Fexinidazole induced cytotoxicity is distinct from related anti-trypanosome nitroaromatic drugs

Nitroaromatic drugs are of critical importance for the treatment of trypanosome infections in Africa and the Americas. Fexinidazole recently joined benznidazole and nifurtimox in this family when it was approved as the first oral therapy against Human African trypanosomiasis (HAT). Nitroaromatic prodrugs are bioactivated by the trypanosome-specific type I nitroreductase (NTR) enzyme that renders the compounds trypanocidal. A caveat to the specificity of NTR activation is the potential for drug resistance and cross-resistance that can arise if NTR expression or functionality is altered through mutation. The outcomes of NTR bioactivation of nitroaromatic compounds is variable but can include the formation highly reactive open chain nitriles that can damage biomolecules including DNA. A proposed mechanism of action of nitroaromatic compounds is the formation of reactive oxygen species (ROS) resulting in the formation of trypanocidal levels of DNA damage. Fexinidazole made its way to clinical approval without a significant interrogation of its effects on trypanosome biology and a limited understanding of its mechanism of action. Early reports mentioned fexinidazole potentially affects DNA synthesis but without supporting data. In this study, we evaluated and compared the cytotoxic effects of nifurtimox, benznidazole, and fexinidazole on Trypanosoma brucei using in vitro analyses. Specifically, we sought to differentiate between the proposed effects of nitroaromatics on DNA damage and DNA synthesis. Toward this goal we generated a novel {gamma}H2A-based flow cytometry assay that reports DNA damage formation in conjunction with cell cycle progression. Here we report that fexinidazoles cytotoxic outcomes are distinct from the related drugs nifurtimox and benznidazole. Specifically, we show that fexinidazole treatment results in a pronounced defect in DNA synthesis that reduces the population of parasites in S phase. In contrast, treatment with nifurtimox and benznidazole appear accumulate DNA damage early in cell cycle and result in a defective G2 population. The findings presented here bring us closer to understanding the anti-trypanosomatid mechanisms of action of nitroaromatic compounds, which will promote improved drug design and help combat potential drug resistance in the future. Our findings also highlight DNA synthesis inhibition as a powerful anti-parasitic drug target.