Neutralization of Pathogenic Fungi with Small-Molecule Immunotherapeutics

Article abstract of DOI:10.1002/anie.201707536

Systemic fungal infections represent an important public health concern, and new antifungal agents are highly desirable. Herein, we describe the design, synthesis, and biological evaluation of a novel class of antifungal compounds called antibody-recruiting molecules targeting fungi (ARM-Fs). Our approach relies on the use of non-peptidic small molecules, which selectively bind fungal cells and recruit endogenous antibodies to their surfaces, resulting in immune-mediated clearance. Using the opportunistic fungal pathogen Candida albicans as a model, we identified a highly specific bifunctional molecule able to mediate the engulfment and phagocytosis of C. albicans cells by human immune cells in biologically relevant functional assays. This work represents a novel therapeutic approach to treating fungal illness with significant potential to complement and/or combine with existing treatment strategies.

Full text of DOI:10.1002/anie.201707536

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Accepted Article
Title: Neutralization of pathogenic fungi with small-molecule
immunotherapeutics
Authors: Egor Chirkin, Viswanathan Muthusamy, Paul Mann, Terry
Roemer, Philippe G. Nantermet, and David Spiegel
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201707536
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Angewandte Chemie International Edition
10.1002/anie.201707536
COMMUNICATION
Neutralization of pathogenic fungi with small-molecule
immunotherapeutics
§
[a]
§[a]
[b]
[c]
[b]
Egor Chirkin, Viswanathan Muthusamy, Paul Mann, Terry Roemer, Philippe G. Nantermet,
[
a]
and David A. Spiegel*
Abstract: Systemic fungal infections represent an important public
health concern, and new antifungal agents are highly desirable.
Herein, we describe the design, synthesis, and biological evaluation
of a novel class of antifungal compounds called Antibody-Recruiting
Molecules targeting Fungi (ARM-Fs). Our approach relies on the use
of non-peptidic small molecules, which selectively bind fungal cells
and recruit endogenous antibodies to their surfaces, resulting in
immune-mediated clearance. Using the opportunistic fungal
pathogen Candida albicans as a model, we identified a highly
specific bifunctional molecule able to mediate the engulfment and
phagocytosis of C. albicans cells by human immune cells in
biologically relevant functional assays. This work represents a novel
therapeutic approach to treating fungal illness with significant
potential to complement and/or combine with existing treatment
strategies.
(ABT) recognizes and recruits anti-DNP antibodies, which are
[
9]
already present in the bloodstream in humans. It has been
suggested that endogenous antibodies are present in
immunocompromised patients, who are especially prone to
fungal infection, thus extending the applicability of our
[
10]
strategy.
Upon binding the pathogenic target cell, the
bifunctional species recruits endogenous antibodies in the
bloodstream, resulting in redirection of immune cells toward the
target, which had until then evaded immune surveillance. Indeed,
our lab has previously reported similar strategies for
[
11]
immunotherapy of cancer and HIV.
Compared to antibodies,
however, small-molecule immunotherapeutics are expected to
exhibit low immunogenicity, high thermal stability, excellent
penetration properties, and have the potential to be orally
bioavailable. Furthermore, because they are unlikely to enter the
fungal cytosol, ARMs are unlikely to be susceptible to the known
mechanisms of drug resistance, such as increase of drug efflux
[
1]
Fungal infections represent a global public health threat.
[3]
and metabolic adaptation.
Systemic invasive mycoses are associated with a high mortality
rate in immunocompromised individuals; immunosuppression
arising from HIV infection, organ transplant and cancer therapy
has led to a threefold increase in the incidence of fungal sepsis
[
2]
over the past three decades. These factors, coupled with the
rapid spread of resistance to available antifungals, make novel
[
3]
therapeutic strategies to fight fungi highly desirable.
Fungi are eukaryotic, and therefore difficult to discriminate
[4]
from human cells. The fungal cell wall, however, is absent from
mammalian systems; thus, its components might constitute
[5]
attractive targets for the development of selective therapies.
Chitin, an amino-polysaccharide highly specific to the fungal cell
wall is particularly compelling, and has already been employed
[
6]
for diagnosis of fungal infections and specific delivery of drugs
into fungal cells.
[7]
Figure 1. Design of antifungal antibody-recruiting small molecules (ARM-F)
targeting chitin.
Herein we report the design, construction, and evaluation of
Antibody-Recruiting Molecules (ARMs) to mediate killing of
fungal cells by human effector cells. As shown in Figure 1, these
ARMs are bifunctional small molecules consisting of two primary
domains. The target-binding terminus (TBT) interacts with chitin,
a fungal cell wall component consisting of repeating -1,4-linked
Our design of a candidate antifungal ARM (called ARM-F)
started with calcofluor white as the TBT (Figure 1). Calcofluor
white is a well-studied fungal whitening agent known to possess
high specificity for chitin and antifungal properties at high
[
8]
N-acetylglucosamine units. The antibody-binding terminus
[12]
doses.
Because ARM-F needs to bind simultaneously to
fungal chitin and anti-DNP antibodies, we sought a site for linker
attachment on calcofluor white that would not compromise
chitin-binding activity. We, therefore, prepared three candidate
ARM-F derivatives: compound 6 resulted from the replacement
of diethanolamine moieties of calcofluor whereas 7 and 8 were
obtained by linker incorporation into aniline functionalities.
Synthesis of compounds 6–8 proved relatively straightforward
and followed the three-step route illustrated in Scheme 1. Thus,
trans-4,4’-diaminostilbene 2,2’-disulfonic acid (1) was first
allowed to react with cyanuric chloride followed by gradual
substitution on the triazine core of 2 with amines 3 or 4 (Scheme
[
a]
Dr. E. Chirkin, Dr. V. Muthusamy, Prof. Dr. D.A. Spiegel
Department of Chemistry
Yale University
225 Prospect Street, New Haven, CT 06511, USA
E-mail: david.spiegel@yale.edu
P. Mann, Dr. P.G. Nantermet
Merck & Co., Inc.
[
[
b]
c]
2000 Galloping Hill Road, Kenilworth, NJ 07033, USA
Dr. T. Roemer
Prokaryotics, Inc.
1000 Morris Ave, STEM Bldg., Suite 5-13, Union, NJ 07083, USA
§
These authors contributed equally
1). The degree of substitution was fine-tuned by controlling
Supporting information for this article is given via a link at the end of
the document
temperature and pH of the reaction, thus allowing the selective
incorporation of ABT into the stilbene scaffold.
[7],[13]
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Angewandte Chemie International Edition
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COMMUNICATION
R2
H
H
H2N
Cl
N
N
R1
N
N
SO3H
SO H
Cl
N
3
SO3H
HN
a)
b,c)
N
N
N
N
N
N
N
N
SO3H
Cl
SO H
3
NH
SO3H
NH2
N
Cl
N
H
N
R1
1
H
2
R2
R1
NO2
H
N
H
N
O
R2 = H
NO2
6
7
8
9
=
,
(14%)
(20%)
9
NH2
NO2
H
H
N
N
H
N
NO2
R
H
O
R1 = C l, R2
H
=
N
O
N
NH2
9
O
9
O
O
NO2
NO2
9
NO2
OH
OH
O2N
HN
R1
, R2
H
H
N
3
=
=
(11%)
HN
HN
N
4
R =
O
O
NO2
9
9
O
O
NO2
5
R = Boc
OH
OH
R1
=
R2
H
N
H
N
,
=
(23%)
Boc
Scheme 1. a) cyanuric chloride, H
2
O, MEK, 0°C, pH 4-5; b) 6: aniline, H
2 2 2
O, MEK, 45°C, pH 6-7; 7-8: 4, H O, MEK, 45°C, pH 6-7; 9: 5, H O, MEK, 45°C, pH 6-7;
c) 6: 3, H O, MEK, 85°C, pH 8-9; 7-9: DEA, H
2
2
O, MEK, 85°C, pH 8-9. Boc = tert-butyloxycarbonyl, DEA = diethanolamine, MEK = methyl ethyl ketone.
A flow-cytometry based assay was developed to assess the
As expected, 9 did not exhibit any antibody-recruitment activity
antibody recruitment ability of candidate ARM-Fs to isolates of C. (Figure 2A). ARM-F (8) showed a dose-dependent recruitment
albicans. We chose this organism for our studies because it is
of anti-DNP antibodies with maximal levels at 1 µM and
decreased responses at higher doses (Figure 2B). This bell-
shaped dose-response curve is indicative of a “prozone” effect,
which is expected in three-body complexes as previously
both a known human pathogen, and readily tractable for
[
14]
experimental manipulations. Ternary complexes formed from
ARM-F, fungal cells and anti-DNP antibodies were detected
using an anti-DNP secondary antibody conjugated to Alexa
Fluor 647 fluorophore. Among the ARM-F candidates prepared,
[
15]
described.
Importantly, the viability of C. albicans was not
affected by ARM-F (8) at concentrations used in this assay
compound
8
demonstrated the highest antibody-recruiting
(Figure 2B).
activity, with 35-45% of C. albicans cells exhibiting AF647
fluorescence (Figure 2A). In addition to demonstrating that ARM-
F could induce ternary complex formation, these results also
provided insights into the structure-activity relationships for
functionalizing the calcofluor scaffold. Thus, para substitution of
linker functionality on the aniline moiety is well-tolerated while
derivatization of the diethanolamine substituent substantially
To confirm engagement of chitin target by ARM-F (8), we
developed an enzyme-linked immunosorbent assay (ELISA)
system. Here solubilized polymeric chitin was incubated with
varying concentrations of 8 followed by anti-DNP antibody.
Dose-dependent antibody recruitment was observed with a
maximal response at 10 nM (Figure 3A). The sigmoidal nature of
the dose-response curve indicates specific binding of ARM-F (8)
to plate-bound chitin. We hypothesize that the hundred-fold
increase in potency of 8 in this assay compared to flow-
cytometry experiments shown above can be attributed to higher
density and free accessibility of plate-bound chitin.
decreases binding affinity.
structurally similar to 8 but lacking the ABT was synthesized
Scheme 1) and used as a negative control.
A non-functional compound 9
(
Demonstration that 8 is capable of binding chitin in cells was
obtained using a Saccharomyces cerevisiae model wherein both
pharmacologic and genetic modulation of chitin levels are
[
16],[17]
possible.
The yeast -factor pheromone regulates chitin
synthase enzymes responsible for chitin synthesis in S.
[
16]
cerevisiae. Treatment with 100 µM of -factor led to a modest
increase in antibody recruitment by 8 (Figure 3B). In a genetic
counterpart to this experiment, we used a chitin synthase triple
deletion mutant S. cerevisiae Chs 1, 2, 3 strain which entirely
[
17]
lacks chitin.
observed by ARM-F (8) to the mutant S. cerevisiae Chs 1, 2, 3
Figure 3B).
As expected, no antibody recruitment was
(
Figure 2. Antibody recruitment to fungal cell surface by flow cytometry A.
Screening of potential ARM-F candidates. B. Dose-dependent recruitment of
anti-DNP antibodies by ARM-F (8) with no loss of viability.
We further evaluated the activity of 8 in C. albicans by
enhancing access to chitin using the antifungal caspofungin.
Caspofungin and other echinocandin antifungals disrupt the
integrity of fungal cell wall by inhibiting the synthesis of its major
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COMMUNICATION
component -1,3-glucan. Increased chitin production is the
salvage mechanism of the fungal cell wall and is associated with
[
18]
resistance to echinocandins.
At 1 µM concentration of 8,
antibody recruitment positively correlated with caspofungin
dosage. At 1.25 ng/ml treatment with caspofungin, 90% of the
fungal cells recruited antibodies and were alive, while higher
concentrations resulted in reduced viability (Figure 3C). In dose
ranging studies, 1 µM of 8 elicited a maximum response in
caspofungin treated cells. A hundredfold reduced dose (10 nM)
was able to elicit antibody recruitment comparable to the
maximum possible in the non-caspofungin model (Figure 3D).
Compound 9 did not recruit antibodies to caspofungin-treated C.
albicans cells, ruling out artefactual “sticky” effects of
caspofungin resulting in enhanced recruitment by 8 (Figure 3D).
These observations establish that treatment of fungi with sub-
inhibitory concentrations of caspofungin enhances both the
functionality and potency of 8. These results also suggest that
combinations of echinocandins and ARM-F could have
synergistic effects in clinical settings.
Taken together, these experiments suggest that ARM-F (8)
binds to fungal chitin in a dose-dependent manner and recruits
antibodies to the surface of fungal cells. These conclusions
prompted us to perform functional assays evaluating the ability
of 8 to mediate immune clearance of fungal pathogens by
redirecting human immune cells.
Figure 3. Binding of ARM-F (8) is dependent on cell wall chitin. A. Binding of 8
to immobilized chitin in ELISA. B. Chitin dependent activity of 8 in a S.
cerevisiae chitin synthase knock-out model. C. Effect of caspofungin treatment
on antibody recruitment by 8. D. Dose-dependent antibody recruitment to C.
albicans pretreated with 1.25 nM caspofungin.
In the physiological state, neutrophils mediate immune
[
19],[20]
clearance of antibody-opsonized fungal cells.
To mimic this
In conclusion, we discovered a novel bifunctional small
molecule that can target pathogenic fungi toward immune-based
clearance. The high-affinity molecule ARM-F (8) demonstrated a
specific, dose-dependent binding to fungal chitin, recruited anti-
DNP antibodies, and consequently mediated dose-dependent
phagocytosis of the fungal cells by human immune cells. The
utilization of the host’s own immune system, as opposed to
cytotoxic agents, for the elimination of pathogens makes this
approach desirable from a therapeutic standpoint. Furthermore,
we believe that our ARM-F strategy could be applied to drug-
resistant strains of Candida; ARM-F has the advantage of
circumventing known resistance mechanisms and may be more
effective against echinocandin-resistant fungi, wherein there is
enhanced chitin production in the fungal cell wall. Although our
ARM-F approach was developed using the opportunistic
pathogen C. albicans as a model, our chitin-targeting therapeutic
platform could potentially be effective against a number of
pathogenic fungi, including Aspergillus, since chitin is an
essential structural component present in the fungal cell wall.
Thus, ARM-F provides a novel therapeutic strategy that could be
used solely or in combination with current antifungal
interventions to combat C. albicans and other pathogenic fungi.
mechanism, we developed a functional assay to analyse ARM-
F-mediated opsonophagocytosis of fungal cells. The assay is
based on two color flow cytometry and operates on the principle
of differentiating phagocytosed fungal cells (intracellular) from
[21]
non-phagocytosed cells (extracellular). Thus, FITC-labeled C.
albicans were treated with ARM-F (8) and co-incubated with
human neutrophils in the presence of anti-DNP antibodies. After
completion of incubation, polyclonal rabbit anti-Candida
antibodies in combination with AF647-labeled anti-rabbit
immunoglobulin G (IgG) were used to fluorescently tag the free
non-phagocytosed fungal cells. Upon selective osmotic
disruption of neutrophils, the single (FITC only) labeled
phagocytosed cells were released and could be easily
differentiated from the dual (FITC and AF647) labeled non-
phagocytosed fungal cells by flow cytometry (Figures 4A and B).
As anticipated, the treatment with ARM-F (8) induced a drastic
increase in number of phagocytosed fungal cells compared to
the untreated cells. ARM-F (8) induced a dose-dependent
phagocytosis of C. albicans cells with the maximal response at
10 µM again showing the bell-shaped curve characteristic of
ternary complexes (Figure 4C).
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Figure 4. Antibody-dependent cellular phagocytosis assays. A. Schematic depiction of flow cytometry-based functional phagocytosis assay. B. Representative
flow cytometry plots illustrating the distribution of C. albicans cell populations post phagocytosis in the presence/absence of 8. C. ARM-F (8) induces a dose-
dependent
opsono-phagocytosis
of
C.
albicans.
[
[
10] R.T.C. Sheridan, J. Hudon, J.A. Hank, P.M. Sondel, L.L. Kiessling
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D.A.S. and E.C. gratefully aknowledge the grant LKR#144525
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for the generous gift of chitin-deficient S. cerevisiae strain.
Keywords: antibodies • antifungal agents • cell recognition •
drug discovery • immunology
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Egor Chirkin, Viswanathan Muthusamy,
Paul Mann, Terry Roemer, Philippe G.
Nantermet, and David A. Spiegel*
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Neutralization of pathogenic fungi
with small-molecule
immunotherapeutics
Taking aim at chitin. Design and synthesis of non-peptidic small-molecule
immunotherapeutics for treatment of fungal infection is reported. A drug candidate
was able to recruit endogenous antibodies, thus promoting the dose-dependent
phagocytosis of fungal pathogens by human immune effector cells.
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