Dual-Stage Picolinic Acid-Derived Inhibitors of Toxoplasma gondii

Article abstract of DOI:10.1021/acsmedchemlett.0c00267

Toxoplasma gondii causes a prevalent human infection for which only the acute stage has an FDA-approved therapy. To find inhibitors of both the acute stage parasites and the persistent cyst stage that causes a chronic infection, we repurposed a compound library containing known inhibitors of parasitic hexokinase, the first step in the glycolysis pathway, along with a larger collection of new structural derivatives. The focused screen of 22 compounds showed a 77% hit rate (>50% multistage inhibition) and revealed a series of aminobenzamide-linked picolinic acids with submicromolar potency against both T. gondii parasite forms. Picolinic acid 23, designed from an antiparasitic benzamidobenzoic acid class with challenging ADME properties, showed 60-fold-enhanced solubility, a moderate LogD7.4, and a 30% improvement in microsomal stability. Furthermore, isotopically labeled glucose tracing revealed that picolinic acid 23 does not function by hexokinase inhibition. Thus, we report a new probe scaffold to interrogate dual-stage inhibition of T. gondii.

Full text of DOI:10.1021/acsmedchemlett.0c00267

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Letter
Dual-Stage Picolinic Acid-Derived Inhibitors of Toxoplasma gondii
Muhammad M. Khalifa,# Bruno Martorelli Di Genova,# Sarah G. McAlpine, Gina M. Gallego-Lopez,
David M. Stevenson, Soren D. Rozema, Neil P. Monaghan, James C. Morris, Laura J. Knoll,
and Jennifer E. Golden*
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ABSTRACT: Toxoplasma gondii causes a prevalent human infection for which only the acute stage has an FDA-approved therapy.
To find inhibitors of both the acute stage parasites and the persistent cyst stage that causes a chronic infection, we repurposed a
compound library containing known inhibitors of parasitic hexokinase, the first step in the glycolysis pathway, along with a larger
collection of new structural derivatives. The focused screen of 22 compounds showed a 77% hit rate (>50% multistage inhibition)
and revealed a series of aminobenzamide-linked picolinic acids with submicromolar potency against both T. gondii parasite forms.
Picolinic acid 23, designed from an antiparasitic benzamidobenzoic acid class with challenging ADME properties, showed 60-fold-
enhanced solubility, a moderate LogD7.4, and a 30% improvement in microsomal stability. Furthermore, isotopically labeled glucose
tracing revealed that picolinic acid 23 does not function by hexokinase inhibition. Thus, we report a new probe scaffold to
interrogate dual-stage inhibition of T. gondii.
KEYWORDS: Toxoplasma gondii, picolinic acid, parasite hopping, inhibitor, multistage
Toxoplasma gondii is responsible for a commonly acquired
parasitic infection that is estimated to affect 30−50% of the
human population.1 Mainly contracted through consumption
of contaminated food or water,2 T. gondii parasites rapidly
proliferate in the tachyzoite stage, causing an acute infection
that recruits a robust host immune response. Chemo-
therapeutic or immune pressure induces tachyzoite parasites
to differentiate into metabolically quiescent bradyzoite cysts
that evade the host immune system and are impenetrable to
current first-line therapeutics.2,3 Failure to clear parasites from
the host results in a lifelong opportunistic chronic infection
with significant risk of fatal complications by reactivation in
immunocompromised patients.4 While immunocompetent
individuals who become infected are typically asymptomatic
or may suffer self-limiting flulike illness, ocular lesions have
been reported.5 Maternal infection during pregnancy can lead
to miscarriage or visual or motor disabilities in newborns.6−8
The acute infection, caused by T. gondii tachyzoites, is
frequently treated with a synergistic combination of the
antifolate drugs pyrimethamine and sulfadiazine.9,10 Unfortu-
nately, significant side effects11−13 and resistance14 have been
reported with use of these drugs, and persistent bradyzoite
parasites evade this therapy.15,16 Despite new develop-
ment17−24 and drug repurposing efforts,25−28 effective treat-
ments for T. gondii, especially those capable of clearing the
bradyzoite stage of the parasite, remain an important unmet
medical need.15,29−31 We previously described a series of
benzamidobenzoic acids, exemplified by 1 (Figure 1), that
inhibited the glycolytic hexokinase enzyme of the kinetoplastid
parasites Trypanosoma brucei and Leishmania major.32
Through broader screening, we determined that unrelated
parasites may be inhibited by these same compounds, though
Received: May 19, 2020
Accepted: October 7, 2020
https://dx.doi.org/10.1021/acsmedchemlett.0c00267
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A

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The synthesis of several of the benzamidobenzoic acids32 has
been previously disclosed, and new analogues in that series
followed similar protocols (see the Supporting Information).
The sequence of steps was modified, but the picolinic acid
derivatives were generally prepared by benzoylation of 3-
amino-5-chloropicolinic acid esters followed by Suzuki
coupling when a bromine atom was installed at C6 (Scheme
1). Hydrolysis of the ester functionality revealed the desired
Figure 1. Probe 1 and next-generation aza analogues.
the mechanism may not be shared. For instance, when our
compound library was assessed against the glucokinase of the
pathogenic free-living protozoa Naegleria fowleri, a neuro-
invasive amoeba that causes a rare but highly lethal brain
infection, hits were identified, and distinct structure−activity
relationships (SARs) emerged from testing these compounds
against various parasites.33,34 This parasite-hopping ap-
proach26,35−37 reveals opportunities for therapeutic interven-
tion and divergent scaffold optimization due to differences in
SARs across a spectrum of familial and distantly related
parasites. With this in mind, we curated a small library of 22
compounds from our collection that included compound 1,
several related analogues, and a collection of new picolinic and
nicotinic acid derivatives that evolved from the benzamido-
benzoic acids (Table 1S).
a
Scheme 1. Synthesis of Picolinic Acid Derivatives
Compounds were evaluated against both tachyzoite38 and
bradyzoite39 forms of the T. gondii parasite in separate in vitro
screens. Briefly, human foreskin fibroblast (HFF) host cells
harboring either T. gondii tachyzoites or in vitro-differentiated
bradyzoites were treated with each of the compounds at a
single compound concentration of 10 μM.40 Pyrimethamine,
which is known to be active against the tachyzoite stage but
not the bradyzoite stage, was included as a control (Figure
2).41 Compound 1 displayed >98% growth inhibition for both
a
Reagents and conditions: (a) 2.6 M Br2 in AcOH, 2 M H2SO4, rt, 4−
72 h, 51−55%; (b) ArCOCl, Et3N or i-Pr2EtN, THF or CH3CN, rt to
150 °C, 30−89%; (c) PhB(OH)2, Na2CO3, Pd(PPh3)2Cl2, 10:1
CH3CN/H2O, 100 °C, 0.5 h, 67%; (d) 1.3 M aq LiOH, 3:2 THF/
H2O, rt, 12−48 h, 31−93%.
acids (compounds 2−6 and 10−14). In some cases,
replacement of the carboxylic acid with a bioisostere was
surveyed by using the appropriately substituted picolinic
derivative and employing the same benzoylation procedure
(compounds 7−9). Nicotinic acid analogues 15 and 16 were
generated by similar methods (see the Supporting Informa-
tion).
In parallel with compound 1, these second-generation aza
analogues 2−16 were subsequently evaluated in dose−
response-formatted growth inhibition assays for each stage of
the T. gondii parasite.38,39 General cytotoxicity was assessed in
uninfected HFF cells over 72 h for comparison with the
parasitic assays performed in the same cell line (Table 1). Our
benchmark, compound 1, inhibited both tachyzoites and
bradyzoites with similar submicromolar potency. Integration of
the nitrogen atom into the core to generate the analogous
picolinic acid 2 resulted in comparable tachyzoite and
bradyzoite inhibition (EC50 = 0.56 and 0.41 μM, respectively).
Across the aza series, inhibition was generally congruent
between tachyzoites and bradyzoites for a given compound,
except in four cases in which inhibition for one stage was
significantly favored. The effort revealed that the C6-phenyl
ring appended to the picolinic acid core was not necessary to
achieve submicromolar inhibition, and removing this moiety
helped to reduce the lipophilicity. Conversely, the C5-chloro
substituent of the aza analogue set was important for retaining
activity (compounds 4−6). Compound 5 showed the best
tachyzoite inhibition (EC50 = 0.24 μM) with commensurate
bradyzoite activity; however, some apparent toxicity in HFF
cells was also noted, affording selectivity indices of 10.4 to 7.1,
respectively. The methyl ester derivative 7 showed activity
Figure 2. Percent growth inhibition of T. gondii tachyzoites and
bradyzoites with compounds 1−22 at 10 μM.
T. gondii parasite stages at 10 μM, and several of the related 1-
arylbenzamidobenzoic acids (compounds 18−22) also resulted
in high levels of inhibition (see Figure 2 and Table 1S). These
compounds provided some initial SAR insights; however, their
high lipophilicity (cLogD7.4 = 5.0−7.4) limited their solubility
and development potential. We were encouraged to see that
many of the picolinic acid derivatives were also active, as these
compounds comparatively had improved physiochemical
properties.
B
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Table 1. T. gondii Tachyzoite and Bradyzoite Inhibition and Cytotoxicity Studies with Compounds 1−16
a
Compounds were minimally tested in triplicate (n ≥ 3) in HFF cells unless otherwise indicated. SI is the selectivity index, given by SI = CC50/
b
c
EC50. HFF cell line, 72 h exposure. Tested in duplicate (n = 2).
against both parasite forms, though hydrolysis to reveal an
active acid metabolite (compound 5) is likely, especially for the
bradyzoite assay, which involves base-mediated maturation of
parasites. Notably, though, this analogue did not manifest the
same levels of toxicity toward HFF cells as observed with
compound 5. Other isosteric replacements of the carboxylic
acid were also surveyed, revealing that a primary amide
isosteric replacement of the acid was inactive; the toxicity of
tetrazole 9 confounded the interpretation of any inhibitory
effect.
The necessity of the 4-tert-butyl appendage of the
benzamide moiety was also evaluated. Exchange of the tert-
butyl group of 5 with a methyl group (compound 10) or a
hydrogen atom (compound 11) eroded the potency against
both parasitic forms in a trend that suggests that hydrophobic
interactions may be important in this region of the scaffold.
This hypothesis was further supported by the more
pronounced loss of potency when a more polar 4-
methoxyphenyl group was installed in place of the 4-tert-
butylphenyl moiety (compound 12). Migration of the methoxy
group to other positions on the phenyl ring was not
advantageous for potency over other structural modifications
already discussed (compounds 13 and 14). Nicotinic acid
derivatives 15 and 16 were devoid of inhibitory activity against
the bradyzoite form. The benzamidobenzoic acid series,
represented by compound 1, possessed challenging physi-
ochemical properties. For instance, lipophilic probe 1 had an
experimental LogD7.4 of >5, along with modest solubility and
microsomal stability (Table 2). The aza analogues were
designed to address these liabilities. Selected picolinic
analogues and compound 1 were evaluated for LogD7.4,
solubility, microsomal and plasma stability, and brain
permeability (PAMPA). Compound 5 had a desirable
LogD7.4 value of 2.7 and the most improved solubility and
microsomal stability relative to compound 1. Despite the
presence of the carboxylic acid, the BBB PAMPA assay
predicted that the compound should achieve brain exposure. A
pharmacokinetic mouse study was planned; however, we found
that compound 5 was difficult to formulate into a solution for
proper administration. To enhance the solubility of 5, the
sodium salt of the carboxylic acid was generated to afford
analogue 23 (Table 2). Parasite growth inhibition was verified,
C
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four T. gondii-infected mice 30+ days postinfection, purified,
and transferred to fibroblast cells (HFF). Compound 23 was
independently added to the cells at three separate concen-
trations (1, 3, or 6 × EC50) followed by incubation for 3 days.
Monolayers were fixed and incubated with an anti-BAG1
antibody that recognizes a bradyzoite-specific antigen, BAG1,
which is absent in tachyzoites.42 Fluorescence imaging
(Incucyte) of the infected cells showed that compound 23
reduced the fluorescence signal corresponding to BAG1 in a
dose -dependent manner compared with the DMSO control,
thus demonstrating that compound 23 inhibits ex vivo
bradyzoite cysts (Figure 3).
Table 2. ADME Results for Selected Compounds
1
2
R = NHCO-4-tert-butylphenyl. Selectivity index, given by SI =
3
CC50/EC50. CC50 in HFF cells after 72 h = cytotoxic concentration
4
at which cell viability is reduced by 50%. Experimentally determined.
5
6
Kinetic aqueous solubility at pH 7.4. Mouse microsomes; value
7
reported as percent parent remaining after 1 h of exposure Mouse
8
plasma; percent parent remaining after 1 h of exposure. BBB
PAMPA, Pe (10−6 cm/s). ND = not determined.
revealing EC50 values for salt 23 comparable to those for the
parent compound 5 against both tachyzoites and bradyzoites.
Notably, the solubility of salt 23 was improved 5-fold over that
of 5, which enabled us to find a particle-free formulation for
pharmacokinetic evaluation.
Figure 3. Ex vivo harvested T. gondii cysts from mouse brains in HFF
cells incubated with BAG1 antibody and fluorescently imaged after 3
day treatment with (A) DMSO control or (B−D) compound 23 at
(B) 1 × EC50, (C) 3 × EC50, or (D) 6 × EC50. Antibody-labeled cysts
appear in green. The experiment was performed twice, each time in
triplicate, but only one experiment is shown for clarity (see Figure 1S
and Table 2S).
To assess the plasma and brain exposure of compound 23, a
snapshot pharmacokinetic experiment in mice was conducted.
Compound 23 was administered to male BALB/c mice at 15
mg/kg via ip injection as a clear solution. Plasma
concentrations were assessed 30 min and 2 h postadministra-
tion, and brain exposure was measured at the 2 h time point.
Terminal mean plasma and brain exposure (AUC2h = 7883 ng/
mL and AUC2h = 816 ng/g, respectively) were determined,
affording a brain-to-plasma (B/P) ratio of 0.1. These values
translate to 22 μM in plasma and 2.3 μM in brain 2 h
postadministration, though because of high protein binding
(99.5%), the free fraction is substantially reduced. Encourag-
ingly, compound 23 demonstrated exposure at a low dose in
both plasma and brain; however, additional work will be
required to improve the selectivity index and increase the
exposure to permit suitable dosing in mice for efficacy
assessment. The ester of compound 7 may offer potential
insight into addressing these characteristics.
A biological target of the picolinic acids is currently
unknown. However, the benzamidobenzoic acids from which
these compounds were designed, represented by 1, were
originally developed as inhibitors of the T. brucei parasite
glycolytic enzyme hexokinase 1 (TbHK1).32 T. gondii
possesses a single copy of its hexokinase (T. gondii hexokinase,
TgHK),43,44 and like T. brucei parasites, T. gondii bradyzoites
are glycolysis-dependent; however, the tachyzoite stage can use
alternative metabolic pathways when necessary. To determine
whether compound 23 inhibited TgHK, T. gondii-infected
HFF cells were treated with DMSO control or with compound
23 at either 2 or 6 × EC50 4 h postinfection. After 5 h, the
medium was changed to glucose-free RPMI medium
supplemented with isotopically labeled 13C6-glucose.45 After
10 min of labeling, infections were rapidly quenched,
processed, and subjected to broad-spectrum mass spectrometry
analysis. Data were evaluated by the Metabolomics Analysis
and Visualization Engine (MAVEN)46 software. Analysis of
13C6-labeled metabolite production from the initial glycolysis
reactions (Figure 4A) revealed two notable outcomes related
to the absence or presence of compound 23.
Contrary to the tachyzoites, the in vitro-derived bradyzoites
used in the primary screen and SAR assessment were not
susceptible to pyrimethamine as a control, indicating that the
bradyzoites were differentiated. Nonetheless, we evaluated
whether the observed inhibition of in vitro-derived bradyzoites
was recapitulated with ex vivo-derived bradyzoite cysts using
compound 23. Bradyzoites were harvested from the brains of
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more study; however, these results show that 23 impacts the
glycolytic pathway, although inhibition of the hexokinase is not
the primary mechanism of action.
Herein we have described the results of a parasite-hopping
approach revealing a novel picolinic acid template that inhibits
both tachyzoite and bradyzoite T. gondii parasites. Generally, a
good correlation between the in vitro potencies toward the two
parasite forms was found, and structure−activity relationships
were identified for a preliminary collection of 18 compounds,
several of which showed submicromolar inhibition. Compared
with the first-generation benzamidobenzoic acids, the second-
generation picolinic acid derivative 23 was more balanced in
terms of its lipophilic character and demonstrated a >30%
improvement in mouse microsomal stability and a 60-fold
improvement in solubility. Some cellular toxicity in HFF cells
was observed, and additional work will be required to enhance
the exposure, especially in brain tissue, where the bradyzoite
burden is significant. As current FDA-approved therapies are
ineffective against T. gondii bradyzoites, these compounds
represent an opportunity to study the acute and chronic stages
of T. gondii infection with a new probe scaffold.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00267.
Assay, experimental details, synthetic procedures, and
NMR spectra for all new compounds reported in this
work (2−23) (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Jennifer E. Golden − Pharmaceutical Sciences Division, School
of Pharmacy, University of Wisconsin-Madison, Madison,
Wisconsin 53703, United States; orcid.org/0000-0002-
6813-3710; Email: jennifer.golden@wisc.edu
Authors
Muhammad M. Khalifa − Pharmaceutical Sciences Division,
School of Pharmacy, University of Wisconsin-Madison, Madison,
Wisconsin 53703, United States
Bruno Martorelli Di Genova − Department of Medical
Microbiology and Immunology, University of Wisconsin-
Madison, Madison, Wisconsin 53706, United States;
orcid.org/0000-0001-9983-1856
Sarah G. McAlpine − Eukaryotic Pathogens Innovation Center,
Department of Genetics and Biochemistry, Clemson University,
Clemson, South Carolina 29634, United States
Gina M. Gallego-Lopez − Department of Medical Microbiology
and Immunology, University of Wisconsin-Madison, Madison,
Wisconsin 53706, United States; Morgridge Institute for
Research, Madison, Wisconsin 53715, United States;
orcid.org/0000-0001-7872-5595
David M. Stevenson − Department of Bacteriology, University
of Wisconsin-Madison, Madison, Wisconsin 53706, United
States of America
Soren D. Rozema − Pharmaceutical Sciences Division, School of
Pharmacy, University of Wisconsin-Madison, Madison,
Wisconsin 53703, United States
Neil P. Monaghan − Eukaryotic Pathogens Innovation Center,
Department of Genetics and Biochemistry, Clemson University,
Clemson, South Carolina 29634, United States
Figure 4. (A) Schematic of initial T. gondii glycolysis reaction
pathways. (B, C) Mass spectrometry analyses showing the
percentages of the total label-free or 13C6-labeled (B) glucose-6-
phosphate and (C) fructose-6-phosphate in T. gondii-infected HFF
cells treated with DMSO (white bars) or with compound 23 at 2 ×
EC50 (gray bars) or 6 × EC50 (black bars). Error bars = 95%
confidence interval. Asterisks indicate a significant difference in 13C6-
labeled fructose-6-phosphate mass ion (p = 0.02, t test corrected for
multiple comparison using the Holm−Sidak method) between the
DMSO and 6 × EC50 results. Shown is one representative experiment
of two, each performed in triplicate.
In the presence of compound 23, 13C6-glucose-6-phosphate,
the product of the hexokinase, did not change significantly in
treated parasites compared with the DMSO control, suggesting
that the hexokinase was not appreciably inhibited (Figure 4B).
In contrast, 13C6-fructose-6-phosphate accumulated in a
statistically significant amount compared with DMSO-treated
tachyzoites (Figure 4C). An impact of compound 23 on other
pathway metabolites was not observed (data not shown).
Elucidating the biological target of compound 23 that leads to
the observed increase in 13C6-fructose-6-phosphate will require
E
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James C. Morris − Eukaryotic Pathogens Innovation Center,
Department of Genetics and Biochemistry, Clemson University,
Clemson, South Carolina 29634, United States
Laura J. Knoll − Department of Medical Microbiology and
Immunology, University of Wisconsin-Madison, Madison,
Wisconsin 53706, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00267
Author Contributions
#M.M.K. and B.M.D.G. contributed equally. The manuscript
was written through contributions of all authors. All of the
authors approved the final version of the manuscript.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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The authors acknowledge the assistance of Dr. Gang Yan and
Claire Caschetta in obtaining PAMPA assay data as well as Dr.
Gene Ananiev in the UW-Madison Screening Facility for
determining HFF cytotoxicity data. The authors thank Dr.
Daniel Amador-Noguez for use of the Thermo Fisher
Vanquish Horizon UHPLC joined to a Thermo Scientific Q
Exactive Orbitrap high-resolution mass spectrometer. This
work made use of the instrumentation at the UW-Madison
Medicinal Chemistry Center and Analytical Instrumentation
Center funded by the UW School of Pharmacy. M.M.K.
gratefully acknowledges support from the National Science
Foundation Graduate Research Fellowship Program (DGE-
1256259) and the American Foundation for Pharmaceutical
Education Pre-Doctoral Fellowship in Pharmaceutical Sciences
Program. Work from the laboratory of J.C.M. was supported in
part by the NIH Center for Biomedical Excellence (COBRE)
Grant P20GM109094. L.K.J. acknowledges support from the
University of Wisconsin Food Research Institute. B.M.D.G.
and G.M.G.-L. were supported by Morgridge Metabolism
Interdisciplinary Fellowships from the Morgridge Institute for
Research. J.E.G. acknowledges institutional support from the
Office of the Vice Chancellor for Research and Graduate
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ABBREVIATIONS
■
AUC, area under the curve; BAG1, T. gondii bradyzoite specific
heat shock protein; BBB, blood−brain barrier; CC50, cytotoxic
concentration that results in death of 50% of the cells; cLogD,
calculated partition coefficient for an ionizable compound at a
given pH; EC50, effective concentration of compound
necessary to inhibit 50% of the parasites; HFF, human foreskin
fibroblasts; PAMPA, parallel artificial membrane permeability
assay; SAR, structure−activity relationships; SI, selectivity
index; TbHK1, Trypanosoma brucei hexokinase 1; TgHK, T.
gondii hexokinase.
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G.; Li, Y.; Lu, H.; Chen, Z.; Thomas, S. B. Discovery of Potent and
Selective Leads against Toxoplasma gondii Dihydrofolate Reductase
via Structure-Based Design. ACS Med. Chem. Lett. 2016, 7 (12),
1124−1129.
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Identification of Small-Molecule Inhibitors of Nucleoside Triphos-
phate Hydrolase in Toxoplasma gondii. Antimicrob. Agents Chemother.
2002, 46 (8), 2393−2399.
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