Unveiling Benznidazole's mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi

Abstract

Benznidazole (BZ) is the most commonly used drug for the treatment of Chagas disease. Although BZ is known to induce the formation of free radicals and electrophilic metabolites within the parasite Trypanosoma cruzi, its precise mechanisms of action are still elusive. Here, we analyzed the survival of T. cruzi exposed to BZ using genetically modified parasites overexpressing different DNA repair proteins. Our results indicate that BZ induces oxidation mainly in the nucleotide pool, as heterologous expression of the nucleotide pyrophosphohydrolase MutT (but not overexpression of the glycosylase TcOgg1) increased drug resistance in the parasite. In addition, electron microscopy indicated that BZ catalyzes the formation of double-stranded breaks in the parasite, as its genomic DNA undergoes extensive heterochromatin unpacking following exposure to the drug. Furthermore, the overexpression of proteins involved in the recombination-mediated DNA repair increased resistance to BZ, reinforcing the idea that the drug causes double-stranded breaks. Our results also show that the overexpression of mitochondrial DNA repair proteins increase parasite survival upon BZ exposure, indicating that the drug induces lesions in the mitochondrial DNA as well. These findings suggest that BZ preferentially oxidizes the nucleotide pool, and the extensive incorporation of oxidized nucleotides during DNA replication leads to potentially lethal double-stranded DNA breaks in T. cruzi DNA.

Nitro/Nitrosyl-Ruthenium Complexes Are Potent and Selective Anti-Trypanosoma cruzi Agents Causing Autophagy and Necrotic Parasite Death

“…Given the substantial dedication to identifying optimal drug combinations for the treatment of Chagas disease (49,50), a combination of ruthenium complex 3 and benznidazole would offer a potential therapy to reduce the benznidazole dosage required to cure infection. This is supported by the fact that combination would target T. cruzi at two different modes of action: NO release and autophagy induction mediated by complex 3 and nitroreductase inhibition and oxidative stress induction mediated by benznidazole (51,52). In vitro, combinations of complex 3 and benznidazole were synergic in killing trypomastigotes.…”

Benznidazole Biotransformation and Multiple Targets in Trypanosoma cruzi Revealed by Metabolomics

“…Here we used 20 µM and 50 µM over a 6 h incubation period with parasites to detect induced changes. A recent report shows that DNA damage, including double strand breaks, was found in T. cruzi after treatment with 240 µM Bzn for up to 72 h [44]. 8-oxoguanine was encountered in T. cruzi DNA after Bzn exposure and augmented levels of oxidized deoxyguanosine triphosphate (8-oxodGTP) are proposed as the main cause for the observed DNA damage.…”

“…It has been widely agreed that Bzn exerts activity through induction of reductive stress, involving covalent modification of cellular macromolecules by intracellular reduced metabolites of the parent compound [12], [13]. Bzn can arrest protein, RNA and DNA synthesis, as well as promote damage to both nuclear and kinetoplast DNA and macromolecule degradation [41], [42], [43], [44]. Here we used 20 µM and 50 µM over a 6 h incubation period with parasites to detect induced changes.…”

Benznidazole induces in vitro anaerobic metabolism in Trypanosoma cruzi epimastigotes

“…Although one of the proposed mode of actions of benzinidazole is to be mediated via reduced intermediates which interfere in macromolecules such as lipids, DNA and proteins [16,26,27], the concentrations of benzinidazole used in our study did not interfere in the proteins catabolism due to the maintenance of urea, creatinin and fumarate levels after the exposure to the drugs. It is important to highlight that other factors that interfere in the protein turnover or in the translation inhibition were not evaluated.…”