mg/mL solution in PBS and delivered intraperitoneally.
Supplemental Chemical Synthesis Methods
(Jean-Baptiste Langlois and Stephen Buchwald)
General reagent information
Commercial materials were purchased and used as received unless otherwise noted.
Acetonitrile (anhydrous, 99.8%), carbon tetrachloride (anhydrous, 99.5%) and NN-
153
dimethylformamide (anhydrous, 99.8%) were purchased from Sigma-Aldrich. Acetone
(ACS reagent, 99.6%) was purchased from Acros Organics. Tetrahydrofuran was purified
and dried using a solvent purification system consisting of successive passage through
alumina and Q5 reactant-packed columns. Deionized water was employed; this solvent
was degassed by sonication under slight vacuum for 5 minutes, followed by backfilling
with argon. Potassium carbonate (ACS reagent, 99.0%), tribasic potassium phosphate
(reagent grade, 98%) and sodium azide (ReagentPlus®, 99.5%) were purchased from
Sigma-Aldrich. Ammonium chloride (ACS reagent, 99.6%) was purchased from Acros
Organics. Phosphorus (V) oxychloride (ReagentPlus@, 99%), hydrazine monohydrate
(N 2H 4 64-65%, reagent grade, 98%), N-chlorosuccinimide (98%), N-bromosuccinimide
(ReagentPlus@,
99%),
bromoacetonitrile
(97%),
2,2'-azobis(2-methylpropionitrile)
(98%), 2-methylpyrazine (99%) and dimethylsulfate (99%) were purchased from SigmaAldrich. (Trimethylsilyl)diazomethane solution (2M in hexanes) was purchased from
Sigma-Aldrich and kept in a fridge at -5 'C. 2-fluoro-5-(trifluoromethyl)-phenylboronic
acid and 7-fluoro-1H-indazole
were purchased from Combi-Blocks.
2-fluoro-3-
methylbenzoic acid was purchased from Oakwood Chemicals. XPhos ligand was
purchased from Strem and precatalyst P was prepared following a reported procedure.'
Compounds described herein were purified by flash chromatography using Silicycle
SiliaFlashP60 (230-400 mesh) silica gel.
General analytical information
All compounds described herein were characterized by 'H,
154
1C
and ' 9F NMR (when
applicable).
1H,
13
C and 19F NMR spectra were recorded on BrUker 400 MHz
spectrometers. The samples were prepared in CDCl 3, and chemical shift (6) are given in
parts per million (ppm) relative to the residual solvent peak (7.26 ppm for 1H and 77.16
ppm for
for
13 C).
13 C).
Few samples were prepared in d6 -DMSO (2.50 ppm for 'H and 39.51 ppm
19F NMR were performed with proton decoupling. Peak multiplicity is reported
as: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets),
dt (doublet of triplets), ddd (doublet of doublets of doublets), td (triplet of doublets), br
(broad), app (apparent). Coupling constants (J) are given in Hertz (Hz). Mass
spectrometry analyses were performed for both chemical probes (X1 and X2). HRMS
data were obtained using a direct analysis in real time (DART) ionization method on a
BrUker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance Mass
Spectrometer (FT-ICR-MS). The relative purities of chemical probes X1 and X2 were
determined using LC-MS analysis on an Agilent 1260 Infinity Chromatograph coupled
with an Agilent 6120 Quadrupole Mass detector. The analyses were performed using an
Eclipse XDB-C18 column (5
tm, 4.6x150 mm) using an isocratic mobile phase
composed of 90% of acetonitrile, 9.9% of water and 0.1% of acetic acid (flow rate = 0.8
ml/min).
Preparation of chemical probe X1
155
0
0
OH
Me 2SO 4 , K 2CO 3
0.
Acetone, r.t.
Me
Scheme
1.
Me
Preparation of methyl 2-fluoro-3-methylbenzoate 2.
Methyl 2-fluoro-3-methylbenzoate 2: 2-fluoro-3-methylbenzoic acid 1 (2 g, 13 mmol, 1
eq) was charged into a 100-ml round-bottomed flask and dissolved in acetone (20 ml).
Potassium carbonate (2.15 g, 15.6 mmol, 1.2 eq) was added in portions and the resulting
suspension was stirred until the gas evolution ceased (10 min). Then, dimethylsulfate
(1.23 ml, 13 mmol, 1 eq) was added and the reaction mixture was stirred for 1 hour at
room temperature. The reaction was monitored by TLC (Hexanes/AcOEt 9/1, Rf() = 0.1,
Rf(2 ) = 0.7). After complete consumption of the starting material, the reaction mixture
was diluted with acetone (10 ml) and filtered on a plug of celite. The solid was rinsed
twice with acetone (2x 10 ml) and the filtrate was concentrated using a rotary evaporator.
The crude residue was triturated with hexanes (10 ml) and the resulting suspension was
filtered on a plug of celite. The filtrate was concentrated using a rotary evaporator to
yield the desired product as a pale yellow liquid (2.2 g, 99%). 'H NMR (400 MHz,
CDCl 3 ): 6 7.66-7.70 (m, 1H), 7.52-7.57 (m, 1H), 7.18-7.22 (t, J= 7.7 Hz, 1H), 3.84 (s,
3H), 2.26-2.27 (d, J= 2.4 Hz, 3H).
13C
NMR (125 MHz, CDCl 3): 6 164.2-164.2 (d, J=
3.2 Hz), 158.0-160.5 (d, J= 256.5 Hz), 136.1-136.2 (d, J= 5.9 Hz), 129.1, 125.9-126.1
(d, J= 17.5 Hz), 124.0-124.0 (d, J= 4.5 Hz), 117.9-118.0 (d, J= 10.9 Hz), 52.3, 14.114.1 (d, J= 4.6 Hz).
156
0
0
0/
F
Me 2
N'NH2
N 2 H 4.H 20
MeOH
60 *C, 1.5 hr
F
H
Me
Scheme 2. Preparation of 2-fluoro-3-nethylbenzohydrazide 3.
2-fluoro-3-methylbenzohydrazide 3: methyl 2-fluoro-3-methylbenzoate 2 (2.2 g, 13
mmol, 1 eq) was charged into a 100-ml round-bottomed flask and dissolved in methanol
(20 ml). Hydrazine monohydrate (3.15 ml, 65 mmol, 5 eq) was added and the flask was
equipped with a reflux condenser. The flask was then placed in an oil bath preheated at
60 'C and the reaction mixture was refluxed for 1.5 hours. After this time, the reaction
mixture was cooled to room temperature and volatiles were removed using a rotary
evaporator. The crude residue was triturated in hexanes (20 ml) and the supernatant
solution was removed with a pipet. The operation was repeated two times and the
resulting gummy solid was dissolved in AcOEt, dried with Na2 SO 4 , filtered and
concentrated using a rotary evaporator. The crude reaction mixture was dried in vacuum
for 12 hours to give the desired product as a white solid (1.8 g, 83%). 'H NMR (400
MHz, d -DMSO): 6 9.48 (brs, 1H), 7.32-7.40 (m, 2H), 7.12-7.15 (t, J = 7.6 Hz, 1H),
4.51 (brs, 2H), 2.25-2.25 (d, J= 2.3 Hz, 3H).
3
1
C NMR (125 MHz, d6 -DMSO): 6 163.6,
156.2-158.7 (d, J= 248.1 Hz), 133.2-133.3 (d, J= 5.2 Hz), 127.3-127.3 (d, J= 3.2 Hz),
124.9-125.0 (d, J= 17.7 Hz), 123.9-123.9 (d, J= 4.2 Hz), 122.9-123.1 (d, J = 16.1 Hz),
14.1-14.2 (d, J= 4.2 Hz).
157
OH
0
N' NH2
H
N 2H4.H20
sealed tube
140 *C, 15 hr
Me 3
NN
H
Me
Scheme 3. Preparation of 7-methyl-I H-indazol-3-ol 4.
7-methyl-1H-indazol-3-ol 4: 2-fluoro-3-methylbenzohydrazide 3 (1.5 g, 9 mmol, 1 eq)
was charged into a sealed tube and suspended in hydrazine monohydrate (6 ml). The tube
was sealed with a Teflon screw valve and placed in an oil bath preheated at 140 'C. The
reaction mixture was placed behind a protective shield and stirred for 15 hours. After this
time, the reaction mixture was cooled to room temperature and the protective shield was
removed. The cooled mixture was poured into crushed ice and the mixture was stirred for
10 min. A white precipitate formed and was filtered off the suspension with a fritted
funnel. The solid was washed twice with water (5 ml) and subsequently dried in vacuum
for few hours to afford the desired product as a white solid (1 g, 75%). 'H NMR (400
MHz, d -DMSO): 6 10.52-11.12 (brm, 2H), 7.42-7.44 (d, J= 8.0 Hz, 1H), 7.07-7.09 (d,
J = 6.9 Hz, 1H), 6.86-6.90 (dd, J = 6.9 and 8.0 Hz, 1H), 2.38 (s, 3H). '3 C NMR (125
MHz, d -DMSO): 6 156.6, 142.5, 127.0, 119.9, 118.9, 117.6, 112.3, 16.1.
OH
I
Me
NN
N
H
Cl
POC1 3
sealed tube
140 *C, 5 hr
4
N
-N
Me
H
5
Scheme 4. P~reparation of 3-chloro-7-rnethyl- I H1-indazole 5.
158
3-chloro-7-methyl-1H-indazole 5: 7-methyl-1H-indazol-3-ol 4 (500 mg, 3.38 mmol, 1
eq) was charged into a sealed tube and suspended in phosphorus (V) oxychloride (2 ml).
The tube was sealed with a Teflon screw valve, placed in an oil bath preheated at 140 'C
and the reaction mixture was stirred at this temperature for 5 hours. After this time, the
reaction mixture was cooled to room temperature and poured into crushed ice. The
mixture was stirred for 30 min and an off-white precipitate formed. The solid was filtered
off the suspension with a fritted funnel. The residual water was removed by azeotropic
evaporation with toluene using a rotary evaporator. The resulting solid was dried in
vacuum for few hours to afford the desired product as an off-white solid (565 mg, 99%).
'H NMR (400 MHz, CDC1 3): 6 7.47-7.49 (d, J= 8.1 Hz, 1H), 7.17-7.19 (d, J= 7.0 Hz,
1H), 7.09-7.13 (dd, J= 7.0 and 8.1 Hz, 1H), 2.53 (s, 3H).
13 C
NMR (125 MHz, CDC 3 ):
6 141.6, 135.3, 128.1, 122.1, 120.7, 120.2, 117.0, 16.6.
C1
Me
N'
H
N
C1
BrCH 2CN, K2CO3
DMF, 90 *
2 hr
5
N
Me
6
N
CN
Scheme 5. Preparation of 2-(3-chloro-7-methyl-IH-indazol-1-yl)acetonitrile 6.
2-(3-chloro-7-methyl-1H-indazol-1-yl)acetonitrile
6: 3-chloro-7-methyl-1H-indazole 5
(300 mg, 1.81 mmol, 1 eq) and potassium carbonate (500 mg, 3.62 mmol, 2 eq) were
charged into a test tube and suspended in DMF (5 ml). The tube was capped with a
Teflon screw-cap septum. Bromoacetonitrile (0.25 ml, 3.62 mmol, 2 eq) was added via
159
syringe through the septum and the tube was placed in an oil bath preheated at 90 'C. The
reaction was monitored by TLC (Hexanes/AcOEt 6/4, Rf(6 )
=
0.8). After complete
consumption of the starting material (2 hours), the reaction mixture was cooled to room
temperature, quenched with an aqueous solution of HCl (IM) and extracted with Et2 O.
The organic layer was washed with brine, dried with Na 2 SO 4 , filtered and concentrated
using a rotary evaporator. The crude residue was adsorbed onto silica gel, dried in
vacuum and purified by flash chromatography (Hexanes/AcOEt 6/4, Rf(6)
=
0.8) to afford
the desired product as an off-white solid (350 mg, 94%). 'H NMR (400 MHz, CDC13 ): 6
7.54-7.57 (ddq, J= 0.7, 1.3 and 8.0 Hz, 1H), 7.26-7.28 (in, 1H), 7.17-7.21 (dd, J= 7.1
and 8.0 Hz, 1H), 5.43 (s, 2H), 2.79 (s, 3H).
13 C
NMR (125 MHz, CDC13 ): 6 140.8, 136.5,
130.9, 123.3, 123.2, 120.3, 118.4, 114.6, 39.9, 18.6.
F3 C
B(OH) 2
F
CI
I
N
F
CF3
P (5 mol %), K 3PO4
M CNTHF/degassed water
40 OC, 2hr
CN
Me 6
6
I
N
PCy2
i-Pr
i-Pr
FPd-XPhos
N
Me 7
7
NH 2
C
CN
P
XPhos
Scheme 6. Preparation of 2-(3-(2-fluoro-5-(trifluoromethyl)phenyl)-7-methyl-1H-indazol-1-yl)acetonitrile
2-(3-(2-fluoro-5-(trifluoromethyl)phenyl)-7-methyl-1H-indazol-1-yl)acetonitrile
7.
7: 2-
(3-chloro-7-methyl-1H-indazol-1-yl)acetonitrile 6 (100 mg, 0.49 mmol, 1 eq), ), 2-fluoro5-(trifluoromethyl)-phenylboronic acid (152 mg, 0.73 mmol, 1.5 eq), tribasic potassium
phosphate (212 mg, 1.0 mmol, 2 eq) and XPhos precatalyst P (18 mg, 0.025 mmol, 0.05
160
eq) were charged into a test tube, which was capped with a rubber septum. The vessel
was successively evacuated and backfilled with argon. This operation was repeated three
times and anhydrous THF (1 ml) was added via syringe through the septum followed by
freshly degassed water (2 ml). The rubber septum was then replaced by a Teflon screwcap septum and the tube was placed in an oil bath preheated at 40 'C. After 2 hours, the
reaction mixture was cooled to room temperature, quenched with water and extracted
with AcOEt. The organic layer was dried with Na 2SO 4 , filtered and concentrated using a
rotary evaporator. The crude residue was adsorbed onto silica gel, dried in vacuum and
purified by flash chromatography (Hexanes/AcOEt 8/2, Rf(7) = 0.5) to afford the desired
product as a yellow solid (147 mg, 90%). 'H NMR (400 MHz, CDC1 3 ): 6 8.06-8.08 (dd,
J= 2.4 and 6.4 Hz, 1H), 7.70-7.74 (m, 1H), 7.63-7.66 (dd, J= 3.4 and 8.1 Hz, 1H), 7.347.39 (m, 1H) 7.25-7.28 (m, 1H) 7.18-7.22 (dd, J= 7.0 and 8.1 Hz, 1H), 5.58 (s, 2H), 2.86
(s, 3H). 13C NMR (125 MHz, CDC1 3 ): 6 160.6-163.1 (d, J = 255.6 Hz), 141.3, 140.5,
130.1, 128.9-129.0 (m), 127.7-127.9 (m), 127.3-127.6 (m), 124.3, 123.1, 121.2-121.4 (d,
J= 16.0 Hz), 120.1, 120.0, 119.7-127.9 (q, J= 272.1 Hz), 117.1-117.3 (d, J= 23.3 Hz),
114.9, 40.1, 18.8. 9F NMR (376 MHz, CDC13 ): 6 -62.0 (s, 3F), -107.9 (s, IF).
F 3C
F3C
F
Me
NH 4CI, NaN 3
F.IN
N
15
hr
Me
8
TMSCH 2N 2
THF,
N
DMF, 100 *C
NN
F3 C
NH
N:N
0
O
C to r.t.
15
hr
Scheme 7. Preparation of chemical probe X1.
161
N
NN
Me
Xi
N me
N:N
3-(2-fluoro-5-(trifluoromethyl)phenyl)-7-methyl-1-((2-methyl-2H-tetrazol-5yl)methyl)-1H-indazole
X1:
2-(3-(2-fluoro-5-(trifluoromethyl)phenyl)-7-methyl-1H-
indazol-1-yl)acetonitrile 7 (100 mg, 0.3 mmol, 1 eq), ammonium chloride (32.4 mg, 0.6
mmol, 2 eq) and sodium azide (39 mg, 0.6 mmol, 2 eq) were charged into a test tube and
suspended in DMF (1 ml). The tube was sealed with a Teflon screw-cap septum and
placed in an oil bath preheated at 100 'C. The reaction mixture was stirred at this
temperature for 15 hours. After this time, the reaction mixture was cooled to room
temperature, quenched with an aqueous solution of HCl (IM) and extracted with Et2 0.
The organic layer was dried with Na2 SO 4 , filtered and concentrated using a rotary
evaporator to recover compound 8 as a yellow oil. The crude mixture (100 mg, 0.27
mmol, 1 eq) was charged into a test tube, which was subsequently closed with a Teflon
screw-cap septum. The vessel was successively evacuated and backfilled with argon.
This operation was repeated three times and anhydrous THF (2 ml) was added via syringe
through the septum. The reaction mixture was cooled to 0 'C with an ice bath and a
solution of (trimethylsilyl)diazomethane in hexanes (2M, 0.2 ml, 0.41 mmol, 1.5 eq) was
added dropwise via syringe through the septum. Then, the ice bath was removed and the
yellow reaction mixture was stirred for 15 hours. After this time, the excess
(trimethylsilyl)diazomethane was quenched with few drops of tetrafluoroboric acid.
When the gas evolution ceased, the reaction mixture was concentrated using a rotary
evaporator. The crude residue was adsorbed onto silica gel, dried in vacuum and purified
by flash chromatography (Hexanes/AcOEt 6/4, Rf(xl) = 0.7) to afford the desired product
as a white solid (23 mg, 22% from 8). 'H NMR (400 MHz, CDC1 3 ):
162
8
8.06-8.08 (dd, J=
2.1 and 6.4 Hz, 1H), 7.65-7.69 (in, 1H), 7.59-7.62 (dd, J= 3.3 and 8.0 Hz, 1H), 7.30-7.35
(appt, J= 9.1 Hz, 1H), 7.18-7.20 (dt, J= 1.2 and 6.9 Hz, 1H), 7.11-7.14 (dd, J= 7.0 and
8.1 Hz, 1H), 6.14 (s, 2H), 4.28 (s, 3H), 2.83 (s, 3H). "C NMR (125 MHz, CDC1 3 ): 6
163.4, 160.6-163.2 (d, J= 256.0 Hz), 140.6, 139.9, 129.3, 129.1-129.3 (m), 127.0-127.4
(in, 2C), 123.8, 119.8-127.9 (q, J= 272.2 Hz), 122.2, 122.0-122.1 (d, J= 16.1 Hz), 120.7,
119.5-119.6 (d, J= 7.2 Hz), 116.8-117.1 (d, J= 23.5 Hz), 46.8, 39.7, 19.4. "F NMR
(376 MHz, CDCl3 ): 6 -61.9 (s, 3F), -108.0 (s, IF). LC-MS: RT(x1) = 2.3 min (>98%
purity). HRMS (m/z): [M+H] calculated for C 18H 15F 4N 6, 391.1289; found, 391.1277.
Comment: The low isolated yield obtained after the methylation step was due to the
concomitant formation of two additional regioisomers that were separated during the
purification by flash chromatography.
Preparation of chemical probe X2
CI
N
F
/
H
N-chlorosuccinimide
CH 3CN, 70 *C, 15 hr
F
9
N
H
N
10
Scheme 8. Preparation of 3-chloro-7-fluoro-J H-indazole 10.
163
3-chloro-7-fluoro-1H-indazole 10: 7-fluoro-lH-indazole 9 (500 mg, 3.7 mmol, 1 eq)
and N-chlorosuccinimide (500 mg. 3.7 mmol. 1 eq) were charged into a test tube
equipped with a stir bar. Anhydrous acetonitrile (5 ml) was added and the tube was sealed
with a Teflon screw-cap septum. The tube was then placed in an oil bath preheated at
70 'C and stirred for 15 hours. After complete consumption of the starting material,
monitored by TLC (Hexanes/AcOEt 8/2, Rf(l) = 0.5; Rf(2 ) 0.7), the reaction mixture was
cooled to room temperature and acetonitrile was removed using a rotary evaporator. The
solid residue was dissolved in AcOEt and washed twice with an aqueous solution of
NaOH (2M). The organic layer was dried with Na 2 SO 4 , filtered and concentrated using a
rotary evaporator. The crude residue was adsorbed onto silica gel, dried in vacuum and
purified by flash chromatography (Hexanes/AcOEt 8/2, Rf(2 )= 0.7) to afford the desired
product as a white solid (540 mg, 85%). 'IH NMR (400 MHz, CDC1 3 ): 6 10.78 (brs, lH),
7.46-7.50 (m, 1H), 7.12-7.19 (m, 2H). 13C NMR (125 MHz, CDC1 3 ): 6 146.7-149.1 (d, J
=
249.0 Hz), 136.2-136.3 (d, J= 4.0 Hz), 131.2-131.3 (d, J= 16.1 Hz), 124.2-124.3 (d, J
4.3 Hz), 122.3-122.4 (d, J= 5.3 Hz), 115.5-115.6 (d, J= 4.4 Hz), 112.2-112.4 (d, J=
15.5 Hz). '9F NMR (376 MHz, CDC 3 ): 6 -132.4 (s, IF).
Comment: The isolated product was contaminated with a side-product stemming from
additional chlorination in position 5 of the indazole (4% according to 19F NMR).
164
c1
-~N'
(;
F
H
N
K 2 CO3 ,
80 *C,
Ci
Br
DMF
1 hr
10
N
F
N
12
N
Scheme 9. Preparation of 3-chloro-7-fluoro- I -(pyrazin-2-ylrnethyl)- I H-indazole 12.
Comment: The preparation of compound 12 requires the use of alkylation reagent 11,
which is prone to decomposition in the contact of air at room temperature. To circumvent
this problem, the following procedure has been devised to prepare reagent 11 as a
solution in DMF that can be directly employed in the alkylation event.
3-chloro-7-fluoro-1-(pyrazin-2-ylmethyl)-1H-indazole 12: N-Bromosuccinimide (6.1 g,
34.5 mmol, 15 eq), and 2,2'-azobis(2-methylpropionitrile) (377 mg, 2.3 mmol, 1 eq) were
charged into a 100-ml round-bottomed flask and suspended in carbon tetrachloride (30
ml). 2-methylpyrazine was added and the flask was equipped with a stir bar and a reflux
condenser. The flask was then placed in an oil bath preheated at 80 'C and refluxed for 3
hours. After this period of time, a black precipitate formed. The clear supernatant was
diluted with hexanes (20 ml), cooled to room temperature and filtered on a plug of celite.
The dark solid was rinsed two times with hexanes (2x20 ml). DMF (20 ml) was added to
the filtrate and low boiling solvents were removed using a rotary evaporator (hexanes and
carbon tetrachloride). The resulting solution of 11 in DMF was transferred into a 1 00-ml
round-bottomed flask containing potassium carbonate (3.8 g, 27.6 mmol, 12 eq) and 3chloro-7-fluoro-1H-indazole 10 (400 mg, 2.3 mmol, 1 eq). The flask was equipped with a
reflux condenser and placed in the oil bath preheated at 80 'C. After 2 hours, the reaction
165
mixture was cooled to room temperature, quenched with water and extracted with Et 2 0.
The organic layer was dried with Na2SO4 . filtered and concentrated using a rotary
evaporator. The crude residue was adsorbed onto silica gel, dried in vacuum and purified
by flash chromatography (Hexanes/AcOEt 7/3, Rf(4) = 0.4) to afford the desired product
as a yellow solid (466mg, 77%). 1H NMR (400 MHz, CDC13 ): 6 8.49-8.52 (brm, 2H),
8.39 (brs, 1H), 7.44-7.49 (in, 1H), 7.08-7.16 (in, 2H), 5.82 (s, 2H). '3 C NMR (125 MHz,
CDC13 ): 6 152.0, 146.9.149.4 (d, J= 248.3 Hz), 144.4, 144.2, 143.4, 135.1-135.1 (d, J=
3.0 Hz), 130.9-131.0 (d, J= 13.3 Hz), 125.5-125.5 (d, J= 4.0 Hz), 122.3-122.4 (d, J=
5.7 Hz), 116.0-116.0 (d, J
4.4 Hz), 112.8-112.9 (d, J= 16.9 Hz), 54.6-54.7 (d, J= 4.2
Hz). 'F NMR (376 MHz, CDC13 ): 6 -133.6 (s, IF).
Comment: The 2,5-dichloro-7-fluoro-1H-indazole
initially present in substrate 10
underwent the alkylation reaction with similar efficiency to afford a side-product that we
did not manage to remove from product 12 (96% pure according to 19F NMR).
B(OH) 2
F 3C
F
C
IFN
N
F 12
NHPCy
,H 2
SF
CF3
P (5 mol %), K PO 4
THF/degassed 3water
40 *C, 2 hr
N
IN
NN
F X2
N
Nz
\/
C
P
N
Scheme 10. Preparation of chemical probe X2.
166
i-Pr
i-Pr
Pd-XPhos
i-Pr
XPhos
2
7-fluoro-3-(2-fluoro-5-(trifluoromethyl)phenyl)-1-(pyrazin-2-ylmethyl)-1H-indazole
X2: 3-chloro-7-fluoro-1-(pyrazin-2-ylmethyl)-1H-indazole
12 (400 mg, 1.52 mmol, 1 eq),
2-fluoro-5-(trifluoromethyl)-phenylboronic acid (475 mg, 2.28 mmol, 1.5 eq), tribasic
potassium phosphate (645 mg, 3.04 mmol, 2 eq) and XPhos precatalyst P (55 mg, 0.076
mmol, 0.05 eq) were charged into a 100-ml Schlenk tube, which was capped with a
rubber septum. The vessel was successively evacuated and backfilled with argon. This
operation was repeated three times and anhydrous THF (8 ml) was added via syringe
through the septum followed by freshly degassed water (16 ml). The rubber septum was
then replaced by a Teflon screw valve and the tube was placed in an oil bath preheated at
40 'C. After 2 hours, the reaction mixture was cooled to room temperature, quenched
with water and extracted with AcOEt. The organic layer was dried with Na2 SO 4 , filtered
and concentrated using a rotary evaporator. The crude residue was adsorbed onto silica
gel, dried in vacuum and purified by flash chromatography (Hexanes/AcOEt 8/2, Rf(xl)=
0.4) to afford the desired product as a white solid (482 mg, 81%). 1 9F NMR analysis of
the isolated product revealed the presence of the side-product carried out during all this
synthetic sequence. The product was consequently further purified by preparative HPLC
to yield pure compound X2 (384 mg, 65%). The purification was performed on an
Agilent 1260 Infinity instrument coupled with a diode-array detector. An Agilent prepC18 column (5 pm, 212x150 mm) was employed with an isocratic mobile phase
composed of 90% of acetonitrile, 9.9% of water and 0.1% of acetic acid (flow rate = 20
ml/min, RT(x2) = 3.0 min). 'H NMR (400 MHz, CDC13 ): 8 8.47-8.60 (brm, 2H), 8.348.46 (brs, 1H), 8.09-8.11 (dd, J= 2.3 and 6.6 Hz, 1H), 7.67-7.77 (m, 1H), 7.57-7.60 (ddd,
J= 1.1, 3.5 and 7.8 Hz, 1H), 7.32-7.37 (ddd, J= 0.9, 8.7 and 9.6 Hz, 1H), 7.08-7.18 (m,
167
2H), 5.99 (s, 2H). "C NMR (125 MHz, CDC 3 ): 6 160.5-163.0 (d, J= 255.5 Hz), 152.4,
147 2-149 6 (d, J= 2473 Hz), 144.4 144.1, 143.3, 140.3, 130.7-130.9 (d, J= 12.8 Hz),
128.8-129.0 (m), 127.5-127.7 (m), 127.2-127.9 (qd, J= 3.2 and 32.9 Hz), 126.8-126.8 (d,
J= 3.7 Hz), 122.4-122.5 (dd, J= 1.4 and 5.5 Hz), 121.4-121.5 (d, J= 15.8 Hz), 119.7127.7 (q, J= 270.1 Hz), 117.6-117.8 (dd, J= 4.4 and 8.0 Hz), 117.0-117.2 (d, J= 23.4
Hz), 111.9-112.1 (d, J = 16.9 Hz), 54.8-.54.8 (d, J = 4.4 Hz). 'F NMR (376 MHz,
CDC13 ): 6 -62.0 (s, 3F), -108.1 (s, IF), -134.1 (s, IF). LC-MS: RT(x2)
=
3.0 min (>99%
purity). HRMS (m/z): [M+H] calculated for C19 H 12 F 5N 4 , 391.0977; found, 391.0971.
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Applications. CSH Protoc. 2008;2008:pdb.prot5080.
171
B
A~
\
Fluconazole
resistant
C. albicans
einI
F~..conaw
1654 screen hits
i
1014 Non-toxic to Fibroblasts
CHpk~
622 active with IC50 < 1pM
48hr
296 Non-toxic alone, active vs Caci-8
300.000
Compounds
30 Non-Promiscous Scaffolds
3 Candidates for SAR
J'
8
E
.1
C
S
I
I.
-S
I
Inz-266
C
:5
I-
High NaCl
Aureobasidin
BPS
Cerulenin
Peroxide
Calcofluor White
Tunicamycin
Caspofungin
Simvastatin
AA
D
I
Inz-268 concentration (pM)
A
->
A
A
ARf IRA
Glucose
S. cerevislae
Glycerol
Glucose
C. albicans
Glycerol
Figure 1. Discovery of Inz-266 by high-throughput screening.
A) Schematic of the high throughput screen for inhibitors of azole tolerance. B) Triage
of hits from the screen. C) Profiling of fungal growth in stress conditions in the presence
of Inz-266 and other small molecules. D) Effect of Inz-266 on growth of S. cerevisiae
and C. albicans in media containing glucose or glycerol as carbon source.
172
A
Parental
WT
lnz-266Resistant #1
lnz-266Resistant #2
C
B
Growth In 8-lycerol Media
Cytochrome B Enzyme Actvity
1.0-
1o
--
cytocwome C
RedUction
+-
~-Type
Relative
WC-T9pe
CtB F90Y
Growth
COS F90Y
0.1
0.I
00.
-1
IN
0.4
0~In-W
0
1
logonz-2661 p
.1
1a
2
[Fluconazole] pjg/mL
D
0 0.12 0.25 0.5 1.0 2.0 4.0 8.0
WT
WT+tnz-266
riplt/A
0-00 0.25 0-50 0.75 1-00
F
E
CyEoclwome B
Enzyme Assay
Cell Prollferatlon
0.
401.4
+
5,
Human
C. albcans
Relatlv
ATP
L"vl
- HepG2
- 3T3 Fibroblast
10
-w
C. abicans
.
O.S.
0.4
lo [
.1
0
1
109 onz-2"s
"e Pn&-2661
2
Figure 2. Inz-266 is a fungal-selective inhibitor of mitochondrial cytochrome bcl.
A) Cytochrome B mutation in two Inz-266-resistant isolates, visualized by the Integrative
Genomics Viewer (IGV). B) Inhibition of yeast cytochrome B enzymatic activity by Inz266 in both wild-type and F90Y mutant mitochondria. C) Resistance of F90Y mutant to
growth inhibition by Inz-266. D) Fluconazole tolerance in C. albicansCaCi-2 WT,
treated with Inz-266, or riplz/A deletion mutant. E) Comparison of inhibition of
cytochrome B activity in C. albicans and human (HEK293) mitochondria. F)
Comparison of the effect of Inz-266 on proliferation of HepG2 and NIH-3t3 cells under
respiring conditions (DMEM-galactose) with inhibition of C. albicansin presence of 8
ptg/mL fluconazole.
173
B
A
P270(L/
-
Qi pocket
M7-
Qo pocket
Bovine= Brown
Yeast = Green
D
C
Nnmal Inhbitory Concentradon
WT
I
L275F
Cytochrome
B
P27OL
Mutation
LIGSOF
1147V
G143A
4
i
431
-f
Inz-266, pM
.
Figure 3. Structural analysis of Inz-266 binding to cytochrome bcl. A), Structure of
yeast cytochrome bc 1 indicating location of Qi and Qo pockets. B) Structural aligment
of yeast and bovine cytochrome bcl and indication of location of mutations that confer
resistance to Inz-266. C) MIC of Inz-266 under respiratory growth conditions against
mutants of the Qo pocket. D) Computational docking of Inz-266 onto Qo pocket of yeast
cytochrome bc
174
0.005
0.007
0.016
0.020
1.25x
Atovaquone
3.79
1.88
>32
12.72
<0.5x
Inz-266
8.092
45.32
1.655
>32
>19.3x
JBL-202
2.972
35.98
1.013
>32
>31.5x
JBL-22
1.058
8.655
0.722
>32
>44.3x
JBL-84
0.1448
4.622
0.305
>32
>104.9x
JBL-94
1.005
21.28
0.398
>32
>80.3x
JBL-140
0.026
1.044
0.080
>32
>398.4x
JBL-218
5.423
83.81
25.87
>32
>1.2x
22.63
109
4.987
>32
>2.4x
JBL-36
0.141
10.78
0.758
12.98
17.1x
JBL-182
0.289
6.507
0.381
10.59
27.7x
Antimycin A
JBL-114
N"-
C
Figure 4. Optimization of Inz-266 through medicinal chemistry. Compounds were
synthesized and tested as described in materials and methods.
175
A
DMSO
Fluconazole
DMSO
JBL-182
B
Time Kill Analysis
Fluconazole +I- JBL-182
Proliferation
(CFU/mL)
1071
+ Wild-type
10
+
ip1A/A
+ Wild-Type + JBL-1 82
Wild-Type + FCZ
riplA/A+FCZ
+ Wild-Type+ FCZ + JBL-182
1o
10
10
-Ing
0
24
Time (Hours)
48
Figure 5. JBL-182 renders fluconazole cidal and reduces the emergence of
resistance. A) 106 C. albicans wild-type (SC5314) cells were plated on media containing
64 mg/L fluconazole, IOpm JBL-182, or a both compounds in combination. Photos were
taken after 4d incubation. B) 5x10 3 C. albicans SC5314 cells were incubated in the
presence of 32 mg/L fluconazole, 10 [tM JBL-182, or both compounds, and C. albicans
rip1iz/ were incubated in the presence and absence of 32 mg/L fluconazole. Dilutions
were plated on YPD after 24 and 48 hr.
176
A
2% Glucose
Amino acids
Galactose
Serum
Lactate
Acetate
B
Wild Type
+ DMSO
Wild Type
+INZ-182
npN
30'
120
C
D
Fungal Survival In Co-culture
Filawentatlon In Macrophages
Percent
PerentPercent
(1201
wrf
Hyphalof
225
DMSO
Inz-182
tiplAiA
DMSO
Inz-182
riplIA
Figure 6. Inhibition of cytochrome bcl by JBL-182 prevents adaptation to
suboptimal carbon sources. A) Growth of_10 4 C. albicans SC5314 cells on agar plates
containing various carbon sources in the presence of a filter disc containing DMSO or
10pg JBL- 182. B) Co-culture of BFP-labeled C. albicans and GFP-tubulin-labeled
mouse bone-marrow-derived macrophages. C) Quantification of effects observed in (B)
D) Fungal survival quantified by alamar blue dye reduction after 14 hr co-culture at MOI
of 0.125 in the presence of bone marrow macrophages.
177
Mouse Tall Vein Infection
A
CaCi-2
CaCi-2 rplA/A
--CaCi-2 niplA/A
+ RIP1
Percent
survival Is*
2S.
A
A5
I*
S
0
2S
Days
Kidney and Brain Colonization
No Drug Treatment
B
Total
Recovered
CaCi-2
CaCi-2 riplA/L
CaCi-2 rip1A/A
+ RIPI
2.0o10
CFU
2.00101
*90
0
0
2.01021
_:.
2.0010
el
13 flulifts
LnWoIDOeoon
(20CR))
Brain
Kidneys
Recurrence of Fluconazole-Resistant infection
C
- CaCi-2
- CaCi-2 rip 1/A
- CaCi-2 riplA/A
1-09
Percent
survival
+ RIPI
2
0cilL5
24 mngazg
Fluconazote
D
Days
Kidney and Brain Colonization
After Fluconazole Treatment
Total
2.03,101
* CaCi-2
CFU
Recovered
CaCi-2 riplA/A
2.001
2.0a1
2.031
Brain
Kidneys
(20 CFUJ1
Figure 7. Loss of cytochrome bcl severely reduces fungal virulence and fluconazole
tolerance, but increases brain colonization. A) Tail-vein infection of Balb/C mice with
5x105 fungal cells of indicated strains. N = 7 mice per strain. B) Viable fungal burden
isolated from brain 4d after infection with 6x104 cfu of indicated strains. N = 6-7 mice
per strain. C) Survival of mice infected with 106 cfu of CaCi-2 then treated for 3d with
24 mg/kg fluconazole. N = 6-10 mice per strain. D) Mice were infected with 6x104 cfu of
the indicated strains, treated for 3d with 24 mg/kg fluconazole, monitored for 4 more
days, then sacrificed and fungal burden in kidney and brain was quantified.
178
A
PASIVPEWYLLPFYAILRSIPDKLLGVIT
PPSIVPEWYLLPFYAILRSIPDKLGGVIA
PPAIVPEWYLLPFYAILRSIPNKLLGVIA
PASIVPEWYLLPFYAILRSIPDKLGGVIA
PPSIVPEWYLLPFYTILRSIPNKLLGVVG
PPHIKPEWYFLFAYTILRSVPNKLGGVLA
Saccharomyces cerevisiae
Candida albicans
Aspergillus fumigatus
Rhizopus oryzae
Cryptococcus neoformans
Homo Sapiens
B
294
266
RPMI,
0.2% Glucose
YNB,
2% Glucose
YNB,
2% Glvcerol
Rhizopus
oryzae
Aspergillus
terreus
Scedosporium
prolificans
Figure 8. Broad-spectrum activity of JBL-182. A) Alignment of a contiguous
segment of Qo site of cytochrome bel from indicated species. Mutations conferring
resistance to indazoles are indicated in bold; residues conserved in all of these fungi but
not humans are indicated in red. B) Activity of JBL-182 against indicated species.
ATCC reference strain of each species was grown on potato-dextrose-agar plates, then
4
resuspended in water with 0.1% Tween, counted, and 10 cells were plated on indicated
media with filter discs containing DMSO or I Ogg JBL-182 and incubated at 35*C.
Images were taken after 24 hr (Rhizopus) or 48 hr (Aspergillusand Scedosporium).
179
Chapter Five:
Conclusions and Future Directions
180
AMPHOTERICIN B AND THE CONSTRAINTS OF EVOLUTION
In this thesis, I investigated the mechanisms that promote and restrict the
evolution of resistance to antifungal drugs in the clinic. My work establishes that
mutations that confer resistance to AmB impose a strict requirement for high levels of
Hsp90 function, sensitize Candidato stresses encountered in the host environment, and
disarm the virulence factors of this pathogen.
I suggest it is the sum of these effects that
has prevented the emergence of resistance to AmB in the clinic. These specific
mechanisms further our understanding of the role of stress responses in the rapid
evolution of new traits. They also might inform the design of novel resistance-refractory
antimicrobial agents. Here I discuss the broader implications of my main conclusions as
well as future directions to extend this work.
Expanding the role of Hsp90 and stress responses in the evolution of new traits
Hsp90 is a highly abundant chaperone whose activity governs the folding and
function of diverse metastable proteins, especially those involved in signal transduction
(including kinases, steroid hormone receptors, and ubiquitin ligases) [1]. Importantly, its
activity is intimately linked to environmental conditions. Although Hsp90 is normally
produced far in excess of cellular demands, stressful conditions can overburden Hsp90's
folding capacity. This protein folding burden modulates the output of the pathways in
which Hsp90 client proteins function. These properties position Hsp90 to have powerful
effects on the emergence of new traits.
At least five different ways in which Hsp90 can affect rapid evolutionary change
have been described. First, Hsp90 directly binds and stabilizes novel mutated proteins
181
that might otherwise misfold and be degraded, enabling them to have immediate
phenotypic consequences [2,3]. Second, Hsp90 buffers pre-existing genetic variation,
keeping it cryptic until it is released by the broad effects of stress on the proteome [4-6].
Third Hsp90 can regulate epigenetic factors [7]. Fourth, overburdening of Hsp90 can
release transposons [8]. Fifth, Hsp90 enables the evolution of drug resistance by
supporting stress responses that mitigate drug toxicity [9].
Our studies on the emergence of resistance to AmB now provide evidence for a
sixth mechanism by which Hsp90 mediates evolutionary change: empowering cells to
respond to, and withstand, the stressful consequences of disruptive new mutations. The
ergosterol biosynthesis mutations that enable AmB resistance severely perturb membrane
physiology by altering the structure of a key lipid that regulates fluidity, permeability,
and transmembrane protein functions. As such, the survival of AmB-resistant mutants
becomes strictly dependent on the capacity of Hsp90 to mature certain stress response
proteins, including calcineurin. This mechanism is distinct from previous studies of
Hsp90's role in azole and echinocandin resistance. For azole and echinocandin
resistance, the mutations resistance mutations are not the source of stress; instead stress
comes as a direct effect of toxicity induced by the drug. Our findings indicate that Hsp90
may have an even broader role in in evolutionary processes: allowing cells to tolerate the
drastic and disruptive physiological changes inherent in novel traits.
182
Extending the evolutionary breadth of the AmB resistance-virulence tradeoff
One major question that was not addressed in our work is how broad the tradeoff
between resistance to AmB and virulence is across evolutionarily diverse species. Our
work focused primarily on C. albicans for several reasons. First, C. albicans has been
the most common fungal pathogen in hospitals for many decades, and certainly has been
given broad enough exposure to AmB for resistance to evolve. Second, ample evidence
is available from large surveillance studies of this species to establish that clinical
resistance to AmB is extremely rare [10]. Third, as this species is genetically tractable,
we were able to demonstrate with genetically matched strains that every mutation known
to confer strong resistance completely abrogates virulence in mice. With this foundation,
it is now important to determine how broadly our findings extend to fungal pathogens
other than C. albicans.
One of the main mechanisms underlying the avirulence of AmB-resistant C.
albicansis the loss of filamentation in resistant mutants. However, many pathogenic
fungi are not filamentous, but grow as yeasts. Are these pathogens more likely to evolve
resistance to AmB? Isolates of the evolutionarily distant non-filamentous fungus
Candidaglabratawith ergosterol biosynthesis mutations have been described in a
handful of cases [11]. It is not known if these mutants are merely bystanders that
survived amphotericin resistance, or in fact retain virulence. However, given the haploid
nature of C. glabrata,if loss of function mutations in ERG2 or ERG6 did not carry major
fitness costs, nearly every population would be expected to rapidly evolve resistance to
AmB. Another non-filamentous species of interest is the extremely distant yeast
Cryptococcus neoformans. Amphotericin B resistance has been reported in isolates from
183
several HIV patients with cryptococcosis, but resistance still remains exceedingly rare
[12,131. Thus, it appears likely that even in non-filamentous fungi, the modifications of
ergosterol biosynthesis that confer AmB resistance carry severe selective disadvantages.
These defects may include the deficiencies in stress tolerance and host adaptation that we
demonstrated for C. albicans.
Although AmB has at least moderate inhibitory activity against virtually every
fungus, some species are intrinsically more resistant than others. Aspergillus terreus is
generally considered to be insusceptible to AmB (but is killed by high concentrations of
the drug). Previous work has suggested that both heightened oxidative stress responses
and limited drug accessibility to the plasma membrane could contribute to the intrinsic
AmB resistance of A. terreus [14,15]. Further study is warranted to elucidate how this
fungus eludes killing by AmB, especially in light of the new "sterol sponge" model of
AmB action [16].
Deepening our understanding of the tradeoff between resistance and virulence
Returning to C. albicans, another interesting avenue of research will be to exploit
the availability of genetic tools and mouse models of infection to further probe the fitness
costs of resistance to AmB in vivo. One intriguing possibility is that AmB-resistant
mutants lose systemic virulence but retain the potential to colonize specific host niches.
Many of the strains tested in antimicrobial susceptibility surveillance studies come from
cases of thrush. Given the low rate of AmB resistance in these studies, AmB-resistant
fungi appear unlikely to cause disease in the oropharynx. However, it is possible that
AmB-resistant strains could persist as commensals of the gastrointestinal tract or other
184
niches, depending on their selective disadvantage compared to AmB-sensitive strains. It
is tempting to speculate that resistance to AmB merely shuts down the capacity of this
fungus for virulence, but not for other forms of host colonization. As such, the selective
pressure to evolve AmB resistance would remain low, since it would only prove
beneficial during the treatment of a terminally ill patient (and lead to death of the host,
for that matter). If this is true, the resilience of AmB????may be attributed to the
tendency of the drug to select for an avirulent population, indirectly achieving the desired
goal of approaches that seek to directly inhibit virulence factors [17].
It may also be interesting to determine if there are mechanisms that bypass the
virulence defects of amphotericin-resistant C. albicans. Recent work has described
several fluconazole-resistant erg3 mutants that retain fully wild-type filamentous growth
and virulence, unlike previous isolates lacking erg3 that exhibited moderate fitness
defects [18]. This result demonstrates that it is possible for cells to buffer the fitness
costs of certain ergosterol mutations. However, the fitness costs of AmB-resistant erg2,
erg6, and erg3/erg] mutants were much more severe than those observed for
fluconazole-resistant erg3 mutants in the strains we constructed. One approach to
determining the potential of new mechanisms to buffer these mutations would be to
passage an amphotericin-resistant deletion mutant through several rounds of infection in
a mouse. If such passaging selected for mutants that increased its fitness and virulence, it
would be possible to sequence the genome of the resulting strains and identify the
mutations responsible for buffering the parental phenotype.
185
Lessons from AmB for the Design of Resistance-Refractory Therapeutics
The ability to design drugs that are inherently refractory to resistance would have
extraordinary value in the treatment of many types of infections. Our work points to one
such novel design strategy. Specifically, searching for compounds for which resistance
can evolve only via mechanisms that bear substantial fitness costs. Predicting apriori
whether a specific compound or drug target will evade resistance is extremely difficult.
To shed light on new ideas for the design of resistance-refractory agents, it is important to
first put our findings with amphotericin in the context of other antimicrobial drugs, in
particular antibacterial agents, for which resistance currently presents an enormous
societal challenge. Admittedly, comparisons of resistance rates and mechanisms between
antibacterial and antifungal agents are difficult to make. Bacterial drug resistance often
involves the rapid and efficient horizontal transfer of resistance mechanisms, while
antifungal drug resistance spreads much more slowly, and must often evolve de novo.
Nevertheless, it is useful to first examine how AmB compares with different
antibacterials with regards to the mechanisms and costs of resistance.
As most antibacterials are natural products, mechanisms to detoxify them have
previously evolved in the natural world, sometimes in the species that produces the
antibiotic itself. The most prevalent and worrisome resistance mechanisms involve the
activity of enzymes that modify the drug itself, such as aminoglycoside acetyltransferases
or beta-lactamases [19]. In general, such drug detoxification mechanisms carry only
miniscule fitness costs. However, if a particular antibiotic is kept out of use for many
years, the cost of maintaining the genes encoding these resistance determinants
(especially if they are encoded on a plasmid) tends to decrease their prevalence [20].
186
Remarkably, although AmB is a natural product, no drug-detoxifying enzymes are known
to act upon it. This is a possible consequence of the unusual polyene structure of AmB,
which does not resemble any cellular metabolites. Thus, there are no enzymes acting on
similar molecules that could evolve the ability to degrade AmB through subtle changes in
substrate specificity. Indeed, synthetic antibacterials that do not closely resemble cellular
metabolites, including oxazolidinones and fluoroquinolones, remain generally
insusceptible to resistance by enzymatic modification [19].
Another common mechanism of antibiotic resistance that must be evaded in
bacteria and fungi alike is pump-mediated efflux. Overexpression of multidrugresistance (MDR) efflux pumps in certain species, including Pseudomonas aeruginosa,
does appear to have some negative consequences for virulence in certain models [21].
However, the frequent isolation of MDR Pseudomonas (and other gram negative
bacteria) in the clinic suggests that these costs can either be buffered by compensatory
mechanisms, or still permit virulence in humans. Drug efflux is also a major mechanism
of antifungal drug resistance, to the azoles. As AmB does not need to enter cells to be
active, it is not susceptible to such a mechanism of resistance. Thus, although targets on
the cell surface are few in number, they present this highly attractive benefit of evading
efflux.
Yet another important mechanism of resistance is changes in the drug target
These can occur through, amino acid substitutions in the drug-binding site of the target
protein, post-translational modification of the target protein (typically methylation), or
alteration of the structure of non-protein targets such as lipids [19]. Modifications of
amino acid side chains can readily occur without significant fitness costs in most cases,
187
and single mutations that confer resistance must be avoided. One approach to designing
agents that are not susceptible to resistance through point mutation is to design inhibitors
that fit within the "substrate envelope" of the target. Using this approach, mutations that
abrogate drug binding also abrogate substrate binding and thereby enzymatic function
[22]. Cellular components that present fewer opportunities for modification than proteins
(and ribosomal RNA) are better targets for resistance-refractory agents. Importantly,
when such cellular components are central and highly conserved, their modification bears
more severe fitness costs for the pathogen.
Advantages and limitations of targeting Lipids
Combining all of these previous observations with my own results with AmB
suggests that lipids are ideal targets for resistance-refractory antimicrobial agents. First,
they are less amenable to modification than other biomolecules. Their relatively small
and simple structures are not readily modified without severe consequences for cellular
membrane structure. As many lipids are abundant and conserved, membrane proteins
have evolved to function in concert with their structures. As such, even small
modifications in lipid structure can have drastic consequences for the function of many
such proteins. Second, cell-surface lipids can be targeted without the potential for
resistance mediated by efflux. Finally, as drugs targeting lipids can rapidly kill cells
through immediate permeabilization, the pathogen may have insufficient time to deploy
enzymatic modification of such compounds prior to cell death.
Available antibacterial drugs targeting membrane phospholipids have also
remained effective over time, albeit not quite as refractory to resistance as AmB. The
188
polymyxin-based drug colistin is regaining clinical use against gram negative bacteria,
which it kills by binding to the outer membrane. However, resistance has repeatedly
emerged through modification of the structure of outer membrane lipopolysaccharide
[23]. This mechanism may reflect that the bacterial outer membrane acts mainly as a
sieve from the exterior world, separated from the bacterial cytoplasm by a cell wall and
inner membrane. As such, the structures of outer membrane lipids, which are already
much larger and more complex than sterols, can be readily modified without the
phenotypic consequences inherent in modifying the eukaryotic plasma membrane.
Daptomycin acts on the membrane of gram positives (interior to the cell wall), and is
generally refractory to resistance, but is slowly losing efficacy. Resistance to daptomycin
appears to occur through complex modifications of phosphatidylglycerol that are still
poorly understood [24].
The ability of bacteria to evolve resistance to these two agents may reflect their
greater tolerance for perturbation of membrane structure compared to fungi. It may also
reflect a greater diversity of large, complex, and modifiable lipid structures in bacterial
membranes than in fungal membranes. Another possibility is that the resistancerefractory nature of AmB is a property of targeting sterols, not phospholipids; these two
classes of lipid have highly distinct structures and functions. Unfortunately, bacterial
membranes are generally devoid of sterols. Nevertheless, the further characterization of
bacterial membrane components might serve to identify a similarly irreplaceable target,
and thus present a promising target for intervention.
189
FUTURE PROSPECTS
The evolutionary history of life on earth suggests that over time, populations will adapt to
nearly any challenge they encounter. However, ecological studies suggest that focused
adaptation to one extreme condition often diminishes the ability of an organism to
tolerate other stresses. Our work suggests that AmB exploits this evolutionary
conundrum by imposing a tradeoff between resistance and adaptation to the host
environment. Finding new ways to impose evolutionary conflicts on pathogens is a
promising strategy to stop the global challenge posed by drug resistance.
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15. Blum G, Perkhofer S, Haas H, Schrettl M, Wurzner R, et al. (2008) Potential basis for
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16. Anderson TM, Clay MC, Cioffi AG, Diaz KA, Hisao GS, et al. (2014) Amphotericin
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191
Appendix A:
Selective Targeting of the Mitochondrial
Phosphate CarrierYields an Antifungal
Therapeutic Strategy that Evades
Resistance
*This chapter is currently in preparation for submission.
192
Selective Targeting of the Mitochondrial Phosphate Carrier Yields an Antifungal
Therapeutic Strategy that Evades Resistance
Benjamin M. Vincent" 2 , Willmen Youngsaye 3 , Catherine Hartland3 , Alex K. Lancaster2
Ruth Scherz-Shouval 2, Luke Whitesell 2, Susan Lindquist24
Affiliations
1 Microbiology Graduate Program, Massachusetts Institute of Technology, 77
Massachusetts Avenue, Cambridge, Massachusetts, 02139, United States of America,
2 Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge,
Massachusetts, 02142, United States of America,
3 Chemical Biology Platform and Probe Development Center, Broad Institute of MIT and
Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA
4 Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of
Technology, Cambridge, Massachusetts, 02139, United States of America
ABSTRACT
Novel approaches to the treatment of drug-resistant fungal infections are sorely
needed. Here we describe the discovery of FT-350, a highly potent thiohydantoin
derivative that kills fungi by targeting the mitochondrial phosphate carrier. A single
residue of the mitochondrial phosphate carrier (Asni 84) is responsible for a >500-fold
difference in potency of FT-350 against fungal and human cells. Resistance to FT-350
emerges primarily through deletions in mitochondrial DNA that disable respiration, but
render cells hypersensitive to widely-used azole antifungals. These data suggest that
selective targeting of mitochondrial phosphate transport could provide an avenue to the
development of an antifungal combination therapy that evades resistance.
INTRODUCTION
Although many complex natural products that selectively kill fungi have been
described, tractable synthetic compounds with potent and selective antifungal activity are
exceedingly rare [1,2]. Indeed, azole antifungals targeting Erg IIp are the only agents of
purely synthetic origin to have gained broad clinical use against fungal infections (with
193
the exception of topical agents). Although small, synthetic compounds with useful
antifungal activity are very difficult to find, they have great advantages over natural
product-based agents. First, their chemical tractability allows for repeated rounds of
optimization for important therapeutic parameters, including potency, selectivity,
spectrum of activity, off-target effects, in vivo half-life, and oral bioavailability. Second,
small synthetic agents are better than large natural products at penetrating hard-to-reach
niches in which fungal pathogens can hide and resist treatment [3]. Third, scale-up and
production of synthetic agents is generally a much simpler and more predictable process
than it is for natural products. Most importantly, a small synthetic agent would be an
ideal candidate for an oral combination therapy with azoles that could clear fungal
infections more rapidly and prevent the emergence of resistance.
RESULTS
Identification of a Thiohydantoin Derivative with Potent Antifungal Activity
In a high-throughput screen for small molecules that reverse azole resistance in C.
albicans (described in detail in chapter 4), we identified a thiohydantoin-based scaffold
(Figure IA, left panel) with potent antifungal activity as a single agent (MIC = 0.3 pM).
This compound was a hit in less than 1% of other screening assays in which it was tested
(by other investigators), suggesting it does not act promiscuously [4]. Initial structureactivity relationship studies performed around this scaffold identified a fluorinated
derivative with 5-fold improved potency, which we named FT-350 (Figure 1A, right
panel). Under standard susceptibility testing conditions, FT-350 exhibited surprisingly
strong antifungal activity against the azole-resistant strain CaCi-2 as a single-agent (MIC
0.0625-0.125ptM) (Figure IB). Antifungal activity was even more potent in the presence
194
of fluconazole (MIC 0.015-0.03pM). Extensive details on the testing and structureactivity relationships of the thiohydantoin scaffold are available online in the indicated
reference (MLPCN probe ML316) [4].
Initial studies with FT-350 showed an unusual pattern of antifungal activity
against S. cerevisiae and C. albicans. FT-350 potently inhibited the growth of S.
cerevisiae on media containing glycerol, but not glucose, as a carbon source (Figure 1 C).
This result indicated that it might act as an inhibitor of mitochondrial respiration.
However, FT-350 inhibited growth of C. albicans under both fermenting and respiring
conditions (glucose and glycerol) with equal potency (Figure ID). Although S.
cerevisiae represses respiration in the presence of glucose and C. albicansdoes not,
respiration is not required for growth of C. albicans [5]. Taken together, these results
indicated that FT-350 did not act as a straightforward inhibitor of the electron transport
chain.
To determine the fungal selectivity of FT-350, we tested it against two
mammalian cell lines, mouse NIH-3T3 fibroblasts and human HepG2 hepatocellular
carcinoma cells. As our fungal data suggested that this agent targets respiration, we
tested the toxicity of FT-350 under forced respiration conditions, using media with
galactose as the primary sugar and quantifying cellular ATP levels [6]. Under these
conditions, we noted no toxicity to the HepG2 cells at 32 pM, and only mild toxicity to
NIH-3T3 fibroblasts at 16 and 32 pM (Figure lE). These data suggest that FT-350
exhibits a therapeutic index of at least 500-fold in cell culture.
195
Loss of Mitochondrial Complex IV Confers Resistance to FT-350
In an attempt to identify the target of FT-350, we selected mutants resistant to this
compound to be used for whole-genome sequencing. Selection of resistant mutants in C.
albicans on media containing 2% glucose and 5pM FT-350 resulted in a high rate of
emergence (~ 10-) of slow-growing colonies that, upon retesting, were completely
unaffected by treatment with FT-350. Genome sequencing of one of these mutants along
with the parental strain revealed that it contained a large deletion in the mitochondriallyencoded core enzymatic subunit of cytochrome C oxidase, COX (Complex IV of the
election transport chain) (Figure 2A). Two other mutants with the same phenotype also
harbored deletions in the mitochondrial COX gene (data not shown). Cytochrome C
oxidase is a highly conserved protein complex that acts directly after cytochrome bc 1
(ubiquinol cytochrome C reductase, described in chapter four) in the electron transport
chain. However, since cytochrome C oxidase is not required for fungal growth, and its
deletion confers resistance to FT-350. Thus, FT-350 cannot kill fungi through inhibition
of FT-350. Rather, loss of cytochrome C oxidase appears to detoxify the effects of FT350. Indeed, we also noted that deletion of RIP] of complex III also conferred resistance
to FT-350 (data not shown). Further analysis is needed to determine which mitochondrial
proteins and complexes must be present and functional for FT-350 to be toxic.
The finding that loss of mitochondrial respiration is a major mechanism of
resistance to FT-350 is particularly interesting because loss of mitochondrial respiration
renders fungi hypersensitive to azole antifungals (chapter four). To demonstrate pursue
the potential of these two compounds in combination, we monitored their effects on the
CaCi-2 fluconazole-resistant strain and the FT-350-resistant isolate derived from CaCi-2.
196
We tested these two strains on media containing fluconazole, FT-350, or both
compounds, as well as under respiring conditions (glycerol media) (Figure 2B). As
expected, the FT-350-resistant cytochrome C oxidase mutants were unable to grow on
glycerol media and were also acutely hypersensitive to fluconazole. Thus, as the
dominant mechanism of resistance to FT-350 leads to hypersensitivity to fluconazole,
these two agents could be used in a combination therapy that poses very high barriers to
resistance.
A Point Mutation in the Mitochondrial Phosphate Carrier Confers Resistance to
FT-350
Our selections for resistance to FT-350 on glucose-containing media allowed for
the emergence of respiration-deficient mutants and thus did not reveal the target. To
identify mutants that bypass this mechanism of resistance, we selected for colonies on
media containing FT-350 with glycerol as the sole carbon source, so that cells that lose
respiration would be unable to grow, and only respiration-competent FT-350-resistant
mutants could emerge. Under these conditions, mutants emerged at a much lower rate
(<108), and were not slow-growing. Genome sequencing of three independently-selected
mutants resistant to FT-350 revealed that they all carried an identical heterozygous
mutation in MIR], which encodes the mitochondrial phosphate carrier protein [7,8]
(Figure 3A). This mutation replaced asparagine 184 of the protein with threonine
(henceforth referred to as Ni 84T).
Miri p is localized to the inner membrane of mitochondria and transports
phosphate into the mitochondrial matrix [9,10]. It is the sole source of phosphate for the
production of ATP from ADP by ATP synthase. Intriguingly, no small molecule
197
inhibitors of this protein have been identified, and only low-affinity, non-selective, thiolreactive agents (such as mersalyl and N-ethylmaleimide) have been used to inhibit its
activity in vitro [11]. Mirip is required for respiration of S. cerevisiae, but not for
fermentation, and it has not been studied in C. albicans or other fungal pathogens [7].
We compared the sequence surrounding the resistance-determining N 184 residue
of the mitochondrial phosphate carrier protein homologs across various fungi as well as
the human homolog of the protein. Intriguingly, asparagine is present at position 184 in
all species sensitive to FT-350, but threonine is present in this position in all species
resistant, including humans (human homolog is named SLC25A3) (Figure 3B). These
data suggest that the presence of asparagine and not threonine at position 184 of the
Mirip protein dictates the activity of FT-350.
To confirm that the Ni 84T mutation is critical for the activity of Miri p, we
cloned it into an integrating C. albicans expression vector and transformed the construct
into a wild-type strain [12]. Indeed, transformants expressing the Ni 84T mutation
exhibited a 512-fold increased MIC for FT-350 (Figure 3C). Importantly, while cell
culture assays for mammalian cell cytotoxicity are liable to artifacts, the extremely strong
resistance conferred by replacing a single residue of fungal Mirip with the corresponding
residue of the human homolog provides compelling evidence for the fungal selectivity of
this compound.
To further this analysis, we perturbed the copy number of MIR] in C. albicans,
and tested its effects on growth inhibition by FT-350. Deletion of one copy of the MIR]
gene caused a 2-fold decrease in the MIC for FT-350, supporting that it may be the target
of the compound on the principle of induced haploinsufficiency [13,14] (Figure 3C).
198
Adding one additional copy of the MIR] gene resulted in a ~2-fold increase in resistance
to FT-350, also supporting that Mirlp could be the drug target. However, demonstration
that FT-350 inhibits mitochondrial phosphate uptake in mitochondria purified from wildtype C. albicans, but not strains expressing the Ni 84T mutant, would be required to
confirm Mirlp as the target.
DISCUSSION
FT-350 presents a novel and intriguing mechanism of action for a small molecule.
The questions raised by our unexpected findings with this compound may lead to a
deeper understanding of the poorly studied mitochondrial phosphate carrier. An initial
question concerns why this drug is potently toxic to C. albicans as a single agent, when
other inhibitors of mitochondrial respiration complexes do not kill this species. Several
hypotheses could explain this result. First, inhibition of the phosphate carrier could
depolarize the inner membrane proton gradient, as this protein co-transports phosphate
with protons. Evidence for such a mechanism of killing by FT-350 would be bolstered
by demonstrating that treatment with the compound directly depolarizes the
mitochondrial membrane potential (using potential-sensitive dyes), and that other
uncouplers such as FCCP or 2,4 DNP also kill C. albicans. Second, it is possible that
inhibition of phosphate transport into mitochondria would induce an osmotic shock that
renders mitochondrial membranes swollen and permeable, and could trigger pathways of
programmed cell death. Third, inhibition of phosphate accumulation in the mitochondrial
matrix could cause a rapid and severe buildup of ADP, which might be toxic on its own.
Finally, it is important to note that while mitochondrial ATP production is not required
199
for survival, mitochondria perform other essential activities in the cell, including lipid
biosynthesis and redox regulation. Thus, FT-350 could kill cells by disrupting one of
these other activities.
Nevertheless, the finding that loss of electron transport chain components (in
particular cytochrome C oxidase) confers resistance to FT-350 bolsters a toxic "gain of
function" mechanism for its antifungal activity. Whatever the mechanism, FT-350
provides the first chemical tool for studying this poorly understood yet broadly conserved
protein, and reveals an unexpectedly vital role of the phosphate carrier in cell survival.
Moreover, the potent, single-agent activity of FT-350 and its multilevel synergy with
fluconazole suggest that this compound or others like it may hold great promise for the
treatment of drug-resistant Candidainfections.
MATERIALS AND METHODS
Fungal Growth Assays in Liquid Culture
Toxicity of FT-350 to fungal cells was tested as described in chapter four, with the
following specifications. For glucose/glycerol comparison, media consisted of yeast
nitrogen base and complete amino acid mix (Difco) supplemented with 2% glucose or
2% glycerol. Growth was determined by OD600 at 24 hr (C. albicans glucose or
glycerol, S. cerevisiae glucose) or 48hr (S. cerevisiae glycerol). Relative growth was
normalized to a no-drug control. MIC testing and growth inhibition of strains with
varying levels of MIR] was performed in RPMI media with 0.2% glucose, testing for
24hr.
Mammalian Cell Cytotoxicity
200
Toxicity to HepG2 and NIH-3t3 cell lines was tested exactly as described in chapter four,
recording results at 72hr, and normalizing to DMSO control.
Genome sequencing and polymorphism discovery
Genome sequencing and polymorphism discovery were performed as described in [15]
and chapter four.
Spot assays
Fungal growth on agar plates was performed by standard methods as previously
described [15].
Cloning of MIRI and Transformation of C. albicans
The MIR] ORF was cloned from wild-type or Ni 84T mutant strains into the pDIS3
integrating vector [12] using the following oligonucleotides: MIR1-975up-f,
tattatcgatTGGACAGGACTAACCCCAAC; MIRi-950dn-r,
tatacccgggCAACAATCTTGAATCCGGGTA. MIR] heterozygotes were generated by
deleting one copy of MIRI using the HIS3 marker [16] and the following
oligonucleotides: MIR I-updel-fATTTGCCTGCCAGAATCAAA; MIRI-updel-r,
CACGGCGCGCCTAGCAGCGGGAGGGAATTTTTGGTTGGAA; MIRi-dndel-f,
GTCAGCGGCCGCATCCCTGCCCATGAATGAATAAGGGAAAGC, MIRI-dndel-r,
CCATGAATGAATAAGGGAAAGC.
Transformation of C. albicanswas performed
using standard protocols [16].
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3. Odds FC, Brown AJ, Gow NA (2003) Antifungal agents: mechanisms of action.
Trends Microbiol 11: 272-279.
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Metabolic Requirements for Fungal Virulence - Probe 1. Probe Reports from
the NIH Molecular Libraries Program. Bethesda (MD).
5. Sun N, Fonzi W, Chen H, She X, Zhang L, et al. (2013) Azole susceptibility and
transcriptome profiling in Candida albicans mitochondrial electron transport
chain complex I mutants. Antimicrob Agents Chemother 57: 532-542.
6. Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y (2007) Circumventing the
Crabtree effect: replacing media glucose with galactose increases
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7. Murakami H, Blobel G, Pain D (1990) Isolation and characterization of the gene
for a yeast mitochondrial import receptor. Nature 347: 488-491.
8. Wohlrab H, Briggs C (1994) Yeast mitochondrial phosphate transport protein
expressed in Escherichia coli. Site-directed mutations at threonine-43 and at
a similar location in the second tandem repeat (isoleucine-141).
Biochemistry 33: 9371-9375.
9. Phelps A, Wohlrab H (2004) Homodimeric mitochondrial phosphate transport
protein. Transient subunit/subunit contact site between the transport
relevant transmembrane helices A. Biochemistry 43: 6200-6207.
10. Phelps A, Briggs C, Mincone L, Wohlrab H (1996) Mitochondrial phosphate
transport protein. replacements of glutamic, aspartic, and histidine residues
affect transport and protein conformation and point to a coupled proton
transport path. Biochemistry 35: 10757-10762.
11. Phelps A, Wohlrab H (1991) Mitochondrial phosphate transport. The
Saccharomyces cerevisiae (threonine 43 to cysteine) mutant protein
explicitly identifies transport with genomic sequence. J Biol Chem 266:
19882-19885.
12. Gerami-Nejad M, Zacchi LF, McClellan M, Matter K, Berman J (2013) Shuttle
vectors for facile gap repair cloning and integration into a neutral locus in
Candida albicans. Microbiology 159: 565-579.
13. Xu D, Jiang B, Ketela T, Lemieux S, Veillette K, et al. (2007) Genome-wide fitness
test and mechanism-of-action studies of inhibitory compounds in Candida
albicans. PLoS Pathog 3: e92.
14. Roemer T, Boone C (2013) Systems-level antimicrobial drug and drug synergy
discovery. Nat Chem Biol 9: 222-231.
15. Vincent BM, Lancaster AK, Scherz-Shouval R, Whitesell L, Lindquist S (2013)
Fitness trade-offs restrict the evolution of resistance to amphotericin B. PLoS
Biol 11: e1001692.
16. Noble SM, Johnson AD (2005) Strains and strategies for large-scale gene deletion
studies of the diploid human fungal pathogen Candida albicans. Eukaryot Cell
4: 298-309.
202
A
U*
*
-
FT350: MIC=0.06 pM
Hit: MIC=0.3 pM
B
Relative
Growth1.00
FT-350 Alone
FT-350 + 8 pg/mi Fluconazole
0.25
0.c 02 0.004 0.006 0.016 0.031 0.063 0.125 0.250 0.500 1.000
[FT-350, IMI
C
Relative
Growth
0.75
- S. cerevisiae, Glucose
- S. cerevisiae, Glycerol
0.50
0.004
0.250
0.063
0.616
1.000
[FT-350], pM
D
Relative
Growth -*
0.75,
C. albicans, Glucose
C. albicans, Glycerol
0.50'
0.2&
0.004
0.250
0.063
0.016
1.000
[FT-350, pM
E
Relative
Growth 1.00.
(Galactose
Media) 0.7&
- Human HepG2
- Mouse 3t3 Fibroblasts
0.50'
0.25
0.5 0.50
1
2
a
4
16
32
[FT-350, 1 M]
Figure 1. A thiohydantoin derivative with potent, fungal-selective growth
inhibition. A) Structures of initial hit compound and derivative FT-350. B) Growth
inhibition of C. albicans CaCi-2 strain in RPMI media by FT-350. C-D) Growth
inhibition of S. cerevisiae and C. albicans on glucose or glycerol media. E) 72 hr growth
inhibition of HepG2 hepatocellular carcinoma cells or NIH-3T3 mouse fibroblasts by FT350 in DMEM-galactose media, quantified by Cell titer-glo ATP assay.
203
A
FT350-R
COX(mtDNA)
I
Glucose
+
B
Glucose
Glycerol
Fluconazole
FCZ-R
DMSO
FT350-R
FCZ-R
0.25 gM
FT350
FT350-R
Figure 2. Loss of cytochrome oxidase confers resistance to FT-350 but sensitivity to
fluconazole. A) Genome sequence and alignment of a FT-350-resistant mutant with a
large deletion in the mitochondrial COX gene. B) Serial dilution spot assay of CaCi-2
("FCZ-R") and the FT350-resistant mutant sequenced above. Plates were photographed
at 48hr after spotting yeast. FT-350 was added at 0.25 pM; fluconazole was added at 16
pg/mL.
204
A
WT
R2
R3
MIR1
B
173
S. Cerevisiae
C.
C.
C.
C.
A.
Glabrata
Albicans
albicans-R
Neoformans
Funigatus
H. Sapiens
188
FTPILFKQIPYNIAKF
FTPILFKQIPYNIAKF
FTPILFKQIPYNIAKF
FTPILFKQIPYTIAKF
FGPILFKQVPYTMAKF
FGPILFKQIPYTMAKF
GVAPLWRQIPYTMMKF
[FT-350 pM
C
0 0.0010.003 0.007 0.015 0.0310.062 0.125 0.25 0.5O 1
2
4
8
16
MIR1/MIR1
MI/mirlA
MIR1/MIRI+pMIR1
MIR1/MIRI+mir1N184T
0.00 0.25 0.50 0.75 1.00
Figure 3. The mitochondrial phosphate carrier Mirip is a potential target of FT350. A) Alignment of MIR] sequence from wild-type and three independent FT-350resistant mutants, indicating heterozygous N184T mutation in mutants. B) Alignment of
protein sequence of amino acids 173-188 from the Mirip homologs of indicated species.
Sequences were obtained by BLAST search of the S. cerevisiae Mirip sequence against
the non-redundant protein database for each of the indicated species. C) Genetic
manipulation of MIR] determines susceptibility to FT-350 in C. albicans. Strains
indicated are SN152 wild-type transformed with pDIS3 vector (empty), SN152 wild-type
with one copy of MIR] deleted and transformed with pDIS3 vector, SN 152 wild-type
transformed with a wild-type copy of MIR] in the pDIS3 vector, and SNI 52 wild-type
transformed with the pDIS3 containing the mirJNl84Tmutation. Growth inhibition in
media containing a serial dilution of FT-350 was assessed at 24hr and normalized to a nodrug control.
205
CURRICULUM VITAE
Benjamin Matteson Vincent
benjvinc(agmail.com
EDUCATION
Massachusetts Institute of Technology, Cambridge, MA
Ph.D. Candidate, Microbiology
Thesis Advisor: Professor Susan Lindquist
September 2008 - December 2014
Stanford University, Stanford, CA
Bachelor of Science, Biology
September 2004-June 2008
AWARDS AND FELLOWSHIPS
NSF Graduate Research Fellowship, 2009-2012
Deans Award for Academic Accomplishment, Stanford University, 2008
Weinstein Award, Department of Biology, Stanford University, 2008
PUBLICATIONS
Vincent, B.M., Youngsaye W., Hartland, C., Lancaster, A.K., Whitesell, L., and
Lindquist, S. Selective inhibition of the mitochondrial phosphate carrier yields an
antifungal therapeutic strategy refractory to the evolution of resistance. In preparation.
Vincent, B. M., Langlois J. B., Youngsaye W., Lancaster A. K., Scherz-Shouval R,
Whitesell L, Buchwald S, Lindquist S. A Fungal-Selective Cytochrome bcl Inhibitor
Impairs Virulence and Prevents the Evolution of Drug Resistance. In preparation.
Davis S.A., Vincent B.M., Endo M.M., Whitesell L.W., Marchillo K., Andes D.R.,
Lindquist, S.* and Burke, M.D.* Non-toxic antimicrobials that evade the emergence of
drug-resistance. Submitted.
Vincent, B. M., Lancaster, A. K., Scherz-Shouval, R., Whitesell, L., & Lindquist, S.
(2013). Fitness trade-offs restrict the evolution of resistance to amphotericin B. PLoS
Biology, 10, e1001692.
Youngsaye, W., Hartland, C. L., Morgan, B. J., Ting, A., Nag, P. P., Vincent, B.,
Mosher, C. A., Bittker, J. A., Dandapani, S., Palmer, M., Whitesell, L., Lindquist, S.,
Schreiber, S. L., & Munoz, B. (2013). ML212: A small-molecule probe for investigating
fluconazole resistance mechanisms in Candida albicans. Beilstein Journalof Organic
Chemistry, 1501-1507.
206
Youngsaye, W., Dockendorff, C., Vincent, B., Hartland, C. L., Bittker, J. A., Dandapani,
S., Palmer,. M., Whitesell, L., Lindquist, S., Schreiber, S. L., & Munoz, B. (2012).
Overcoming fluconazole resistance in Candida albicans clinical isolates with tetracyclic
indoles. Bioorganic & Medicinal Chemistry Letters, 9,3362-3365.
&
Youngsaye, W., Vincent, B., Hartland, C. L., Morgan, B. J., Buhrlage, S. J., Johnston, S.,
Bittker, J. A., MacPherson, L., Dandapani, S., Palmer, M., Whitesell, L., Lindquist, S.,
Schreiber, S. L., & Munoz, B. (2011). Piperazinyl quinolines as chemosensitizers to
increase fluconazole susceptibility of Candida albicans clinical isolates. Bioorganic
medicinal chemistry letters, 18, 5502-5505.
CONFERENCES
Keystone Conference: The Chemistry and Biology of Cell Death
Selective targetingof mitochondrialrespirationyields a therapeuticstrategy refractory
to the evolution of resistance
Poster presentation
Santa Fe, NM, February 2014
Gordon Conference: Stress Proteins in Growth, Development and Disease
TargetingStress Responses to Prevent Antifungal Drug Resistance
Oral presentation
Lucca, Italy, July 2011
TEACHING EXPERIENCE
Teaching assistant, Professor Alan Grossman, MIT, Microbial Genetics and Evolution,
Fall 2009.
Teaching assistant, Professor Robert Sapolsky, Stanford University, Human Behavioral
Biology, Spring 2008.
207