Unraveling the antitrypanosomal mechanism of benznidazole and related 2‐nitroimidazoles: From prodrug activation to DNA damage

Nitroheterocycles represent an important class of compound used to treat trypanosomiasis. They often function as prodrugs and can undergo type I nitroreductase (NTR1)‐mediated activation before promoting their antiparasitic activities although the nature of these downstream effects has yet to be determined. Here, we show that in an NTR1‐dependent process, benznidazole promotes DNA damage in the nuclear genome of Trypanosoma brucei, providing the first direct link between activation of this prodrug and a downstream trypanocidal mechanism. Phenotypic and protein expression studies revealed that components of the trypanosome’s homologous recombination (HR) repair pathway (TbMRE11, γH2A, TbRAD51) cooperate to resolve the benznidazole‐induced damage, indicating that the prodrug‐induced lesions are most likely double stand DNA breaks, while the sequence/recruitment kinetics of these factors parallels that in other eukaryotes HR systems. When extended to other NTR1‐activated 2‐nitroimidazoles, some were shown to promote DNA damage. Intriguingly, the lesions induced by these required TbMRE11 and TbCSB activities to fix leading us to postulate that TbCSB may operate in systems other than the transcription‐coupled nucleotide excision repair pathway. Understanding how existing trypanosomal drugs work will aid future drug design and help unlock novel reactions/pathways that could be exploited as targets for therapeutic intervention. Trypanocidal mechanism of 2‐nitroimidazoles. Once nitroimidazole‐based prodrugs (e.g., benznidazole; BNZ) enter the Trypanosoma brucei mitochondrion, they are activated by reaction with a type I nitroreductase (NTR1). This generates several metabolites including some that may access the parasite’s nucleus and promote double stand DNA breaks (DSB). This damage is repaired by the homologous recombination (HR) DNA repair pathway with CSB, a nucleotide excision repair factor, often playing a role in lesion detection.