Benznidazole Biotransformation and Multiple Targets in Trypanosoma cruzi Revealed by Metabolomics

Abstract

BackgroundThe first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.Methodology/Principal findingsParasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, γ-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.Conclusions/significanceOur data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi.

Metabolomics-Based Screening of the Malaria Box Reveals both Novel and Established Mechanisms of Action

“…These untargeted metabolomics techniques have already been used to identify mechanisms of action for novel drug candidates in other protozoan pathogens (26)(27)(28), providing an unbiased, hypothesis-free approach to reveal the actions of novel antiparasitic compounds with no known target, such as those found in the Malaria Box.…”

Bestatin Induces Specific Changes in Trypanosoma cruzi Dipeptide Pool

“…Some gamma-glutamyl-containing dipeptides were raised (see Fig. 3B), and these metabolic alterations were associated with augmented recycling of the observed conjugates (9). No such changes were identified in this experiment, indicating that the changes associated with treatment with bestatin are specific and exclusively related to a failure to degrade dipeptides.…”

“…T. cruzi causes Chagas disease in humans, a neglected disease with only two drugs available for treatment and no immediate prospects for vaccines (5,6). Metabolomics is a powerful tool for unveiling the modes of action of drugs and has been successfully used in trypanosomatids (7)(8)(9)(10)(11)(12)(13). To evaluate the metabolite changes occurring after exposure to a peptidase inhibitor, we performed an untargeted metabolomics analysis of bestatin treated T. cruzi.…”

“…We used a platform consisting of Zic hydrophilic interaction liquid chromatography (HILIC) coupled to high-accuracy mass spectrometry as previously described (9). T. cruzi epimastigotes were treated with 300 M bestatin for 6 h (3 biological replicates).…”

“…One is an N-acetylated dipeptide (N-acetyl leucylleucine), while the other, putatively annotated as gamma-aminogamma-cyanobutanoate, has a retention time (27 min) and degree of unsaturation (C 5 H 8 N 2 O 2 ) more consistent with being an MS-derived fragment of another basic dipeptide (see File S1 [intranet.pasteur.edu.uy/publico/robello/FileS1.xlsb]). When T. cruzi was treated with benznidazole, by contrast, alterations were related primarily to depletion and turnover of low-molecular-weight thiols due to the formation of drug-thiol conjugates (9). Some gamma-glutamyl-containing dipeptides were raised (see Fig.…”