Discovery and Characterization of a Peptoid with Antifungal Activity against Cryptococcus neoformans

Article abstract of DOI:10.1021/acsmedchemlett.6b00338

Studies show there is an increasing rate of fungal infections, especially in immunocompromised patients and treatments for fungal genera, such as Aspergillus, Candida, and Cryptococcus, carry significant cytotoxicity with an increasing prevalence of antifungal resistance. We have previously reported a high-throughput assay for identifying peptoids with antimicrobial properties from combinatorial libraries. Here we report the application of this assay in identifying a peptoid with antifungal properties against Cryptococcus neoformans. Termed AEC5, this peptoid has comparable potency to existing clinical antifungal agents, excellent stability, and minimal cytotoxicity in mammalian cells.

Full text of DOI:10.1021/acsmedchemlett.6b00338

Letter
pubs.acs.org/acsmedchemlett
Discovery and Characterization of a Peptoid with Antifungal Activity
against Cryptococcus neoformans
Ashley E. Corson,† Scott A. Armstrong,† Matthew E. Wright,† Erin E. McClelland,‡ and Kevin L. Bicker*,†
‡
†Department of Chemistry and Department of Biology, Middle Tennessee State University, 1301 East Main Street, Murfreesboro,
Tennessee 37132, United States
S
* Supporting Information
ABSTRACT: Studies show there is an increasing rate of fungal infections,
especially in immunocompromised patients and treatments for fungal genera,
such as Aspergillus, Candida, and Cryptococcus, carry significant cytotoxicity
with an increasing prevalence of antifungal resistance. We have previously
reported a high-throughput assay for identifying peptoids with antimicrobial
properties from combinatorial libraries. Here we report the application of this
assay in identifying a peptoid with antifungal properties against Cryptococcus
neoformans. Termed AEC5, this peptoid has comparable potency to existing
clinical antifungal agents, excellent stability, and minimal cytotoxicity in
mammalian cells.
KEYWORDS: Peptoids, Cryptococcus neoformans, fungal infections, high-throughput screening
Treatment for infections due to organisms such as Aspergillus,
Candida, and Cryptococcus vary depending upon the patient and
species of the infectious organism. Current therapeutics, such as
amphotericin B, flucytosine, and fluconazole, carry their own
risks, including high mammalian cytotoxicity that results in
gastrointestinal complications, vomiting, QT prolongation, and
hepatitis.8−10 Drug resistance has also been observed against
these therapeutics in several fungal species and strains.11
Because of this resistance, current treatments usually include a
cocktail or extended regimen of these drugs and possible
surgery if there is no response to medication, necessitating new
therapeutic options for patients dealing with fungal infections.9
One alternative to combat high toxicity levels and drug
resistance is the use of antimicrobial peptides (AMPs), which
can display minimal toxicity to mammalian cells and may be
able to specifically target fungal cells. Antimicrobial peptides
used against fungi are characterized by their ability to disrupt
the cell membrane structure or form pores that cause
intracellular compounds to leak out of the cell, leading to
cellular death.12 While fungal and mammalian cells share many
similar features, AMPs are able to target key differences, such as
ergosterol, a principal sterol only present in fungal cell
membranes.10 Unfortunately, while showing potency against
pathogenic fungi, AMPs are also easily degraded within the
human body by proteases.13 Their short half-life renders AMPs
ecent studies show an increase in fungal infections
1
Rcompared to previously seen data. For example, diseases
due to Candida species are now the fourth most common
nosocomial fungal infection,2 and infections of the central
nervous system by Cryptococcus neoformans are the most
common form of fungal meningitis.3 Theories regarding the rise
of fungal infections include the increased prevalence of these
illnesses in patients with immunocompromising diseases, the
growth in international travel, changes in land use, and
incorporation of antifungal drugs in agriculture.1 Currently,
nearly 75% of fungal infections isolated from patients in
hospitals are of the species Candida. The remaining 13% and
6% of fungal infections are due to Aspergillus spp. and
Cryptococcus spp., respectively.4
Cryptococcus spp. is a yeast-like fungus with a thick
polysaccharide capsule that results in infections located in the
pulmonary system and central nervous system.3 One species, C.
neoformans, is divided into two serotypes (A and D) and is the
most common infectious agent among transplant, and HIV/
AIDS patients due to the compromised immune system of
these individuals.5 The most recent estimates indicate that
roughly 1 million HIV/AIDS patients contract cryptococcal
meningitis each year and that over 600,000 of these cases will
be fatal.6 The greatest prevalence of these infections is in Sub-
Saharan Africa, where deaths due to cryptococcal meningitis
exceed those due to tuberculosis.6 Another Cryptococcus species
is C. gattii, which is capable of infecting immunocompetent
people.5 An outbreak of C. gattii infections has been observed
recently in the Pacific Northwest regions of Canada and the
United States.7
Received: August 22, 2016
Accepted: October 3, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.6b00338
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Figure 1. Application of the PLAD assay to screen against Cryptococcus neoformans. (Stage 1) PLAD linked peptoids immobilized on beads are
imbedded into soft agar containing a small amount of reducing reagent and inoculated with C. neoformans H99S. (Stage 2) Overnight incubation
resulted in growth of a C. neoformans lawn and release of the beta-strand peptoid from the bead. Peptoids with antifungal properties result in a zone
of inhibition around the bead. (Stage 3) Beads showing zones of inhibition were removed from the plate, alpha-strand peptoids cleaved from the
bead, and the peptoid structure determined by MS/MS.
unsuitable as clinical therapeutics for combating in vivo fungal
infections.
characterization of this compound revealed that it has no
observable unwanted cytotoxicity against mouse fibroblasts,
human hepatocytes, human peripheral lung epithelial cells, or
human erythrocytes at the minimum inhibitory concentration
for C. neoformans H99S.
One alternative to this problem is the employment of peptide
mimics called peptoids. Peptoids, often termed N-substituted
glycines, have the side chain attached at the nitrogen in the
amide backbone, as opposed to the α-carbon in peptides.14
This slight structural difference provides peptoids with
increased in vivo stability and an extended half-life compared
to peptides, while presumably maintaining the same or very
similar modes of actions.14,15 Although there are significant
studies on antibacterial peptoids,16−21 little research has been
performed using peptoids as therapeutic agents against fungi.
Nonetheless, studies have successfully demonstrated that
peptoids may be used against some fungal pathogens such as
Candida albicans,22 Fusarium virguliforme, and Fusarium
lateritium.23
The need in this field of research is for the rapid
identification of antifungal peptoids via high-throughput
screening of combinatorial libraries. Combinatorial libraries
allow for a large cohort of unique compounds to be synthesized
and screened in a relatively short period of time.24−26
Combinatorial libraries produced by split-and-pool synthesis
result in a one-bead−one-compound library, where each bead
contains thousands of copies of a unique peptoid that is
different from any other bead.24−26
We have recently reported a technique for the high-
throughput screening of peptoid combinatorial libraries
known as the Peptoid Library Agar Diffusion (PLAD)
assay.27 This assay relies on a branched linker immobilized
on a solid-phase bead that displays parallel strands of identical
peptoids. Each strand can be released from the bead
orthogonally in response to different chemical stimuli. One
peptoid strand, termed the beta-strand, is released during
screening to identify peptoids with antimicrobial properties,
while the other strand, termed the alpha-strand, is released after
screening to identify the peptoid structure by mass spectrom-
etry (MS). Herein we report the optimization and application
of this assay to identify a tripeptoid, termed AEC5, with
antifungal properties against Cryptococcus neoformans. Further
The PLAD assay was easily amendable to screening for
antifungal peptoids against C. neoformans, given that this
organism forms a robust lawn when grown in inoculated soft
agar (Figure 1). C. neoformans H99S,28 a lab strain of serotype
A, was chosen for these studies. One requirement of the PLAD
assay is that the microorganism being screened must tolerate a
small amount of reducing reagent, which is required to cleave
the disulfide on the PLAD linker and release the beta-strand
peptoid. The tolerance to reducing reagent (dithiothrietol;
DTT) and optimal cell count that would produce a robust,
even lawn was determined experimentally. This was done by
inoculating yeast extract peptone dextrose (YPD) soft agar
containing 0 or 10 mM DTT with varying cell counts,
solidifying the mixture onto a hard agar YPD plate, and
incubating at 37 °C for 20 h. Evaluation of lawn density
indicated that a robust lawn was easily formed at the lowest cell
count used (1 × 106 cells/mL) (Figure S1). Encouragingly, the
addition of 10 mM DTT, which was shown previously to be
sufficient for release of the beta-strand during PLAD screen-
ing,27 had no deleterious effects on C. neoformans growth
regardless of the cell count used. The aforementioned DTT
concentration and cell count were used for all subsequent
PLAD screenings of C. neoformans H99S.
A diverse library of pentapeptoids was synthesized on the
PLAD linker system to give a first generation antifungal peptoid
library (Figure 2A). Comprising 10 different submonomers, this
library had a theoretical diversity of 105 compounds. Split-and-
pool synthesis on a 0.6 g scale using TentaGel Macrobeads
(65,550 beads/g) gave an actual library diversity of 39,300
compounds, which is a moderate but adequate sampling of the
theoretical diversity. The submonomers chosen for this library
(Figure 2B) were aliphatic (isopropylamine (NVal), isobutyl-
amine (NLeu), benzylamine (NPhe)), cationic (1,2-diamino-
ethane (Nae), 1,3-diaminopropane (Nap), 1,4-diaminobutane
B
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Figure 2. (A) General structure for the PLAD linked first generation
antifungal library. (B) Submonomers randomly incorporated into the
library were chosen to display polar, hydrophobic, and cationic side
chains. (C) Image of AEC89 as identified during PLAD screening
against C. neoformans H99S. (D) Structure of antifungal peptoid
AEC89.
Figure 3. (A) General structure for the previously reported PLAD
linked lipopeptoid library. (B) Submonomers incorporated into
specific positions of the tripeptoid library to give hydrophobic and
cationic side chains. (C) Image of AEC5 as identified during PLAD
screening against C. neoformans H99S. (D) Structure of antifungal
peptoid AEC5.
(NLys)) and polar uncharged (ethanolamine (NEol), 3-amino-
1,2-propanediol (NDio), 2-methoxyethylamine (NMeo), and
furfurylamine (NFur)). In general, these submonomers were
chosen to provide a diverse set of hydrophobic, hydrophilic,
and electrostatic properties while maintaining the overall
cationic nature of antimicrobial peptides. Library quality was
evaluated by MS/MS sequencing of peptoids from randomly
chosen beads, which indicated that the alpha-strand of the
PLAD linked peptoid was easily fragmented during MS/MS
analysis, providing the submonomer sequence of the
compound (Figure S2). This entire library was screened via
the PLAD assay against C. neoformans H99S, evaluating 6 mg of
library (∼400 beads) per plate. Unfortunately, screening
yielded only one “hit” or bead displaying a measurable zone
of inhibition. Termed AEC89, the zone of inhibition for this
compound was modest (0.270 mm); however, it did indeed
appear to inhibit fungal growth (Figure 2C).The sequence of
AEC89 was determined by MS/MS to be NPhe-NPhe-Nap-
NPhe-NFur (Figure 2D). This peptoid was synthesized via
solid-phase submonomer methods on Rink Amide resin,
purified by HPLC, and evaluated by a standard broth dilution
assay to determine the minimum inhibitory concentration
(MIC) against C. neoformans H99S. Having an MIC of >200
μg/mL, no further characterization of this compound was done.
We hypothesized that the lack of potent antifungal
compounds identified from our first generation antifungal
peptoid library might be due to the library’s relatively low
lipophilicity. Previous studies have demonstrated the utility of
long alkyl tails in improving peptoid antimicrobial potency.16
To evaluate this hypothesis, we screened a previously
reported27 PLAD linked semicombinatorial library of 18
hydrophobic lipopeptides against C. neoformans H99S (Figure
3A,B). Several replicates of the library were screened and
resulted in several hits with zones of inhibition much larger
than those observed for the first generation antifungal peptoid
library. Although sequencing of all of the hits identified proved
to be problematic, a bead displaying one of the largest zones of
inhibition (0.729 mm; Figure 3C) was successfully sequenced
by MS/MS. The sequence of this tripeptoid, termed AEC5, is
NTri-NLys-NFur (Figure 3D). Given the long 13 carbon alkyl
tail and positively charged side chain, this compound exhibits
the cationic/hydrophobic nature of many naturally occurring
antimicrobial peptides.
To properly characterize the potency of AEC5 against C.
neoformans, this compound was synthesized on Rink Amide
resin, purified by HPLC, and evaluated by standard broth
dilution assays to determine the MIC of AEC5 against C.
neoformans H99S. The MIC against C. neoformans H99S was
6.3 μg/mL (Table 1), which is comparable to fluconazole and
fluocytosine, two common clinical antifungal agents.29 Having
demonstrated the initial antifungal potency of AEC5, we sought
to further evaluate this property by determining the MIC of
AEC5 against other serotypes and strains of C. neoformans;
namely, the serotype D lab strain (ATCC 24067), two serotype
A clinical isolates (B18 and B24),30 and two serotype D clinical
isolates (AD 12−27 and AD 12−30).31 The MIC of AEC5
against all these organisms ranged from 6.3 to 25 μg/mL, while
the IC50 ranged from 0.68 to 12.2 μg/mL (Table 1 and Figure
S3). Given that serotypes A and D are 15% different
genetically32 and the composition of the cell walls are likely
different,33,34 it is not surprising that this compound is not as
potent against clinical isolates of serotype D.
We subsequently determined the fungicidal vs fungistatic
properties of AEC5 at varying concentrations against C.
neoformans H99S. The fungicidal concentration of a compound
is the drug concentration needed to effectively kill a fungal
microorganism, while the fungistatic concentration arrests
fungal growth without killing the microorganism. This property
was determined by preparing an MIC assay with varying AEC5
concentrations, pooling triplicate samples after the 72 h
incubation, washing with PBS to remove AEC5, and spotting
aliquots of the residual cells in PBS onto YPD agar plates. Spots
of cells at concentrations of drug that are only fungistatic will
C
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Table 1. Potency Values for AEC5 against Several Serotypes and Strains of C. neoformans along with Toxicity Values for AEC5
a
against Several Mammalian Cell Types
organism
assay
potency/toxicity (μg/mL)
C. neoformans H99S (serotype A lab strain)
C. neoformans 24067 (serotype D lab strain)
C. neoformans serotype A clinical isolate B18
C. neoformans serotype A clinical isolate B24
C. neoformans serotype D clinical isolate AD 12−27
C. neoformans serotype D clinical isolate AD 12−30
NIH/3T3 mouse fibroblasts
MIC (IC50)
MIC (IC50)
MIC (IC50)
MIC (IC50)
MIC (IC50)
MIC (IC50)
IC50
6.3 (0.68)
6.3 (3.9)
25 (12.2)
12.5 (10.7)
25 (4.0)
25 (6.9)
48.6
HepG2 human hepatocellular carcinoma
HPL1A human peripheral lung epithelial cells
Human erythrocytes
IC50
56.2
IC50
36.3
HC10
68.7
a
MIC = minimum inhibitory concentration; IC50 = inhibitory concentration 50%; HC10 = hemolysis concentration 10%
lead to recovery of the fungal cells, resulting in colony growth
on the YPD agar plate. Spots of cells at concentrations of drug
that are fungicidal results in no colony growth, even after drug
is removed (Figure S5). Using this assay, we determined that
AEC5 is indeed fungicidal at the MIC of 6.3 μg/mL against C.
neoformans H99S. No colony growth was observed at or above
the MIC. Limited colony growth was observed at 3.1 μg/mL,
the first concentration below the MIC, and robust growth was
observed below this concentration and in the no-drug control.
Having established the potency of AEC5 as an antifungal
agent against C. neoformans, we next set out to evaluate the
unwanted cytotoxicity of this compound against mammalian
cells. Specifically, concentrations of AEC5 ranging from 800 to
3.1 μg/mL were tested against NIH/3T3 mouse fibroblasts,
HepG2 human hepatocellular carcinoma cells, HPL1A human
peripheral lung epithelial cells,35 and human erythrocytes.
Although we were concerned about the potential cytotoxicity of
AEC5 given the long 13 carbon alkyl tail, no cytotoxicity was
observed in any of the mammalian lines tested at the C.
neoformans H99S MIC of 6.3 μg/mL. The concentration of
AEC5 resulting in a 50% inhibition of growth (IC50) was
determined against NIH/3T3, HepG2, and HPL1A cells. This
concentration was found to be 50.3 μg/mL for NIH/3T3 cells,
43.6 μg/mL for HepG2 cells, and 36.3 μg/mL for HPL1A cells
(Table 1 and Figure S6). These values are roughly 8, 9, and 6-
fold higher than the MIC against C. neoformans H99S,
respectively. Traditionally, hemolytic activity against human
erythrocytes is defined as the drug concentration that results in
lysis of 10% of the erythrocytes (HC10), due to the sensitivity of
an organism to red blood cell lysis. The HC10 of AEC5 against
human erythrocytes was determined to be 68.7 μg/mL, nearly
11-fold higher than the MIC against C. neoformans H99S
(Table 1 and Figure S6).
To gain a predictive understanding of the therapeutic
potential of AEC5, several physicochemical properties were
determined using ChemAxon’s MarvinSketch Calculator
Plugins.36 The parameters for commonly administered
antifungal agents (amphotericin B, fluconazole, and flucytosine)
and commercial antimicrobial peptides (daptomycin, pexi-
ganan, and colistin) were also determined (Table S1). The
octanol−water partition coefficient (logP), distribution coef-
ficient at pH 7.4 (logD7.4), and water solubility coefficient
(logS) for AEC5 were calculated to be 3.24, −1.18, and −4.13,
respectively. Both logP and logD give a measure of a
compound’s lipophilicity, which plays a role in the bioavail-
ability and absorptivity of a potential drug compound. The
significant difference between logP and logD values shown here
is because logD accounts for the ionization of a compound,
while logP does not. Not surprisingly, with two ionizable amino
groups, the predicted isoelectric point of AEC5 is 12.7, and at
pH 7.4, the predominant species of AEC5 would be doubly
protonated (Table S1), making logD the most accurate
predictor of AEC5 lipophilicity. These data indicate that even
though AEC5 contains a long hydrophobic tail, it still has
excellent aqueous solubility. In fact, we have prepared stock
concentrations of AEC5 in water up to 50 mg/mL. These
physicochemical properties are comparable to the commercial
antifungals and antimicrobial peptides analyzed here. Fur-
thermore, the molecular weight, logD7.4, and polar surface area
are in line with a large number of natural products used to treat
infections,37 indicating that AEC5 may serve as a good lead for
antifungal therapeutic development. Furthermore, the stability
of AEC5 in human serum was determined following well
established protocols.38 No degradation of AEC5 was observed,
even after incubation in human serum for 48 h (Figure S7),
demonstrating the stability of antimicrobial peptoids compared
to antimicrobial peptides.
In summary, the PLAD assay for high-throughput identi-
fication of peptoids with antimicrobial properties has been
adapted and applied to screen two different libraries against the
fungus Cryptococcus neoformans. This screening identified a
relatively simple, biologically stable tripeptoid, AEC5, with
excellent antifungal potency against various serotypes and
strains of C. neoformans. Furthermore, the compound exhibited
no observable cytotoxicity at the MIC and a significant
therapeutic window between C. neoformans MIC values and
IC50/HC10 cytotoxicity values. The reported data regarding
AEC5 are preliminary in scope, but with further in vivo
characterization, AEC5 and other peptoids may prove to be
valuable additions to a field of antifungal agents wrought with
cytotoxicity and drug resistance issues. Work is currently
underway to screen more tailored antifungal peptoid libraries
against C. neoformans and other pathogenic fungi via the PLAD
assay.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00338.
Materials, submonomer synthesis, peptoid synthesis,
screening procedures, assay methods, and supplemental
figures (PDF)
D
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AUTHOR INFORMATION
Corresponding Author
*E-mail: kevin.bicker@mtsu.edu.
Author Contributions
A.E.C., S.A.A., M.E.W., E.E.M., and K.L.B. designed experi-
ments. A.E.C. performed assay development and identified
AEC5. A.E.C. and S.A.A. characterized AEC5. M.E.W.
performed NMR analysis of AEC5. A.E.C., S.A.A., E.E.M.,
and K.L.B. cowrote the manuscript.
■
Funding
This work was supported by start-up and Faculty Research and
Creative Activity Committee (FRCAC) funds provided by
MTSU and by the National Institutes of Health R03 AI112861.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank collaborators Ms. Jeannie Stubblefield and Dr. Iris
Gao for providing the mammalian cell lines tested.
■
ABBREVIATIONS
■
PLAD, Peptoid Library Agar Diffusion; AMP, antimicrobial
peptide; DTT, dithiothrietol; YPD, yeast extract peptone
dextrose; MIC, minimum inhibitory concentration; IC50,
inhibitory concentration 50%; HC10, hemolytic concentration
10%
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