Targeting the Substrate Preference of a Type I Nitroreductase To Develop

Nitroheterocyclic prodrugs are used to treat infections caused by Trypanosoma cruzi and Trypanosoma brucei. A key compo-nent in selectivity involves a specific activation step mediated by a protein homologous with type I nitroreductases, enzymes found predominantly in prokaryotes. Using data from determinations based on flavin cofactor, oxygen-insensitive activity, sub-strate range, and inhibition profiles, we demonstrate that NTRs from T. cruzi and T. brucei display many characteristics of their bacterial counterparts. Intriguingly, both enzymes preferentially use NADH and quinones as the electron donor and acceptor, respectively, suggesting that they may function as NADH:ubiquinone oxidoreductases in the parasite mitochondrion.We ex-ploited this preference to determine the trypanocidal activity of a library of aziridinyl benzoquinones against bloodstream-form T. brucei. Biochemical screens using recombinant NTR demonstrated that several quinones were effective substrates for the par-asite enzyme, havingKcat/Km values 2 orders of magnitude greater than those of nifurtimox and benznidazole. In tests against T. brucei, antiparasitic activity mirrored the biochemical data, with the most potent compounds generally being preferred enzyme substrates. Trypanocidal activity was shown to be NTR dependent, as parasites with elevated levels of this enzyme were hyper-sensitive to the aziridinyl agent. By unraveling the biochemical characteristics exhibited by the trypanosomal NTRs, we have shown that quinone-based compounds represent a class of trypanocidal compound. Over 10 million people are infected with Trypanosoma cruziand Trypanosoma brucei, the causative agents of Chagas ’ dis-ease and human African trypanosomiasis, respectively (8, 52). To