Novel N-benzoyl-2-hydroxybenzamide disrupts unique parasite secretory pathway

Article abstract of DOI:10.1128/AAC.06450-11

Toxoplasma gondii is a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery of N-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range against T. gondii in vitro and in vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be useful as a new scaffold for development of novel and improved inhibitors of T. gondii. Our genome-wide investigations reveal a specific mechanism of resistance to N-benzoyl-2-hydroxybenzamides mediated by adaptin-3β, a large protein from the secretory protein complex. N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs). N-Benzoyl-2-hydroxybenzamide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs. Furthermore, QQ-437 is active against chloroquine- resistant Plasmodium falciparum. Our studies reveal a novel class of compounds that disrupts a unique secretory pathway of T. gondii, with the potential to be used as scaffolds in the search for improved compounds to treat the devastating diseases caused by apicomplexan parasites. Copyright

Full text of DOI:10.1128/AAC.06450-11

Novel N-Benzoyl-2-Hydroxybenzamide
Disrupts Unique Parasite Secretory
Pathway
Alina Fomovska, Qingqing Huang, Kamal El Bissati, Ernest
J. Mui, William H. Witola, Gang Cheng, Ying Zhou, Caroline
Sommerville, Craig W. Roberts, Sam Bettis, Sean T. Prigge,
Gustavo A. Afanador, Mark R. Hickman, Patty J. Lee, Susan
E. Leed, Jennifer M. Auschwitz, Marco Pieroni, Jozef Stec,
Stephen P. Muench, David W. Rice, Alan P. Kozikowski and
Rima McLeod
Antimicrob. Agents Chemother. 2012, 56(5):2666. DOI:
10.1128/AAC.06450-11.
Published Ahead of Print 21 February 2012.
Updated information and services can be found at:
http://aac.asm.org/content/56/5/2666
These include:
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Novel N-Benzoyl-2-Hydroxybenzamide Disrupts Unique Parasite
Secretory Pathway
Alina Fomovska,a Qingqing Huang,*b Kamal El Bissati,a Ernest J. Mui,a William H. Witola,*a Gang Cheng,b Ying Zhou,a
Caroline Sommerville,c Craig W. Roberts,c Sam Bettis,a Sean T. Prigge,d Gustavo A. Afanador,d Mark R. Hickman,e Patty J. Lee,e
Susan E. Leed,e Jennifer M. Auschwitz,e Marco Pieroni,*b Jozef Stec,b Stephen P. Muench,f David W. Rice,g Alan P. Kozikowski,b
and Rima McLeoda
Department of Ophthalmology and Visual Sciences, Pediatrics (Infectious Diseases), Committees on Genetics, Immunology, and Molecular Medicine, Institute of
Genomics and Systems Biology, and The College, The University of Chicago, Chicago, Illinois, USAa; Drug Discovery Program, Department of Medicinal Chemistry and
Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois, USAb; Strathclyde Institute of Pharmacy & Biomedical Sciences, University of Strathclyde, Glasgow,
United Kingdomc; Johns Hopkins School of Public Health, Baltimore, Maryland, USAd; Department of Discovery, Division of Experimental Therapeutics, Walter Reed Army
Institute of Research, Silver Spring, Maryland, USAe; Institute of Membrane and Systems Biology, University of Leeds, Leeds, United Kingdomf; and Department of
Molecular Biology, University of Sheffield, Sheffield, United Kingdomg
Toxoplasma gondii is a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein,
we describe our discovery of N-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range
against T. gondii in vitro and in vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be
useful as a new scaffold for development of novel and improved inhibitors of T. gondii. Our genome-wide investigations reveal a
specific mechanism of resistance to N-benzoyl-2-hydroxybenzamides mediated by adaptin-3␤, a large protein from the secretory
protein complex. N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics
proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs). N-Benzoyl-2-hydroxybenz-
amide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs.
Furthermore, QQ-437 is active against chloroquine-resistant Plasmodium falciparum. Our studies reveal a novel class of com-
pounds that disrupts a unique secretory pathway of T. gondii, with the potential to be used as scaffolds in the search for im-
proved compounds to treat the devastating diseases caused by apicomplexan parasites.
oxoplasma gondii is an apicomplexan, intracellular parasite penicillin-streptomycin-amphotericin B (Fungizone) (IMDM-C) at 33°C
or 37°C and 5% CO2. Toxoplasma gondii parasites were maintained in
that infects one third to one half of the world’s population. It
T
HFF monolayers under the conditions described above. The strains of
parasite used in this study include RH, RH-YFP (kindly provided by Boris
Streipen, University of Georgia), and Prugneaud Fluc (type 2 parasites
stably transfected with luciferase, kindly provided by Seon Kim, Jeroen
Saeij, and John Boothroyd [Stanford University] and Laura Knoll [Uni-
versity of Wisconsin, Madison, Wisconsin]).
can cause eye and brain disease and death, and the presence of
infection has been correlated with a variety of neurologic illnesses.
Moreover, it is the most frequent cause of infectious uveitis world-
wide. Disease can be especially severe in immunocompromised
persons and in those infected congenitally (28).
There is no perfect treatment for T. gondii infection in humans,
as the few available medicines are limited by their side effects and
target only the rapidly proliferating tachyzoite form of the para-
site. Pyrimethamine and sulfadiazine, which are effective against
the tachyzoite form, are currently used to treat active disease.
However, treatment with these medicines can be associated with
toxicity and hypersensitivity (29), and they do not eradicate the
bradyzoite form of the parasite, which remains latent. There are
few secondary medicines, and some of them have a delayed mech-
anism of killing the tachyzoites. No medicines have been reported
to be effective against the latent, encysted bradyzoite stage. T. gon-
dii remains in a person’s body throughout life, leading to a risk for
recurrence of active infection. Novel, effective, and nontoxic anti-
Toxoplasma agents are urgently needed. Herein, we present a se-
ries of experiments to identify new lead compounds effective
against T. gondii and to begin to understand how they act on this
parasite.
High-throughput screen. A high-throughput screen of a library opti-
mized for its absorption, distribution, metabolism, excretion, and toxicity
(ADMET) properties was carried out as described below for in vitro chal-
lenge and toxicity assays.
Synthesis of derivatives of MP-IV-1 and QQ-437. MP-IV-1 and QQ-
437 were synthesized to develop novel inhibitors of T. gondii for this work.
Received 24 December 2011 Returned for modification 7 February 2012
Accepted 13 February 2012
Published ahead of print 21 February 2012
Address correspondence to Rima McLeod, rmcleod@uchicago.edu.
* Present address: Q. Huang, Institute of Drug Discovery and Development, iAIR,
Shanghai Engineering Research Center of Molecular Therapeutics and New Drug
Development, East China Normal University, Shanghai, People’s Republic of China;
W. H. Witola, Department of Agricultural and Environmental Sciences, Tuskegee
University, Tuskegee, Alabama, USA; M. Pieroni, Centro Interdipartimentale
Biopharmanet-Tec, Campus Universitario, Universita di Parma, Parma, Italy.
A.F. and Q.H. contributed equally to this article.
Supplemental material for this article may be found at http://aac.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.06450-11
MATERIALS AND METHODS
Parasites and cell culture. Confluent monolayers of human foreskin fi-
broblasts (HFF) were maintained in Iscove’s modified Dulbecco’s me-
dium supplemented with 10% fetal bovine serum, 1% Glutamax, and 1%
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Benzamides Disrupt Unique Parasite Secretory Pathway
They were repurposed to test against the K1 isolate of P. falciparum with NMR (CDCl3) ␦ 165.2, 156.5, 150.7, 136.2, 128.9, 128.2, 127.6, 121.1,
Leishmania and trypanosomes to compare the structure-activity relation- 119.9, 119.1, 99.6, 28.5, 14.7; HPLC purity, 96.5%.
ship (SAR) for each of those. The details of a number of these syntheses are
(v) N-(4-Diethylaminobenzoyl)-2-methoxybenzenesulfonamide
described elsewhere (42a). The compounds synthesized are shown in Ta- (chg-1-23). To a solution of 2-methoxybenzamide (121 mg, 1 mmol) and
ble 1. Additional details of syntheses and analysis not included in reference 4-dimethylaminopyridine (DMAP) (293 mg, 2.4 mmol) in 2 ml pyridine,
42a are as follows.
4-ethylbenzoyl chloride (1 mmol) was added dropwise at 0°C. The mix-
(i) N-Benzoyl-4-ethylbenzamide (chg-1-13). To a solution of benz- ture was stirred for 10 h at room temperature. White solid was precipi-
amide (121 mg, 1 mmol) in 2 ml pyridine, 4-ethylbenzoyl chloride (1 tated after addition of ethyl acetate (5 ml) and water (5 ml). The solid was
1
mmol) was added dropwise at 0°C. The mixture was stirred for 10 h at collected by filtration to give compound chg-1-23 (127 mg, 40%). H
room temperature, diluted with ethyl acetate, washed with water, and NMR (DMSO-d6) ␦ 11.8 (brs, 1H), 7.90 (d, J ϭ 8.0 Hz, 1H), 7.75 (d, J ϭ
concentrated. The residue was purified by flash chromatography (silica 8.8 Hz, 2H), 7.65 (t, J ϭ 8.0 Hz, 1H), 7.22 (d, J ϭ 8.0 Hz, 1H), 7.13 (t, J ϭ
gel, 25% ethyl acetate [EtOAc]-hexane) to give compound chg-1-13 (177 8.0 Hz, 1H), 6.63 (d, J ϭ 8.8 Hz, 2H), 3.83 (s, 3H), 3.40 (q, J ϭ 6.8Hz, 4H),
mg, 70%). 1H nuclear magnetic resonance (NMR) (CDCl3) ␦ 8.85 (s, 1H), 1.09 (d, J ϭ 6.8 Hz, 6H); 13C NMR (DMSO-d6) ␦ 164.0, 156.8, 151.3,
7.88 (d, J ϭ 7.2 Hz, 2H), 7.81 (d, J ϭ 8.0 Hz, 2H), 7.65 (t, J ϭ 7.2 Hz, 1H), 135.4, 132.0, 130.1, 126.7, 120.6, 116.7, 112.1, 110.4, 56.2, 44.5, 12.4;
7.53 (t, J ϭ 7.2 Hz, 2H), 7.35 (d, J ϭ 8.0 Hz, 2H), 2.75 (q, J ϭ 7.2 Hz, 2H), HPLC purity, 99.1%.
1.29 (t, J ϭ 7.2 Hz, 3H); 13C NMR (CDCl3) ␦ 166.5, 166.2, 149.6, 133.1,
132.5, 130.3, 128.3, 127.9, 127.8, 127.7, 28.5, 14.8; high-pressure liquid (chg-1-24). To a solution of compound chg-1-23 (100 mg, 0.31 mmol) in
chromatography (HPLC) purity, 97.7%. dry dichloromethane was added dropwise a boron tribromide solution in
(vi) N-(4-Diethylaminobenzoyl)-2-hydroxybenzenesulfonamide
(ii) N-Benzoyl-4-diethylaminobenzamide (chg-1-17b). Following dichloromethane (1.1 ml, 1 M in dichloromethane) at Ϫ78°C. The reac-
the same procedure as that for chg-1-13, to a solution of benzamide (121 tion mixture was stirred overnight at room temperature, quenched with 1
mg, 1 mmol) in 2 ml pyridine, 4-diethylaminobenzoyl chloride (1 mmol) N NaHCO3 solution (3 ml) at 0°C, diluted with ethyl acetate, washed with
in pyridine (1 ml) was added dropwise at 0°C. The mixture was stirred for water twice, dried over Na2SO4, and concentrated. The residue was puri-
10 h at room temperature, diluted with ethyl acetate, washed with water, fied through preparative HPLC to give compound chg-1-24 (32 mg, 30%)
and concentrated. The residue was purified by flash chromatography (sil- as white solid. 1H NMR (CD3OD) ␦ 7.89 (d, J ϭ 8.0 Hz, 1H), 7.72 (d, J ϭ
ica gel, 30% EtOAc-hexane) to give compound chg-1-17b (44 mg, 15%). 9.2 Hz, 2H), 7.48 (t, J ϭ 8.0 Hz, 1H), 7.00 (m, 2H), 6.69 (d, J ϭ 8.8 Hz,
1H NMR (CDCl3) ␦ 8.73 (s, 1H), 7.85 (d, J ϭ 7.6 Hz, 2H), 7.80 (d, J ϭ 8.8 2H), 3.45 (q, J ϭ 6.8Hz, 4H), 1.18 (d, J ϭ 6.8 Hz, 6H); 13C NMR (CD3OD)
Hz, 2H), 7.59 (t, J ϭ 7.2 Hz, 1H), 7.49 (t, J ϭ 7.2 Hz, 2H), 6.67 (d, J ϭ 8.8 ␦ 166.6, 155.8, 151.5, 135.0, 130.1, 124.6, 118.9, 117.2, 116.6, 110.1, 44.0,
Hz, 2H), 3.44 (q, J ϭ 6.8 Hz, 4H), 1.23 (t, J ϭ 7.2 Hz, 6H); 13C NMR 11.3; HPLC purity, Ͼ99%.
(CDCl3) ␦ 166.7, 165.0, 150.9, 133.8, 132.2, 130.1, 128.3, 127.5, 118.0,
(vii) N-(4-Ethylbenzoyl)-2-methoxybenzamide (chg-1-27). To a so-
110.1, 44.2, 12.1; HPLC purity, 98.5%.
lution of 2-methoxybenzamide (121 mg, 1 mmol) in 2 ml pyridine, 4-eth-
(iii) N-(4-Ethylbenzoyl)-2-methoxybenzenesulfonamide (chg-1- yl-benzoyl chloride (1 mmol) was added dropwise at 0°C. The mixture
19). To a solution of 5-bromo-2-methoxybenzenesulfonyl chloride (250 was stirred for 10 h at room temperature, diluted with ethyl acetate,
mg, 0.88 mmol) in tetrahydrofuran (THF) (3 ml) was added 1 ml NH4OH washed with water and concentrated. The residue was purified by flash
aqueous solution (28% to 35%). The mixture was stirred at room tem- chromatography (silica gel, 30% EtOAc/Hexane) to give compound chg-
perature for 10 h. The solvent was removed under reduced pressure, di- 1-27 (85 mg, 30%). 1H NMR (CDCl3) ␦ 11.21 (s, 1H), 8.28 (d, J ϭ 8.0 Hz,
luted with ethyl acetate, washed with water twice, dried, and concentrated 1H), 7.84 (d, J ϭ 8.0 Hz, 2H), 7.55 (t, J ϭ 8.0 Hz, 1H), 7.33 (d, J ϭ 8.0 Hz,
to yield 5-bromo-2-methoxybenzenesulfonamide (212 mg, 91%). 1H 2H), 7.10 (t, J ϭ 8.0 Hz, 1H), 7.05 (d, J ϭ 8.0 Hz, 1H), 4.10 (s, 3H), 2.74 (q,
NMR (dimethyl sulfoxide [DMSO]-d6) ␦ 7.79 to 7.73 (m, 2H), 7.25 (s, J ϭ 7.2 Hz, 2H), 1.28 (t, J ϭ 7.2 Hz, 3H); 13C NMR (CDCl3) ␦ 164.8, 162.6,
2H) 7.19 (d, J ϭ 8.8 Hz, 1H), 3.90 (s, 3H).
157.0, 149.4, 134.2, 132.9, 131.1, 128.0, 127.3, 121.6, 120.5, 111.3, 56.2,
A mixture of 5-bromo-2-methoxybenzenesulfonamide (212 mg, 0.8 28.5, 14.8; HPLC purity, Ͼ99%.
mmol), Pd/C (10%, 20 mg) in 5 ml methanol (MeOH) was vigorously
(viii) 4-Ethyl-N-(2-hydroxybenzoyl)benzenesulfonamide (chg-1-
stirred for 10 h under H2 (1 atm) at room temperature. The mixture was 28). To a mixture of 4-ethylbenzenesulfonamide (300 mg, 1.66 mmol),
filtered through a pad of Celite to remove Pd/C. The filtrate was concen- K2CO3 (455 mg, 3.3 mmol) in THF (4 ml) 2-benzyloxybenzoyl chloride
trated to give 2-methoxybenzenesulfonamide (150 mg, 100%). 1H NMR (409, 1.66 mmol) was added, and the solution was heated to 60°C for 5 h,
(DMSO-d6) ␦ 7.74 (d, J ϭ 8.0 Hz, 1H), 7.56 (d, J ϭ 8.0 Hz, 1H), 7.20 (d, diluted with ethyl acetate (30 ml), washed with water twice, and concen-
J ϭ 8.0 Hz, 1H), 7.10 to 7.00 (m, 3H), 3.93 (s, 3H).
trated. The crude intermediate was redissolved with 10 ml MeOH and
To a mixture of 2-methoxybenzenesulfonamide (150 mg, 0.8 mmol), hydrogenated with Pd/C (10%, 10 mg) under H2 (1 atm) for 5 h. Pd/C was
K2CO3 (207 mg, 1.5 mmol) in THF (4 ml) was added 4-ethylbenzoic removed by filtration. The filtrate was concentrated and purified by pre-
chloride (0.15 ml, 1 mmol), and then the solution was heated to 60°C for parative HPLC to afford compound chg-1-28 (152 mg, 30%) as a white
5 h. A white solid was precipitated after addition of ethyl acetate (5 ml) solid. 1H NMR (CDCl3) ␦ 10.85 (brs, 1H), 9.80 (brs, 1H), 8.09 (d, J ϭ 8.4
and water (5 ml). The white solid was collected by filtration to give the Hz, 2H), 7.65 (d, J ϭ 8.0 Hz, 1H), 7.45 (m, 3H), 6.95 (d, J ϭ 8.0 Hz, 1H),
1
compound chg-1-19 (127 mg, 50%). H NMR (CDCl3) ␦ 9.11 (s, 1H), 6.85 (d, J ϭ 8.0 Hz, 1H), 2.75 (q, J ϭ 7.6 Hz, 2H), 1.27 (t, J ϭ 7.6 Hz, 3H);
8.21 (m, 1H), 7.75 (m, 2H), 7.60 (m, 1H), 7.28 (m,1H), 7.18 (m, 1H), 7.02 13C NMR (CDCl3) ␦ 167.7, 161.4, 151.3, 135.9, 134.9, 128.4, 128.2, 127.1,
(m, 1H), 3.95 (s, 3H), 2.65 (m, 2H), 1.25 (m, 3H); 13C NMR (CDCl3) ␦ 119.3, 118.3, 112.3, 28.8, 14.6; HPLC purity, Ͼ99%.
164.2, 156.5, 150.0, 135.5, 131.8, 128.4, 127.9, 127.8, 125.8, 120.2, 111.8,
55.8, 28.5, 14.7; HPLC purity, 98.1%.
(ix) N-(2-Hydroxybenzoyl)-4-isopropoxybenzamide (chg-1-43).
Similar to the synthesis of QQ-437, a mixture of salicylamide (3 mmol)
(iv) N-(4-Ethylbenzoyl)-2-hydroxybenzenesulfonamide (chg-1- and 4-isopropoxybenzoyl chloride (3 mmol) in 6 ml pyridine was refluxed
22). To a solution of compound chg-1-19 (100 mg, 0.31 mmol) in dry for 1 h. Chg-1-43 (628 mg, 70%) was obtained by recrystallization from
dichloromethane, boron tribromide (1.1 mmol, 1 M in dichloromethane) methanol, ethyl acetate, and water. 1H NMR (DMSO-d6) ␦ 11.73 (s, 1H),
was added dropwise at Ϫ78°C. The reaction mixture was stirred overnight 11.58 (s, 1H), 7.87 (m, 3H), 7.46 (t, J ϭ 8.0 Hz, 1H), 7.03 (m, 4H), 4.76 (m,
at room temperature, quenched with 1 N HCl solution (3 ml) at 0°C, 1H), 1.09 (d, J ϭ 4.0 Hz, 6H); 13C NMR (DMSO-d6) ␦ 164.2, 164.2, 161.4,
diluted with ethyl acetate, washed with water twice, dried over Na2SO4, 156.4, 134.2, 131.1, 130.0, 125.4, 120.0, 119.2, 117.1, 115.4, 69.8, 21.7;
and concentrated to give compound chg-1-22 (47 mg, 50%) as a white HPLC purity, Ͼ99%.
solid. 1H NMR (DMSO-d6) ␦ 12.2 (brs, 1H), 10.8 (brs, 1H), 7.85 (m, 3H),
(x) N-(2-Hydroxybenzoyl)-4-pyrrolidin-1-ylbenzamide (chg-1-46).
Similar to the synthesis of QQ-437, a mixture of salicylamide (3mmol)
7.50 (m, 1H), 7.35 (m, 2H), 6.90 (m, 2H), 2.65 (m, 2H), 1.23 (m, 3H); 13
C
May 2012 Volume 56 Number 5
aac.asm.org 2667

TABLE 1 SAR based on inhibitory effects of N-benzoyl-2-hydroxybenzimides on T. gondiia
a The diagram above the table body presents a summary of the SAR of N-benzoyl-2-hydroxybenzimide inhibitors. The table shows the inhibitory effects of N-benzoyl-2-
hydroxybenzimides on T. gondii and the effects on host cells (toxicity). Analysis was also performed for P. falciparum KI and kinetoplastids (42a) for the compounds
shown in the table. The range, where provided, reflects the small variability in the assay when replicate experiments were performed on different days. Also, some
compounds were tested in parallel with QQ-437 but were not tested further upon being identified as inferior.
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Benzamides Disrupt Unique Parasite Secretory Pathway
and 4-pyrrolidin-1-yl benzoyl chloride (3 mmol) in 6 ml pyridine was neal fluid was assessed. Four runs were conducted in total, each consisting
of five groups of mice with four or five mice in each group.
refluxed for 1 h. Chg-1-46 (558 mg, 60%) was obtained by recrystalliza-
tion from methanol and water. H NMR (DMSO-d6) ␦ 11.77 (s, 1H),
1
In the second method, mice were infected intraperitoneally with
20,000 Fluc parasites in 400 ␮l of phosphate-buffered saline (PBS) on day
one. One hour later, mice were injected with either test compound at 20
mg/kg dissolved in 100 ␮l of DMSO or a DMSO control. The test com-
pound was administered daily for 6 days. Parasite burden was assessed by
daily imaging with a Xenogen camera. This experiment was initiated with
4 or 5 mice per group; however, one mouse in the DMSO-only group and
one in the QQ-437 group were omitted due to pregnancy, and one mouse
in the QQ-437 20 mg/kg group died.
11.47 (s, 1H), 7.90 (d, J ϭ 8.0 Hz, 1H), 7.77 (d, J ϭ 8.0 Hz, 2H), 7.46 (t, J ϭ
8.0 Hz, 1H), 7.02 (m, 2H), 6.61 (d, J ϭ 8.0 Hz, 2H), 3.33 (m, 4H), 1.99 (m,
4H); 13C NMR (DMSO-d6) ␦ 164.7, 164.4, 156.7, 151.0, 134.5, 131.5,
130.0, 120.4, 119.6, 119.4, 117.5, 111.5, 47.7, 25.4; HPLC purity, 97.3%.
In vitro challenge and toxicity assays. (i) Initial screen of 6,811 com-
pounds. HFF were cultured in 19 384-well plates as described above. Cells
were inoculated with RH-strain parasites, which express yellow fluores-
cent protein (RH-YFP), at a concentration of 3,500 parasites/ml. After
incubation for 1 h, compounds were added to make the final concentra-
tion 10 ␮M in singleton exemplars using the Tecan Freedom EVO 200
robotic platform. After 72 h, parasite proliferation was assessed using an
ImageXpress Micro automated fluorescence microscope by measuring
parasite fluorescence at a wavelength of 488 nm. Compounds were
deemed effective if in their presence fluorescence was reduced to below
1,000 relative fluorescence units (RFU) at the end of the assay.
(ii)Preparationofcompoundsforchallengeandtoxicityassays. The
initial 6,811-compound library was designed by Chris Lipinski and pur-
chased by Alan Kozikowski at the University of Illinois at Chicago. Dilu-
tions of 10 mM or 1 mM were made in DMSO, and further serial dilutions
were made in IMDM-C. When cells or parasites were treated in vitro, the
DMSO concentration was limited to 0.1%.
(iii) Tachyzoite in vitro challenge assay. RH parasites were separated
from HFF by passage through a 25-gauge needle twice (32). Parasites were
centrifuged at 1,500 rpm for 15 min, resuspended in IMDM-C, and
counted. Confluent HFF in 96-well plates were inoculated with 3,500
parasites in a volume of 100 ␮l per well. Parasites were allowed to infect
cells for 1 h before inhibitory compounds and control medium were
added. After 72 h, parasite burden was assessed with a YFP fluorescence
assay or [3H]uracil incorporation assay.
In a YFP fluorescence assay, the relative fluorescence of parasite sam-
ples, which is directly correlated with parasite viability, was measured
using a Synergy H4 hybrid reader (BioTek) and Gen5 1.10 software.
In a [3H]uracil incorporation assay (32), 25 ␮l of 0.1-mCi/ml [3H]
uracil was added to each well 48 h after initiation of the experiment.
Twenty-four hours later, the contents of the wells were transferred onto
96-well filter plates using a cell harvester, and [3H]uracil incorporation,
which is representative of parasite replication and viability, was measured
using a liquid scintillation counter.
Activity against Plasmodium falciparum. Compound activity against
P. falciparum was assessed using a malaria SYBR green I-based fluores-
cence (MSF) assay. Two laboratory strains of P. falciparum were used: D6
(Centers for Disease Control and Prevention [CDC]/Sierra Leone) and
TM90-C235 (Walter Reed Army Institute of Research [WRAIR], Thai-
land). Parasites were maintained continuously in long-term cultures as
previously described (22). Predosed microtiter drug plates for use in the
MSF assay were produced using sterile 384-well black optical-bottom tis-
sue culture plates containing dilutions of each test compound or chloro-
quine hydrochloride (Sigma-Aldrich Co.) suspended in DMSO. There
were quadruplicate replicate wells for each of the 12 concentrations. There
were serial twofold dilutions starting at 10,000 ng/ml for this dose-re-
sponse test. The final concentration range tested was 0.5 to 10,000 ng/ml
for all assays. Predosed plates were stored at 4°C until used, not to exceed
5 days. No difference was seen in drug sensitivity determinations between
stored and fresh drug assay plates (data not shown). A batch control plate
using chloroquine (Sigma-Aldrich Co.) at a final concentration of 2,000
ng/ml was used to validate each assay run. The Tecan Freedom EVO liquid
handling system (Tecan US, Inc., Durham, NC) was used to produce all
drug assay plates. Based on modifications of previously described meth-
ods (22, 36), P. falciparum strains in the late ring or early trophozoite stage
were cultured in the predosed 384-well microtiter drug assay plates in a
38-␮l culture volume per well at a starting parasitemia of 0.3% and a
hematocrit of 2%. The cultures were then incubated at 37°C with 5% CO2,
5% O2, and 90% N2 for 72 h. Lysis buffer (38 ␮l per well), consisting of 20
mM Tris HCl, 5 mM EDTA, 1.6% Triton X, 0.016% saponin, and SYBR
green I dye at a 20ϫ concentration (Invitrogen), was then added to the
assay plates for a final SYBR green concentration of 10ϫ. The Tecan
Freedom Evo liquid handling system was used to dispense malaria cell
culture and lysis buffer. The plates were then incubated in the dark at
room temperature for 24 h and examined for relative fluorescence units
(RFU) per well using Tecan Genios Plus (Tecan US, Inc., Durham, NC).
Each drug concentration was transformed into log[concentration] and
plotted against the RFU values. The 50% and 90% inhibitory concentra-
tions (IC50s and IC90s, respectively) were then generated with GraphPad
Prism (GraphPad Software Inc., San Diego, CA) using the nonlinear re-
gression (sigmoidal dose-response/variable slope) equation.
(iv) Tachyzoite in vitro toxicity assay. Toxicity to HFF was measured
by means of a [3H]thymidine incorporation assay or a WST-1 cell viability
assay.
In a [3H]thymidine incorporation assay, cells were cultured to ϳ30%
confluence in 96-well plates. Inhibitory compounds and control medium
were added at concentrations equal to those in challenge assays. Forty-
eight hours later, 25 ␮l of 0.1-mCi/ml [3H]thymidine was added to each
well. Twenty-four hours after [3H]thymidine addition, cells were trans-
ferred onto 96-well filter plates using a cell harvester, and [3H]thymidine
incorporation was measured using a liquid scintillation counter.
Toxicity assays also were conducted using WST-1 cell proliferation
reagent (Roche). Confluent HFF were treated with inhibitory and control
compounds at concentrations equal to those being tested in challenge
assays. To assess cytotoxicity, on the day the samples were read, 10 ␮l of
WST-1 was added to each well, cells were incubated for 1 h, and absor-
bance was read using a fluorometer at 420 nm.
Insertional mutagenesis. (i) Transfection of pLK47 plasmid into T.
gondii. The pLK47 vector plasmid (kindly provided by L. Knoll, Madison,
WI) (23) was transfected into T. gondii parasites. Cytomix electroporation
buffer solution, composed of 120 mM KCl, 150 ␮M CaCl2, 5 mM
K2HPO4, 5 mM KH2PO4, 25 mM HEPES, 2 mM EDTA, and 5 mM MgCl2
in water, was utilized. Strain RHdHxgPRT parasites were extracted from
HFF by two subsequent passages through a 25-gauge needle, filtered, cen-
trifuged for 5 min at 25,000 rpm, resuspended in 1 ml of Cytomix solu-
tion, and counted. A volume of Cytomix solution containing 10 ϫ 107
parasites and an equivalent amount of plasmid DNA were mixed to make
up a total volume of 400 ␮l. Immediately after mixing, parasites were
pulsed using Bio-Rad electroporator at the following settings: 1.5 kV, 25
␮F, 100 ⍀.
Tachyzoite in vivo assays. Compound efficacy in vivo in mice was
determined according to two methods. In both cases compounds were
first reconstituted in DMSO.
In the first method, SW mice were infected with 2,000 RH parasites by
intraperitoneal injection. Mice were then treated with control solutions
and with test compounds at various concentrations. Mice were sacrificed
Parasites were grown in IMDM-C medium 37°C and 5% CO2 and
treated with 25 ␮g/ml mycophenolic acid and 50 ␮g/ml xanthine. Me-
dium was changed every other day. Parasites were grown in the presence
5 or 6 days after infection, and the amount of parasites in the intraperito- of mycophenolic acid and xanthine for the duration of 1 month to ensure
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Fomovska et al.
the death of all parasites without the transfected plasmid. After 1 month,
Coverslips were mounted with Antifade (Molecular Probes, Eugene,
parasites were treated with MP-IV-1 at concentrations of 10 ␮M, 0.5 ␮M, OR), and images were analyzed by high-resolution fluorescence using
deconvolution protocols. Microscopy was performed with an inverted
microscope (Nikon) with a 41001 filter (excitation, 480 to 440 nm; emis-
sion, 440 nm) for fluorescein isothiocyanate (FITC), a 41035 filter (exci-
tation, 540 to 525 nm; emission, 620 to 660 nm) for rhodamine, and a
31013v2 filter (excitation, 360 to 340 nm; emission, 460 to 450 nm) for
4=,6=-diamidino-2-phenylindole (DAPI).
and 0.03 ␮M. Parasites were treated with MP-IV-1 for 2 weeks to ensure
the death of all parasites not resistant to MP-IV-1.
(ii) Cloning parasites to isolate individual mutants. A bulk culture of
parasites resistant to MP-IV-1 was diluted to a concentration of 1 parasite/
100 ␮l. A 96-well plate was inoculated with that concentration to yield 1
parasite/well. Parasites were treated with 10 ␮M MP-IV-1 and incubated
for 2 weeks. Wells with a single infection locus were selected and num-
bered A1 to A23. The contents were transferred to individual wells of
12-well plates. These parasites were cultured at 37°C and 5% CO2 in the
presence of 10 ␮M MP-IV-1.
ADMET. Cytochrome P450 (CYP450) inhibition, hERGs, human
liver microsomal stability, solubility, protein binding in human
plasma, and permeability assays were performed as described previ-
ously (45) by Tipparaju et al. ADMET assessments for MP-IV-1 and
QQ-437 were performed by Pharmaron (Louisville, KY) and Cerep
(Redmond, WA), respectively.
(iii) Identification of genes that, when disrupted, confer resistance
to MP-IV-1. MP-IV-1-resistant parasite mutants were propagated in a
T75 flask. DNA was extracted using DNAzol reagent and resuspended in
water. Initial digestion was carried out with the restriction enzymes SalI,
SaclI, and NotI (each separately). Three microliters of restriction enzyme
and 2 ␮l of appropriate buffer were added to 20 ␮l of each DNA sample.
DNA was incubated overnight in a 37°C water bath. After purification of
the DNA via phenol extraction, fragments were religated by adding 4 ␮l
T2 DNA ligase enzyme buffer at a 5ϫ strength and 1.5 ␮l T4 ligase enzyme
and refrigerating overnight at 4°C. After another phenol extraction, frag-
ments were digested with a second set of enzymes paired with the first set
as follows: SalI-NheI, SacI-MluI, NotI-NcoI. Inverse PCR was performed
using forward and reverse primers corresponding to the second set of
restriction enzymes. Primers are as follows: NheI inverse forward, 5=-AA
GCGACGTTGTGTCTCAAAATCRCRGAT-3=; NheI inverse reverse, 5=-
ATGATGGCCGGACAAACAACAGATAA-3=; MluI inverse forward, 5=-
ATTACCGCCTTTGAGTGAGCTGATA-3=; MluI inverse reverse, 5=-AC
ACAGCACCCGAACTTACGGAGCTGGT-3=; NcoI inverse forward, 5=-
ATCGTGGTCTACGAGGCCACACTCA-3=; and NcoI inverse reverse,
5=-TCGCTGTAGCCGTCGCTCATAGCAAT-3=.
RESULTS
Overview of approach and experimental design from lead iden-
tification and optimization to beginning characterization of ef-
fects of N-benzoyl-2-hydroxybenzamides, demonstrating how
the various experiments are used in the critical path of series
selection. Herein, to identify novel leads effective against toxo-
plasmosis, we screened a library optimized for medicinal chemis-
try for compliance with Lipinski’s rule of five (25). This screen of
6,811 synthetic compounds identified 28 primary “hits” with
IC90s of Ͻ10 ␮M. From these hits, MP-IV-1, a synthetic com-
pound belonging to the N-benzoyl-2-hydroxybenzamide class,
stood out as the most attractive lead for our optimization effort.
MP-IV-1 was not toxic at high (10 ␮M) concentrations in vitro
and was active at low (IC90 ϭ 31 nM) concentrations in vitro.
MP-IV-1 was effective in vivo in mice at reducing parasite burden.
N-Benzoyl-2-hydroxybenzamides were synthesized by reaction of
salicylamide with various acid chlorides in refluxing pyridine, pu-
rified by crystallization or preparative HPLC, and further elabo-
rated. QQ-437 was found to be the most effective derivative of
MP-IV-1. These two compounds were also tested against Plasmo-
dium falciparum, a related member of the apicomplexan family
and the causative agent of falciparum malaria. QQ-437 was found
to be superior in efficacy to MP-IV-1, even against chloroquine-
resistant parasites. We noted, however, that N-benzoyl-2-hy-
droxybenzamides may have a different target in malarial parasites,
because there was a substantial dichotomy in susceptibility ob-
served between MP-IV-1 and QQ-437 against P. falciparum that
was not seen with T. gondii.
Next, an insertional mutagenesis library of T. gondii parasites
resistant to MP-IV-1 was created (23) to begin to help us to iden-
tify the molecular target of the N-benzoyl-2-hydroxybenzamides
in T. gondii, Sequencing of four clones resistant to MP-IV-1 iden-
tified an adaptin N-terminal region domain-containing protein.
This suggested that the adapter protein could function in a path-
way which involves the molecular target of N-benzoyl-2-hydroxy-
benzamides.
Adaptin-3␤ is part of a secretory pathway which in T. gondii
transports critical and parasite-specific proteins through the Golgi
apparatus to a series of unique organelles. These organelles in-
clude the micronemes, which secrete their contents for attach-
ment to host cells; rhoptries, which are involved in invasion and
taking over the host cell genome expression; and dense granules,
which contribute their contents to the parasitophorous vacuole
and also interact with host cell molecules. The mechanisms
whereby proteins are directed to these organelles are only partly
understood. Adaptin-1 was found to be involved in biogenesis of
Amplification of fragments was confirmed by means of gel electropho-
resis. Bright bands were cut out, and DNA was extracted from the gel with
a Wizard SV gel and PCR cleanup system kit (Promega). DNA fragments
were subsequently ligated into vector pGEMT and transfected into Esch-
erichia coli. Bacterial colonies were screened to ensure integration of the
plasmid vector by means of gel electrophoresis. Appropriate colonies were
sent for sequencing.
The sequences obtained were analyzed using MacVector and BLAST at
ToxoDB.org.
Electron microscopy experiments. HFF were cultured on carbon-
coated glass coverslips obtained from Juan Jiminez (Albert Einstein Uni-
versity) in 24-well plates. Tachyzoites of the RH strain were treated for
24 h with MP-IV-1 or QQ-437 at the respective IC90. After 24 h, slides
were fixed with 2% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M caco-
dylate buffer at room temperature for 1 h. Samples were sent to be imaged
in 0.1 M cacodylate buffer. They were prepared and imaged at the Albert
Einstein University Analytic Imaging Facility.
Immunofluorescence assay imaging experiments. Wild-type RH
parasites or MP-IV-1-resistant mutants were treated with MP-IV-1 for
either 4 or 24 h at either the IC50 or IC90. After the appropriate treatment
period, slides were fixed with 3% paraformaldehyde. Slides were perme-
abilized with PBS–0.2% Triton X-100 at room temperature and blocked
with PBS–0.2% Triton X-100–3% BSA at room temperature. For this set
of experiments, dense granule antibody GRA1 (mouse) was kindly pro-
vided by Marie-France Cesbron-Delauw and Corinne Mercier (Institut
Jean Roget) (4, 6), rhoptry antibody ROP13 was kindly provided by Peter
Bradley (University of California Los Angeles) (46), and cathepsin anti-
bodies (to visualize the acidocalcisome), cathepsin-like protein (CPL),
and the microneme antibody M2 were kindly provided by Vern Car-
ruthers (University of Michigan) (16), and anti-SAG1 antibody was from
Invitrogen. Samples were stained with primary antibodies (GRA1,
ROP13, CPL, M2, and SAG1) for 1 h and secondary antibody (mouse and
rabbit) overnight.
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Benzamides Disrupt Unique Parasite Secretory Pathway
rhoptries (5, 34). Two other organelles, called the acidocalcisome the wild-type parasites (see Fig. S1C in the supplemental mate-
rial).
Lead compound optimization and SAR. Synthesis and evalu-
and the plant-like vacuole (PLV), are key in calcium regulation
and osmoregulation involving vacuolar ATPases, respectively, but
little is known about protein trafficking to these organelles in T.
gondii.
ation of a wide range of MP-IV-1 derivatives yielded an optimized
compound, QQ-437 (Fig. 1E and F; Table 1). The synthetic pro-
cedures, along with spectral data for this and related 2-N-benzoyl-
2-hydroxybenzamides, are described in detail elsewhere (42a). A
structure-activity relationship (SAR) study for these compounds
was carried out for T. gondii (Table 1). Subsequent analysis of the
potency of these compounds was also conducted against various
strains of P. falciparum and pathogenic kinetoplastid parasites
(Table 2) (42a). Syntheses of many of the derivative compounds
and the subsequent SARs for Leishmania, trypanosomes, and P.
falciparum K1 are described elsewhere (42a). Table 1 demon-
strates the approach to identify an analogue with activity superior
to that of QQ-437, once we determined that QQ-437 was superior
to MP-IV-1. Compound QQ-437 was the most effective derivative
and was more effective than MP-IV-1, demonstrating robust ac-
tivity at 16 nM (IC90) and showing no toxicity at the highest con-
centration tested (10 ␮M) (Fig. 1F and G).
Adaptin-3␤ is known to target proteins to the Arabidopsis vac-
uole homologue of the T. gondii plant-like vacuole (1b, 2, 3, 7, 9,
11, 12, 15, 30, 39, 40, 41, 42, 43, 44). Since T. gondii has many
plant-like features (27, 38), we explored the effects of N-benzoyl-
2-hydroxybenzamides on the unique organelles of the T. gondii
secretory pathway, including the PLV. The PLV can be distin-
guished by immunostaining with a cathepsin-like protein (35).
N-Benzoyl-2-hydroxybenzamide-treated parasites showed dis-
ruption of repartition of micronemes, rhoptries, dense granules,
and the acidocalcisome/plant-like vacuole, which is consistent
with our discovery that resistant mutants had an insertion in
adaptin-3␤. The data reported here demonstrate that QQ-437
meets criteria to be a new lead compound and provide insights
into a mechanism of resistance involving adaptin-3␤, a member of
the secretory pathway of T. gondii.
Since QQ-437 is a closely related derivative of MP-IV-1, it is
possible that the two compounds share a molecular target. Mu-
tants resistant to MP-IV-1 were able to grow in the presence of
QQ-437 which supports this hypothesis (data not shown). Fur-
ther, since both MP-IV-1 and QQ-437 showed potent activity
against tachyzoites we studied them further as representatives of
the N-benzoyl-2-hydroxybenzamide class.
QQ-437 was found not only to be the most effective compound
but also to have the best ADMET properties.
Lead compound identification. (i) N-Benzoyl-2-hydroxy-
benzamides are potent compounds against Toxoplasma para-
sites. A library of 6,811 synthetic compounds was screened against
T. gondii tachyzoites, using a fluorescent parasite screen utilizing
RH-YFP-expressing parasites (see Materials and Methods). The
fluorescent parasite screen identified 28 primary hits against
tachyzoites. This finding was confirmed using a [3H]uracil incor-
poration assay, which also serves as a measure of parasite viability
as uracil becomes incorporated into nucleic acids of T. gondii but
not its mammalian host cells. We discarded hits that displayed
toxicity against host human foreskin fibroblasts (HFF). One com-
pound stood out with robust activity and no toxicity: N-4-ethyl-
benzoyl-2-hydroxybenzamide, referred to as compound MP-IV-1
(Fig. 1A to D; Table 1), which was effective against tachyzoites in
vitro at a concentration of 31 nM (IC90) (Fig. 1B) and showed no
toxicity at the highest concentration tested (10 ␮M) (Fig. 1D).
(ii) Insertional mutagenesis provides a means to identify the
molecular target pathway of MP-IV by selecting a parasite that
grows slowly under compound pressure. An insertional mu-
tagenesis library was created to identify genes which confer resis-
tance to MP-IV-1. This was done by transfecting the plasmid vec-
tor pLK47 (kindly provided by Laura Knoll, Madison, Wisconsin)
into the T. gondii genome, thus disrupting the gene present at the
locus where the plasmid integrated (see Fig. S1 in the supplemen-
tal material). Twenty-three mutants were cloned that were resis-
tant to MP-IV-1 at concentrations of 10 ␮M. The segment of DNA
adjacent to the plasmid in the T. gondii genome was isolated and
sequenced for four clones (see Fig. S1A).
Effects of N-benzoyl-2-hydroxybenzamides on T. gondii. (i)
Effects on T. gondii in vitro. To determine whether MP-IV-1 and
QQ-437 inhibit intracellular parasites or invasion of host fibro-
blasts, parasites were allowed to infect HFF in the presence of
either MP-IV-1, QQ-437, or medium. MP-IV-1 did not prevent
cellular invasion but inhibited the parasite by a different mecha-
nism (Fig. 1H), as inhibition of replication was similar with expo-
sure to the compound before and after invasion, and inhibition
was noted only after incubation of infected cells with the inhibitor.
To determine whether these compounds are parasitistatic or
parasiticidal, HFF infected with tachyzoites were treated with MP-
IV-1 or QQ-437 or with pyrimethamine and sulfadiazine, in one
of four conditions: (i) compound was present for 4 days after
infection and then removed; (ii) compound was present for 4
days, replaced with fresh compound, and removed at 10 days; (iii)
compound was present for 10 days and then removed; (iv) com-
pound was present for the duration of the assay. Fluorescence
measurements of the parasites were conducted daily to determine
inhibition. At 14 days, levels of parasites that had been treated with
MP-IV-1 or QQ-437 were similar to the levels of untreated con-
trols, relative to the levels observed in cells treated with pyrimeth-
amine and sulfadiazine, which remained low (Fig. 1I). Thus, MP-
IV-1 and QQ-437, while inhibiting parasite proliferation, do not
have a lethal effect under these conditions, as the parasite is able to
grow after the compound is removed. Furthermore, the high par-
asite levels observed in assays where the compound was present
throughout the experiment suggest that both QQ-437 and MP-
IV-1 lose efficacy with time.
Each of the four clones contained a disruption in the gene for
an adaptin N-terminal region domain-containing protein, at bp
3170 (see Fig. S1B in the supplemental material). One of the four
clones contained an additional insertion in the region of a putative
dynein-1-alpha heavy-chain flagellar inner arm i1 complex. This
could represent the actual target of the compound or simply mark
the pathway in which the actual target resides at some point in the
parasite life cycle.
To confirm that observation, a standard YFP fluorescence as-
say was used to determine the duration of compound efficacy in
vitro. Parasites were treated by MP-IV-1 and QQ-437 at 1 ␮M and
at each compound’s respective IC50 and IC90 and imaged daily for
The mutant parasites grew more slowly and less robustly than 25 days. On day 7, all parasites treated with MP-IV-1 were at levels
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TABLE 2 Effects on Plasmodium falciparum isolates D6 (Sierra Leone) and C235 (Thailand; chloroquine resistant)
D6
C235
SYBR green IC50
SYBR green IC50
Compound
Structure
␮M
ng/ml
r2
␮M
ng/ml
r2
Chloroquine
3.8
46.1
MP-IV-1
QQ-437
2.25
0.05
604.9
15.87
0.94
0.99
Ͼ2,000
NAa
0.99
0.25
77.19
QQ-421
6.40
1,819
0.81
Ͼ2,000
NA
JS-2-40
JS-2-64
1.64
1.13
476.8
362.3
0.97
0.97
4.26
2.85
1,242
915.2
0.86
0.89
a NA, not available.
equivalent to those of untreated controls, as well as of parasites
treated by QQ-437 at the IC50 and IC90. QQ-437, however, was examined using bioluminescence imaging techniques. Mice were
still effective at 1 ␮M (Fig. 1J). infected intraperitoneally with 20,000 tachyzoites expressing lu-
The effect of MP-IV-1 on T. gondii tachyzoites in vivo was also
To determine how quickly MP-IV-1 and QQ-437 act to inhibit ciferase (Fluc). These luciferase-expressing parasites provide a
parasite proliferation, infected cells were treated for six different means to assess the parasite burden in vivo using a Xenogen cam-
time periods before the compounds were removed and replaced era, without euthanizing the mice. MP-IV-1 inhibited growth of
with medium without compound. The compound incubation du- the parasites at a concentration of 50 mg/kg, demonstrated a dose
rations tested were 1, 2, 4, 24, 48, and 72 h with application of response, and was effective by day 4, with no observable signs of
compound. After 72 h, parasite proliferation was assessed. The toxicity. The DMSO-treated control mice also were observed to
level of parasites in wells treated with MP-IV-1 and QQ-437 for have a reduction in parasite burden by day 6 due to some toxicity
2 h was lower than that of untreated controls, suggesting that these from DMSO needed to dissolve the compound, although less than
two compounds act within the first 2 h to inhibit parasite prolif- with MP-IV-1 (Fig. 2B and C).
eration (Fig. 1K).
The effectiveness of QQ-437 against tachyzoites in vivo and in
(ii) The N-benzoyl-2-hydroxybenzamides MP-IV-1 and QQ- comparison to that of MP-IV-1 was also assessed using biolumi-
437 are potent against Toxoplasma infections in vivo. In order to nescence techniques. After infection with Fluc (Prugneaud type 2
examine the potential for using N-benzoyl-2-hydroxybenzamides stably transfected with luciferase) parasites on day one, mice were
as treatments for T. gondii infections, we tested the efficacy of treated with QQ-437 or MP-IV-1 at a concentration of 20 mg/kg
MP-IV-1 and QQ437 against tachyzoite infections in vivo. Mice or with DMSO as an additional control. Parasite load was assessed
were infected via intraperitoneal (IP) injection with RH strain every other day by using the Xenogen camera. Compound QQ-
parasites and treated daily for 6 days with IP administration of 437 was effective in reducing parasite burden at a concentration of
MP-IV-1 or QQ-437 at a concentration of 1, 10, or 50 mg/kg 20 mg/kg and was more effective than MP-IV-1 (Fig. 2D).
dissolved in DMSO. After 6 days, mice were euthanized, and the
(iii)N-Benzoyl-2-hydroxybenzamidesMP-IV-1andQQ-437
intraperitoneal parasite load was assessed by counting using a he- also are potent in vitro against P. falciparum, with QQ-437 hav-
mocytometer and measurements of fluorescence. Mice treated ing efficacy superior to that of MP-IV-1 against a chloroquine-
with MP-IV-1 at 50 mg/kg demonstrated significantly lower par- resistant Thai parasite. Because many compounds effective
asite counts (Fig. 2A).
against T. gondii have also been found to be effective against P.
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Fomovska et al.
FIG 2 MP IV-1 and QQ 437 protect mice. (A) Effect of MP-IV-1 on intraperitoneal RH tachyzoites. (B) Effect of MP IV-1 on Fluc-expressing Prugneaud
tachyzoites administered intraperitoneally, shown as Xenogen camera images. (C) Effect of MP-IV-1 over time. (D) QQ437 is more effective than MP-IV-1.
falciparum, we assessed the antiplasmodial effect of MP-IV-1, nucleus, cell membrane, and rhoptries were identified in MP-
QQ437, and several derivatives. This was accomplished by using IV-1- and QQ-437-treated parasites and untreated controls.
the malaria SYBR green I-based fluorescence assay, which is a However, a marked difference was seen in several organelles which
microtiter plate drug sensitivity assay that uses the presence of receive proteins from the secretory pathway. Typical dense gran-
malarial DNA as a measure of parasitic proliferation. The interca- ules were not present in parasites treated with MP-IV-1 and QQ-
lation of SYBR green I dye into parasite DNA and its resulting 437. An organelle similar in shape and size to a dense granule was
fluorescence reflects parasite growth. Therefore, a test compound visible in these samples but was much less electron dense. The
that inhibits the growth of the parasite will result in a lower fluo- acidocalcisome/PLV was missing in all MP-IV-1-treated parasites
rescence. Two strains of P. falciparum—D6 (CDC/Sierra Leone, (Fig. 3B) and in many of the QQ-437 treated parasites (Fig. 3C).
chloroquine sensitive) and TM90-C235 (WRAIR, Thailand, chlo-
(v) Immunofluorescence assay imaging studies confirm dis-
roquine resistant)—were used for each compound assessment. torted, improperly localized, and absent secretory organelles in
MP-IV-1 was moderately active against D6 (IC50 ϭ 604.9 ng/ml) T. gondii parasites treated with N-benzoyl-2-hydroxybenz-
and not active against TM91C235 (IC50 Ͼ 2000 ng/ml). QQ-437 amides and adaptin-3␤ mutant parasites treated or not treated
was active against both strains of P. falciparum at much lower with N-benzoyl-2-hydroxybenzamides. Immunofluorescence
concentrations (D6 IC50 ϭ 15.87 ng/ml, C235 IC50 ϭ 77.19 ng/ staining experiments were conducted in order to confirm obser-
ml). These results are summarized in Table 2. Chloroquine was vations of organelle disruption seen in electron microscopy stud-
used as a control for all antiplasmodial studies (Table 2).
ies, as well as to investigate the differences between drug-resistant
(iv) Electron microscopy of N-benzoyl-2-hydroxybenzim- and wild-type parasites, both with and without drug pressure. Our
ide-treated T. gondii tachyzoites reveals disruption of dense imaging experiments were focused on several trafficking organ-
granules, unusual lucent areas, and distortion of secretory or- elles implicated by association with adaptin-3␤: micronemes,
ganelles. An electron microscopy analysis was performed to char- rhoptries, dense granules, and acidocalcisomes/PLVs. Wild-type
acterize the effect of MP-IV-1 and QQ-437 on the morphology of RH parasites and MP-IV-1-resistant mutants were treated with
T. gondii parasites. Tachyzoites were treated for 24 h with either MP-IV-1 for either 4 or 24 h at either the IC50 or IC90 and then
MP-IV-1 or QQ-437 at the IC90 and then fixed and imaged. Un- incubated with antibodies to the following markers: SAG1 (sur-
treated tachyzoites cultured under the same conditions were also face antigen), GRA1 (dense granules), ROP13 (rhoptries), CPL
imaged as a basis for comparison (Fig. 3A). Both MP-IV-1 and (acidocalcisomes/PLVs), or M2 (micronemes). MP-IV-1 caused
QQ-437 appeared to affect parasite morphology in an organelle- fragmentation and then disappearance of the acidocalcisome/PLV
specific manner, rather than simply causing global damage. The and also resulted in marked distortion of immunostaining of
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FIG 3 Electron microscopy images showing loss of dense granule content and acidocalcisomes after treatment with imides. (A) Wild-type, untreated RH
tachyzoite parasites. (Inset) Enlargement. Note normal ultrastructure. DG, dense granules. (B) Tachyzoites treated with MP-IV-1. (C) Tachyzoites treated with
QQ-437. Note the absence of dense granules with normal ultrastructure and density in N-benzoyl-2-hydroxybenzamide-treated parasites. LG, light granules.
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Fomovska et al.
FIG 4 Immunofluorescence staining experiments confirm observations of organelle disruption seen in electron microscopy studies, demonstrating differences
between N-benzoyl-2-hydroxybenzamide-resistant and wild-type parasites, both with and without N-benzoyl-2-hyroxybenzamide pressure. The images show
that the imides target the secretory pathway and that the acidocalcisome/plant-like vacuole is lost with this treatment and other secretory organelles develop
unusual morphology and function. Organelles whose contents are secreted and thereby implicated in association with adaptin-3␤ include rhoptries, acidocal-
cisomes/plant-like vacuoles, micronemes, and dense granules. There is marked distortion of secretory organelles, including micronemes (A), rhoptries (B),
functional dense granules (C), and fragmentation and then disappearance of the acidocalcisome/plant-like vacuole (D). These abnormalities were present at 4 h
and more pronounced at 24 h. Mutant parasites exhibited a similar phenotype, with more pronounced abnormalities being seen in MP-IV-1-treated mutants,
with the exception of dense granule proteins reaching the parasitophorous vacuole in the mutants. These results demonstrate an effect of MP-IV-1 on organelles
that receive proteins via the secretory pathway, with the most profound effect being on the acidocalcisome/plant-like vacuole. Abbreviations for antibody labeling
of parasite surface or organelle: SAG1, surface antigen; GRA1, dense granules; ROP13, rhoptries; CPL, acidocalcisome/plant-like vacuole; and M2, micronemes.
(E) Immunostaining for enoyl reductase, which demonstrates that the plastid may be modestly elongated in the MP-IV-1-treated parasites, especially at 24 h.
Since targeting to the plastid differs from targeting to secretory organelles, this may be a secondary effect. This finding is subtle and not marked as for the
micronemes, rhoptries, dense granules, and acidocalcisome/plant-like vacuole. (F) Higher magnification of merged images of acidocalcisomes/PLVs (immu-
nostained with antibody to CPL), rhoptries (immunostained with antibody to ROP13), and micronemes (immunostained with antibody to MIC2) in wild-type
(WT), N-hydroxy-2-benzamide-treated wild-type parasites (WTϩMP-IV-1), insertional mutants (mut), and mutant parasites treated with N-hydroxy-2-
benzimide (mutϩMP-IV-1).
other secretory organelles, including micronemes, rhoptries, and parasitophorous vacuole of the mutant parasites (Fig. 4). These
functional dense granules. These abnormalities were present at 4 h results demonstrate the effect of MP-IV-1 on organelles that re-
and were more pronounced at 24 h (Fig. 4A to F). The mutant ceive proteins via the secretory pathway, with the most profound
parasites exhibited a similar phenotype, with more pronounced effect being on the acidocalcisome/PLV. An antibody to enoyl
abnormalities being seen in the MP-IV-1-treated mutants. One reductase appeared to demonstrate a subtle increase in size for the
exception was that dense granule proteins may have reached the plastid, especially at 24 h (Fig. 4E).
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Benzamides Disrupt Unique Parasite Secretory Pathway
FIG 4 Continued
Sequence alignment of T. gondii adaptin-3␤ with Arabidop- 3␤, particularly at T. gondii amino acid numbers 17 to 21, 210
sis and other protozoan and human adaptin-3␤s. Multi- to 221, 427 and 428, 446 to 450, 591 to 600, 670 to 674, and 1182
sequence alignments that relate T. gondii adaptin-3␤ to those to 1188 (see Fig. S2). There are some areas in the parasite adaptin-
of other species are shown in Fig. S1 and S2 in the supplemental 3␤s that are relatively conserved compared with the human adap-
material. The T. gondii and Neospora caninum adaptin-3␤s are tin-3␤, including those at T. gondii amino acids 321 to 332 and 689
almost identical. The Arabidopsis adaptin-3␤ has a number of to 691 (see Fig. S2). Overall analysis of the sequence alignment for
similarities (see Fig. S2), some not present in human adaptin- a number of different AP-3␤ sequences shows a low degree of
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Fomovska et al.
FIG 4 Continued
sequence identity across the family, particularly at the C-terminal MP-IV-1 may be metabolized not only by CYP450- and FMO-
region. mediated biotransformation but also by other enzymes such as
Comparison of ADMET properties of MP-IV-1 and QQ-437. hydrolases, including amidases. An in vivo pharmacokinetics (PK)
These promising biological data led to our next experiments de- study was conducted, and similar problems with metabolic stabil-
termining the ADMET and pharmacokinetics properties of MP- ity were observed. In mice, less than 1% of the parent compound
IV-1 (Table 3). MP-IV-1 showed acceptable aqueous solubility remained in the plasma after intravenous injection of 5 mg/kg.
but also showed high plasma protein binding (99.86%) with poor The metabolic stability problems associated with MP-IV-1 are
permeability, as determined by lipid parallel artificial membrane most likely caused by an unstable imide linker in the compound.
permeability assay (PAMPA) (103.77% Ϯ 1.78% recovery), and
Further ADMET assessments were conducted on our most po-
with an apparent log P value of Ϫ4.64. MP-IV-1 also showed tent compound, QQ-437, using metabolic stability assays and P-
inhibition of CYP2C9 (71.32% at 10 ␮M) but not CYP2D6 and cytochrome inhibition assays. We hypothesized that the imide
CYP3A4. No hERG inhibition was observed. Although chemical linker in QQ-437 would be more stable than that of MP-IV-1 due
stability was acceptable, the metabolic stability of this compound to the electron-donating effect of the diethylamine substituent in
was poor. MP-IV-1 was metabolized quickly in liver microsomes ring B, which should contribute to the stabilization of the imide
with a half-life less than 10 min. Only 1.58% remained after 30 linker. QQ-437 was much more stable than MP-1V-1 in a human
min incubation with microsomes. MP-IV-1 was also degraded in liver microsome assay with a half-life of approximately 33 min with
human liver microsomes without NADPH, which suggests that moderate metabolism in human hepatocytes. QQ-437 did not show
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TABLE 3 ADMET-PK assays
Value for:
Assay
Unit(s)
MP-IV-1
QQ-437
Aqueous solubility (pH 7.4)
Plasma protein binding by ultrafiltration
Lipid-PAMPA
␮M
50.82
99.86
4.64 Ϯ 0.06c
103.77 Ϯ 1.78c
71.32
15.50
Ϫ7.69
Ͼ300
100
NDa
ND
ND
ND
22
Ϫ3
Ϫ1
ND
ND
53.6
33
Mean % bound measured
ϪLog P appb
% recovery
% inhibition at 10 ␮M
% inhibition at 10 ␮M
% inhibition at 10 ␮M
IC50 (␮M)
% remaining after 60 min
% remaining after 30 min
Half-life (min)
CYP2C9 inhibition
CYP2D6 inhibition
CYP3A4 inhibition
Cell-based hERG screening
Chemical stability
Human liver microsome stability
1.58
Ͻ10
Human liver microsome stability (without NADPH)
Human hepatocyte stability
Mouse intravenous plasma concn (at 5 mg/kg)
% remaining after 30 min
% remaining after 60 min
ng/ml after 0.083 h
ng/ml after 0.25 h
ng/ml after 0.5
88.2
ND
2,343
335
108
ND
23
ND
ND
ND
ND
ng/ml after 1 h
28.4
a ND, not done.
b ϪLog P app, where P app is the apparent permeability, in cm/s.
c Values are means Ϯ standard deviations.
significant inhibition of any of the three P cytochromes tested that led to our discovery and the development of the compounds
(CYP2C9, CYP2D6, andCYP3A4), whichisanimportantqualityofa in this family is also novel and has not been described previously.
lead medicine candidate. Compared with MP-IV-1, QQ-437 is a su-
perior, more potent inhibitor with a better ADMET profile.
Our genome-wide investigations revealed a specific mecha-
nism of resistance mediated by the adaptin-3␤ protein. Adaptin
proteins, specifically AP-3␤, are found in the trans-Golgi network
and endosomal membranes and have been implicated in the
transportation of proteins between the two compartments (1b, 2,
3, 7, 9, 11, 12, 15, 30, 40, 41, 42, 43, 45). The role of AP3␤ as a cargo
protein prompted an investigation of the effect of MP-IV-1 on
parasite morphology, in particular the trafficking to secretory
pathway organelles. It is not clear at this time what the role of
this protein is in the components of the endomembrane traffick-
ing machinery and pathogenesis. Recent work with Arabidopsis
has provided insight into the interaction of AP-3␤ with other
components of the secretory pathway (summarized in refer-
ence 15).
DISCUSSION
These studies define the N-benzoyl-2-hydroxybenzamides as a
class of compounds that are effective at a low nanomolar range
against T. gondii tachyzoites by interacting with a novel secre-
tory pathway to the acidocalcisome/plant-like vacuole and
dense granules. Our screen of a library of 6,811 compounds against
T. gondii tachyzoite replication identified N-4-ethylbenzoyl-2-hy-
droxybenzamide (MP-IV-1) as an inhibitor of T. gondii tachyzoites,
effective at 31 nM (IC90) in vitro with no toxicity to host fibroblasts at
the highest concentration tested (10 ␮M) and effective in vivo against
T. gondii in mice at 50 mg/kg.
To provide further data on the effects of N-benzoyl-2-hy-
droxybenzamides on T. gondii, electron microscopy studies were
performed which demonstrated that treatment with MP-IV-1 and
QQ-437 had a profound effect on dense granules and acidocalci-
somes/plant-like vacuoles. The effects of these compounds were
localized and organelle specific, as the nucleus, rhoptries, and
membranes remained distinguishable.
Immunofluorescence assays also demonstrated that these
compounds interfere with formation of the acidocalcisome/
plant-like vacuole and interfere with proper targeting to secre-
tory organelles, including rhoptries, micronemes, and dense
granules. This demonstrates the targeting of this secretory
pathway by these novel imides. It will be of interest to further
characterize, with electron micrographs, the ultrastructure of
the mutant parasites along with conditional mutants (20a) of
adaptin-3␤ and other possible molecular targets. Another mo-
lecular target might be identified by click chemistry and pull-
Derivatives of MP-IV-1 were created and tested in vitro, and
compound QQ-437 was found to be more effective than any of
them, being active at 16 nM (IC90) in vitro, with no signs of toxicity
at 10 ␮M. In vivo experiments in mice demonstrated that QQ-437
is effective against tachyzoites at 20 mg/kg. Although the structure
of MP-IV-1 displayed similarities to that of the important enoyl
reductase inhibitor triclosan, enzymatic assays demonstrated that
this compound did not inhibit enoyl reductase (data not shown).
After performing our medium throughput screen, which iden-
tified our lead compound without prior assumptions, we found
that a similar N-benzoyl-2-hydroxybenzamide had been reported
in a patent (36a) as a less-than-optimal inhibitor of P. falciparum.
Coincidently, earlier we had found that salicylhydroxamic acid
(SHAM), a related compound which is partly contained in the
N-benzoyl-2-hydroxybenzamides, was noted to inhibit T. gondii,
P. falciparum, and C. parvum (37a). Nonetheless, N-benzoyl-2-
hydroxybenzamide QQ437 is a novel compound. We synthesized
QQ437 in our SAR and discovered in the experiments described down studies. These data will add to our immunofluorescent-
herein that this is the best of the compounds to inhibit T. gondii as antibody study of the subcellular organelles in the mutant and
well as P. falciparum Thailand and Sierra Leone strains. The SAR compound-treated parasites.
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Fomovska et al.
FIG5 Schematic diagram of the unique adaptin-3␤ secretory pathway that traffics proteins to the plant-like vacuole (PLV) and dense granules identified through
the discovery that novel N-benzoyl-2-hydroxybenzamides can treat T. gondii infections. (Left) Toxoplasma proteins have normal trafficking through the
secretory pathway via adaptin-3␤ to PLV and other organelles that receive proteins from the secretory pathway (R, rhoptries; M, micronemes; DG, dense
granules; PLV, acidocalcisome/plant-like vacuole). This trafficking takes place through the Golgi apparatus (G). N, nucleus; C, conoid; P, plastid. The arrow
shows the addition of N-benzoyl-2-hydroxyhydroxybenzamide or an insertional mutation in adaptin-3␤ that causes the same phenotype. (Right) Secretory
pathway. Adaptin-3␤ has an insertional mutation, or the secretory pathway is interacting with N-benzoyl-2-hydroxybenzamides. QQ-437, the most active of the
N-benzoyl-2-hydroxybenzamides, interacts with and inhibits a protein in the secretory pathway, perhaps adaptin-3␤ or another protein, causing a phenotype
similar to that caused by the mutation in adaptin-3␤. These compounds or an insertional mutation interferes with sorting in the late endosomal compartment.
Disappearance of the PLV and dense granule contents and mistargeting to other secretory organelles occurs. The secretory pathway is disrupted. The red X over
adaptin-3␤ shows the effect of the mutation or interaction of the secretory pathway with N-benzoyl-2-hydroxybenzamide.
Adaptin-3␤ is a 3.6-kbp protein encoded on chromosome 11. slowly than wild-type parasites, and this parasite can survive treat-
In other organisms, adaptins are involved in membrane traffick- ment with the N-benzoyl-2-hydroxybenzamides. This does not,
ing, selecting cargo for incorporation into trafficking vesicles, in however, indicate that adaptin-3␤ is the molecular target of
the form of four distinct adaptin complexes. Adaptin-3 proteins N-benzoyl-2-hydroxybenzamides. It is also possible that the in-
are involved in other organisms in transport between the trans- sertional mutant and the inhibition studies simply reveal the phe-
Golgi network and the endosomes. Adaptin-1 is the only T. gondii notype of a common pathway in which the target molecule func-
adaptin characterized to date and has been found to be important tions. For example, a microneme or other protein traversing the
in T. gondii rhoptry biogenesis (34). Adaptin-3␤ has been found secretory pathway might interact with N-benzoyl-2-hydroxyben-
to be present in Leishmania (10). The adaptin proteins play a crit- zamides.The complex could then obstruct the secretory pathway
ical role in the Apicomplexa for the underlying cell biology of or other molecules interacting with adaptin-3␤. Potentially, phos-
invasion via biogenesis of the rhoptries (i.e., adaptin-1 [34]) and phorylation and ubiquitination may play a role in adaptin-3␤ in-
potentially other aspects involved with membrane trafficking teractions with the secretory pathway as binding sites, as both are
(19). In elegant studies with Trypanosoma brucei, adaptin-3␤ was present in other adaptins (17, 18, 26, 37).
recently reported to be critical for biogenesis of this kinetoplastid’s
Experiments testing N-benzoyl-2-hydroxybenzamides against
acidocalcisome (20), which is consistent with the findings herein two strains of P. falciparum showed that QQ-437, while not as
for T. gondii. The multisequence alignments in Fig. S1 and S2 in effective as the established medicine chloroquine, was able to in-
the supplemental material show differences and similarities of hibit the parasite at low concentrations. MP-IV-1 was less effec-
adaptin-3␤s in different species. Neospora caninum has the most tive, but it did show modest activity against the D6 Sierra Leone
similar adaptin-3␤ to that of T. gondii. Our studies indicate that a chloroquine-sensitive strain but not the TM90-C235 Thailand
mutation in adaptin-3␤ causes the parasite to replicate more chloroquine-resistant strain. This indicates that these inhibitory
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Benzamides Disrupt Unique Parasite Secretory Pathway
compounds may have potential targets in other apicomplexan zoyl-2-hydroxybenzamide and its novel derivative QQ-437 repre-
parasites as well. It does not imply, however, that the molecular sent a family of compounds that affect T. gondii. Resistance to
target is the same. In fact MP-IV-1, has been reported to inhibit P. N-benzoyl-2-hydroxybenzamides occurs when a trafficking pro-
falciparum by targeting heme detoxification protein synthesis tein, adaptin-3␤ is interrupted. This suggests that the molecular
(21). P. falciparum has been described to lack adaptin-3␤ and to target utilizes or is part of this secretory pathway. Definitive target
have a secretory pathway that diverges from that of other apicom- identification will require additional work. The optimization and
plexan parasites (8, 33). The effect of QQ-437 as an antiplasmodial derivatization of these compounds, as well as further exploration
compound has been confirmed in the studies with P. falciparum of their molecular target, holds the promise of a new family of
strain K1, which is also a chloroquine-resistant parasite (42a). In medicines which might be further improved or lead to discoveries
this study, with these compounds, Leishmania, which does have of other novel compounds to treat the devastating diseases toxo-
an adaptin-3␤, was susceptible to another imide (42a). Neospora plasmosis as well as to impact diseases caused by other protozoan
and Eimeria also appear to have adaptin-3␤s, and there is homol- parasites. In our discovery of the molecular target pathway ef-
ogy to the T. gondii and Arabidopsis thaliana adaptin-3␤s, another fected by this new class of compounds, we also identified the en-
plant-like feature of this apicomplexan parasite.
docytic/exocytic hub where sorting of proteins targeted to various
It will be of interest to determine whether QQ-437 has any organelles occurs. In their earlier work, Rohloff et al., Docampo et
effect against bradyzoites. Currently we are developing model sys- al., and others (13, 14, 20, 24, 31, 39) discovered and characterized
tems to test effects of compounds on bradyzoite molecular targets. an important protozoan compartment with these putative or
We are using the following methods for these assays: conditional proven functions. Our work herein indicates that adaptin-3␤ ap-
tetracycline repressor modulation and other means to knock out pears to act in the sorting to this compartment that regulates ions
bradyzoite and tachyzoite targets (20a) in vitro and in vivo, treat- and/or as a post-Golgi sorting compartment for secretory proteins
ment of parasites in tissue culture that are in the encysted brady- destined for the dense granules, micronemes, rhoptries, and aci-
zoite stage, and treatment of parasites in the bradyzoite stage in docalcisomes/plant-like vacuoles in T. gondii.
vivo. If the mechanism of action of the N-hydroxyl-2-hydroxy-
ACKNOWLEDGMENTS
benzamides is the inhibition of the secretory pathway to the plant-
like vacuole, which appears to be essential for withstanding stress
by the parasite, as well as other secretory organelles, it is possible
that the compounds might inhibit bradyzoites better than
tachyzoites. Alternatively, if these compounds only slow replica-
tion and create a stress response by interfering with the secretory
pathway without killing the parasite, it is possible that they would
eliminate only tachyzoites in the presence of a competent immune
response. We demonstrated this type of effect when we eliminated
ribosomal protein S13 (RPS13), leading to G0 arrest and exit from
the cell cycle. In tissue culture, conditional knockdown of RPS13
was not lethal. It resulted in dormant parasites which could persist
for many months in vitro (20a). However, in mice, knockdown of
T. gondii RPS13 created a remarkably effective vaccine (20a).
There was no persistence of the vaccine or challenge organism,
presumably because the immune system eliminated the vaccine
parasites that were arrested in G0 and thereby elicited a robust
immune response sufficient to clear the homologous parent strain
challenge. If the latter was the case for our compounds, they might
be useful only for causing replication arrest in tachyzoites. If this
proved to be the case in future studies in vivo, then perhaps the
compound could be accompanied by a compound that inhibits a
stress-related kinase, such as EIF kinase (43a) or bradyzoite Ape-
tela 2 or other essential bradyzoite transcription factors (1, 1a,
20a). Inhibitors of egress, cell cycle, or stress responses described
previously (23a, 32a, 32b, 34a, 43a, 47) might be compounds that
could work together with this class of compounds. This would
allow elimination and treatment of both tachyzoites and dormant
organisms. We also are studying whether a delivery mechanism
such as the transductive peptides we described earlier (40a) could
facilitate access to the bradyzoites within cysts for additional small
molecules besides triclosan or antisense compounds which could
eliminate or interfere with all molecular targets.
This work was supported by NIAID NIH DMID U01 AI082180, the In-
stitute for Genomics, Genetics, and Systems Biology, University of Chi-
cago, and by gifts from the Mann and Cornwell, Taub, Rooney-Alden,
Engel, Pritzker, Harris, Zucker, and Mussilami families. S.P.M. is sup-
ported by an MRC Career Development fellowship.
We thank Marie-France Cesbron-Delauw and Corrine Mercier for
providing ␣GRA1, Vern Carruthers for providing ␣CPL and ␣MIC, Peter
Bradley for providing ␣ROP13, Laura Knoll for providing the PLK inser-
tional mutagenesis construct, The Albert Einstein Electron Microscopy
Core Facility for preparation of electron micrographs, and Matthias Dean
Carpentier and Daniel Lee for assistance with preparation of figures. We
also very much appreciate the suggestions and advice of NIH Program
Officer John Rogers and SAB members David Jacobus, Clark Eid, John
McCall, and Marie-France Cesbron-Delauw throughout the syntheses,
work, and analyses in these studies.
There are no conflicts of interest.
The opinions of researchers at Walter Reed Army Institute of Research
are their own and do not reflect the views of the U.S. Army or the Depart-
ment of Defense.
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