Prodrugs as new therapies against Chagas disease: In vivo synergy between Trypanosoma cruzi proline racemase inhibitors and benznidazole

Objectives: : Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi , affects approximately 6–7 million people worldwide. There are limited available therapies and they exhibit low eﬃcacy, often high toxicity in chronic cases and some drug resistance. In this study, our objective was to develop ester prodrugs that inhibit proline racemase ( Tc PRAC), a parasitic enzyme previously identiﬁed and characterised as a promising target because of its essential role in the parasite’s life cycle and virulence, and to test their activity against T. cruzi . Methods: : Using structural bioinformatics, we modelled several functional intermediates of the catalytic site between the opened and closed conformations of Tc PRAC based on its crystal structures in complex with its competitive inhibitor, pyrrole-2-carboxylic acid. Guided by these intermediates, which were later validated in cocrystals, we designed and evaluated numerous compounds and tested them enzymatically on live parasites and in mice with our quick and straightforward drug screening method, which is based on state-of-the-art bioluminescent T. cruzi parasites injected subcutaneously. Results: : Some of our novel compounds speciﬁcally inhibited racemase activity, as determined through biochemical assays, and covalently bound to Tc PRAC. Furthermore, the corresponding ester prodrugs were effective in killing parasites in vitro. Bioluminescent T. cruzi assays in mice showed that JR1531, a Tc PRAC inhibitor prodrug, can kill parasites in living animals, with boosted action when combined with low doses of benznidazole. Conclusion: : This approach, based on Tc PRAC inhibitor prodrugs in association with low doses of ben-znidazole, may lead to more effective, speciﬁc and non-toxic therapies against Chagas disease. © 2021


a b s t r a c t
Objectives: : Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi , affects approximately 6-7 million people worldwide.There are limited available therapies and they exhibit low efficacy, often high toxicity in chronic cases and some drug resistance.In this study, our objective was to develop ester prodrugs that inhibit proline racemase ( Tc PRAC), a parasitic enzyme previously identified and characterised as a promising target because of its essential role in the parasite's life cycle and virulence, and to test their activity against T. cruzi .Methods: : Using structural bioinformatics, we modelled several functional intermediates of the catalytic site between the opened and closed conformations of Tc PRAC based on its crystal structures in complex with its competitive inhibitor, pyrrole-2-carboxylic acid.Guided by these intermediates, which were later validated in cocrystals, we designed and evaluated numerous compounds and tested them enzymatically on live parasites and in mice with our quick and straightforward drug screening method, which is based on state-of-the-art bioluminescent T. cruzi parasites injected subcutaneously.Results: : Some of our novel compounds specifically inhibited racemase activity, as determined through biochemical assays, and covalently bound to Tc PRAC.Furthermore, the corresponding ester prodrugs were effective in killing parasites in vitro.Bioluminescent T. cruzi assays in mice showed that JR1531, a Tc PRAC inhibitor prodrug, can kill parasites in living animals, with boosted action when combined with low doses of benznidazole.Conclusion: : This approach, based on Tc PRAC inhibitor prodrugs in association with low doses of benznidazole, may lead to more effective, specific and non-toxic therapies against Chagas disease.
© 2021 The Authors.Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

Introduction
Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi , is one of the most important causes of death from infectious diseases in South and Central America.Nearly 7 million people are infected and another 25 million are at risk.With human migration, the disease has extended to northern countries and has become a global concern [ 1 , 2 ].Moreover, transmission of T. cruzi without an insect vector, by the oral route, has been reported in endemic regions [3] , and T. cruzi is now one of the most prominent emerging foodborne parasitic protozoans [4] .There is no approved vaccine against Chagas disease, and currently only two nitroaromatic drugs (benznidazole and nifurtimox) are available for treatment of Chagas disease [5] .Although these drugs can be effective in the acute phase, their efficacy against chronic forms of the disease is questionable.Altogether, with high toxicity impairing the benefitrisk ratio, poor criteria for cure or treatment monitoring [5][6][7] and a lack of innovative molecules [2] , novel effective treatments are needed and their development is considered a priority.
We have previously identified T. cruzi proline racemase ( Tc PRAC) as a promising target to fight Chagas disease [ 8 , 9 ].This enzyme, present in all stages of the parasite's life cycle, contributes to immune escape and persistence [8] .Indeed, T. cruzi loses viability with Tc PRAC knockdown and gains virulence with Tc PRAC overexpression [10] .The crystal structure of Tc PRAC complexed with a transition analogue, 2-pyrrolecarboxylic acid, revealed tightly closed and highly specific catalytic sites (PDB 1W61).This left almost no space for the modulation of inhibitor candidates but, interestingly, in the hemisaturated Tc PRAC structure (PDB 1W62) the ligand-free catalytic site was opened, showing an accessible surface area of the inhibitor-free monomer.This suggested that the ligand promotes a closure movement of the monomer and, consequently, an opening/closing functional mechanism of the enzyme affording the possibility to model plausible intermediate conformations better suited to efficiently anchor an inhibitor [ 11 , 12 ].This strategy allowed us to enlarge the chemical search space in virtual screening and led us to identify two irreversible inhibitors: (E)-4-oxopent-2enoic acid and its derivative (E)-5-bromo-4-oxopent-2-enoic acid.Optimisation of these molecules by the addition of groups and cyclisation led to a potent Tc PRAC inhibitor, NG-P27 [13] , that exhibited good trypanocidal activity against T. cruzi .
Exploiting these findings, this study aimed: (i) to evaluate the inhibitory activity and specificity for Tc PRAC of three NG-P27derived aryl ketones (JR1530, PU-24 and NG-P62), which further exploit the modelled catalytic site expansion; (ii) to assess the in vitro efficacy and toxicity of these compounds and corresponding prodrugs (JR1531, PU-24CO 2 Me and NG-P65) against epimastigotes and intracellular amastigotes of T. cruzi ; and (iii) to investigate the in vivo activity of the best-characterised prodrug (JR1531), alone or in combination with benznidazole, using bioluminescent virulent parasites and a rapid live-imaging screening method.We found that JR1531 is effective in killing parasites in living animals and that it acts in synergy with benznidazole.Therefore, the association of Tc PRAC inhibitor prodrugs with low doses of benznidazole may lead to specific, non-toxic and thus more effective therapeutic approaches against Chagas disease.

Compound synthesis and analysis
Syntheses of Tc PRAC inhibitors and prodrugs are described in the Supplementary material.Stock solutions of Tc PRAC inhibitors and prodrugs (1 M) and benznidazole (50 mg/mL) were prepared in dimethyl sulfoxide (DMSO).

Cytotoxicity of TcPRAC inhibitor prodrugs in non-infected cells
Cytotoxicity and cell viability were assessed using a CellTox TM Green Cytotoxicity Assay (G8741; Promega) and CellTiter-Glo® Luminescent Cell Viability Assay (G7570; Promega) on non-infected Vero cells (5 × 10 3 cells/well) in 96-well plates incubated with different concentrations of freshly diluted prodrugs at 37 °C with 5% CO 2 for 72 h.Cell morphology changes were evaluated on an Opera® high-content screening system (PerkinElmer) using the same cell, prodrug, temperature, time and CO 2 conditions with 100 nM MitoTracker TM Red CMXRos (M7512; Molecular Probes) and 1 μg/mL Hoechst 33342 staining.

In vitro trypanocidal/trypanostatic activity of TcPRAC inhibitors and prodrugs
Epimastigotes were incubated in 96-well white plates in LIT medium (1 × 10 5 parasites/well) with different concentrations of freshly diluted compounds at 28 °C for 72 h.Live parasite luminescence emitted upon addition of 0.3 mg/mL d -luciferin (122799; PerkinElmer) was quantified using a TECAN luminometer.For intracellular amastigotes, non-infected Vero cells were seeded in a 96-well white plate in supplemented RPMI 1640 medium (5 × 10 3 cells/well) and were infected with 5 × 10 4 trypomastigotes/well at 37 °C for 24 h.Cells were washed and were incubated with different concentrations of freshly diluted compounds at 37 °C under 5% CO 2 for 72 h.The presence of live parasites was measured using a Luciferase Reporter Gene Assay (11814036001; Roche).

In vivo toxicity of TcPRAC inhibitor prodrugs in zebrafish embryos
AB wild-type zebrafish ( Danio rerio ) embryos were raised at 28 °C in embryo water composed of mineral water supplemented with 280 μg/mL methylene blue (M-4159; Sigma-Aldrich) and 30 μg/mL 1-phenyl-2-thiourea (P-7629; Sigma-Aldrich) and were handled according to institutionally approved guidelines.At 72 h postfertilisation, embryos were distributed in 24-well plates (10 embryos/well) in 500 μL of embryo water containing freshly diluted drugs and were incubated at 28 °C for 72 h in the dark.Quantification of live embryos and possible malformations was performed using a stereomicroscope.

In vivo rapid mouse model to test the trypanocidal/trypanostatic activity of TcPRAC inhibitor prodrugs
Six-to eight-week-old male RjOrl:SWISS mice (purchased from Janvier Laboratories) were handled according to the institutional guidelines of the Central Animal Facility at Institut Pasteur.Mice were shaved on the dorsal skin and were injected subcutaneously with 1 × 10 4 blood trypomastigotes in a final volume of 50 μL.

Synthesis and inhibitory activity of (E)-4-aryl-4-oxobut-2-enoic acids
Chagas disease remains one of the most neglected diseases and the development of effective treatments is considered a priority [2] .Expanding the structure of NG-P27, a potent Tc PRAC inhibitor acting both in vitro and in vivo [13] , we produced (E)-4aryl-4-oxobut-2-enoic acids either by the Friedel-Crafts reaction or by crotonic condensation involving aryl methyl ketones.Corresponding esters were obtained by reaction with diethyl sulfate or methyl iodide.All compounds were purified and fully characterised ( Fig. 1 a; Supplementary Table S1).These compounds, named JR1530, PU-24 and NG-P62, irreversibly inhibited recombinant Tc PRAC ( Fig. 1 b).These compounds displayed no inhibition on other tested cysteine enzymes, suggesting that they were specific for Tc PRAC (Supplementary Fig. S1) [13] .

Efficacy against Trypanosoma cruzi and cell viability shown with ester prodrugs
Due to their acidic nature, the Tc PRAC inhibitors were poorly permeable and not effective in killing parasites in vitro ( Fig. 1 ch).To overcome this issue, we synthesised corresponding ester prodrugs following described examples for esterase-activated prodrugs against parasites such as Plasmodium falciparum [15] and Trypanosoma brucei rhodesiense [16] .
The Tc PRAC inhibitor prodrugs JR1531, PU-24CO 2 Me and NG-P65 presented a marked dose-dependent effect on T. cruzi viability, requiring 23-fold lower concentrations than benznidazole to kill epimastigotes.The efficacy against intracellular amastigotes was moderate ( Table 1 ), with doses still similar to that of benznidazole ( Fig. 1 c-h).The dose-dependent cytotoxicity and NOAEL (no observed adverse effect level) of the prodrugs indicated a moderate selectivity index ( Table 1 ).In vivo toxicity tested on zebrafish embryos [17] showed that JR1531 was the prodrug with the lowest dose-dependent toxicity (Supplementary Fig. S2; Table 1 ) and, consequently, it was selected to be tested in the mouse model.

Noticeably favourable synergy of JR1531 with benznidazole in mice
Our rapid mouse model with bioluminescent T. cruzi trypomastigotes allowed us to detect the effect of prodrug JR1531 treatment as soon as 5 days after infection, which is useful for in vivo drug screening ( Fig. 2 a).
Prolonged chemotherapy with benznidazole and nifurtimox produces several adverse effects in almost all treated patients, which correlates with poor quality of life and frequent treatment discontinuation [6] .Hence, as JR1531 showed stronger efficacy than benznidazole on live parasites despite a lower efficacy in vivo ( Fig. 2 b), we tested whether it could have synergistic effects with benznidazole, possibly allowing us to reduce its dose and thus the risk of adverse side effects [18][19][20] .Remarkably, co-administration of JR1531 with suboptimal doses of benznidazole (5 mg/kg benznidazole) was effective in inducing complete clearance of local parasites within 11 days post-infection ( Fig. 2 ).Hence, the Tc PRAC inhibitor prodrug JR1531 was effective in killing parasites in living animals in synergy with benznidazole.
In the absence of an efficient test for cure, especially in the chronic phase [ 1 , 7 ], the quest for better Chagas disease therapies remains a challenge.None the less, the association of Tc PRAC inhibitor prodrugs with low doses of benznidazole may lead to a more effective, specific and non-toxic treatment against Chagas disease.

Fig. 1 .
Fig. 1.Structure and activity of Trypanosoma cruzi proline racemase ( Tc PRAC) inhibitors and prodrugs.(a) Chemical structures of the optimised Tc PRAC inhibitors and prodrugs.(b) Kinetics of 40 mM l -proline racemisation by Tc PRAC alone or in the presence of 5 μM compound, either NG-P27, JR1530, PU-24 or NG-P65.(c-h) Dose-response curves of Tc PRAC inhibitors and their corresponding ester prodrug activity on epimastigotes (c,e,g) and Tc PRAC inhibitor prodrugs on intracellular amastigotes (d,f,h) of T. cruzi (strain CL Brener) in comparison with benznidazole (BNZ): (c,d) JR1530 and prodrug JR1531; (e,f) PU-24 and prodrug PU-24CO 2 Me; and (g,h) NG-P62 and prodrug NG-P65.The grey shaded areas correspond to parasites incubated with dimethyl sulfoxide (DMSO) and the yellow shaded areas correspond to the absence of parasites (medium only).Vertical red lines indicate the estimated NOAEL (no observed adverse effect level) for the Tc PRAC inhibitor prodrugs.Data are expressed as the median and interquartile range.IC 50 ( μM) values of the Tc PRAC inhibitor prodrugs are shown on the graphs.

Fig. 2 .
Fig. 2. In vivo trypanocidal/trypanostatic activity of Trypanosoma cruzi proline racemase ( Tc PRAC) inhibitor prodrugs in a rapid mouse model.(a) Representative images illustrating the two-dimensional bioluminescence signals related to T. cruzi presence in the dorsal region of mice from the different treatment groups at 3, 5, 7 and 11 days post-infection.(b) Efficacy of the 5-day in situ treatment expressed as bioluminescent signals (total flux in photons/s).Lines represent the median (and interquartile range) of the dorsal bioluminescent signals from mice treated with vehicle (DMSO), 50 mg/kg benznidazole (BNZ 50), 5 mg/kg benznidazole (BNZ 5), 50 mg/kg Tc PRAC inhibitor prodrug (JR1531) or the combination of 50 mg/kg Tc PRAC inhibitor prodrug and 5 mg/kg benznidazole (JR1531 + BNZ 5).The green shaded area corresponds to the period of treatment and the grey shaded area corresponds to background signals.

2. 8 .
Ethical statement Animals were housed in the Institut Pasteur animal facilities accredited by the French Ministry of Agriculture for performing experiments on live rodents.Animal experiments were performed in compliance with French and European regulations on the care and protection of laboratory animals (EC Directive 2010/63, French Law 2013-118, 6 February 2013).All experiments were approved by the Ethics Committee #89 and were registered under reference #2013-0047.