PROJECT SUMMARY Fexinidazole has emerged as the front-line treatment against African trypanosomiasis, offering the first oral monotherapy against T. brucei gambiense infections. Because of its current success in African trypanosome treatment, fexinidazole is actively being investigated as a therapy against American trypanosomiasis (Chagas) and Leishmaniasis, which currently infect over 15 million people globally. Fexinidazole joined nifurtimox (NECT combination therapy, HAT) and benznidazole (Chagas disease) as clinically significant nitroaromatic compounds for the treatment of trypanosomatid infections. Nitroheterocyclic compounds function as prodrugs that require nitroreductase (NTR) enzymatic activation to generate cytotoxic species that are thought to damage DNA, lipids, and proteins. Naturally occurring and laboratory resistance and cross-resistance have been documented based on T. brucei NTR mutants and in RNAi-based knock-down studies, respectively. Fexinidazole is a repurposed compound whose success in clinical trials enabled rapid approvals with few publications evaluating the drugs trypanocidal phenotypes, Mechanism of Action (MoA), or potential for drug resistance. Inadequate molecular understanding of fexinidazole’s MoA and modes of drug resistance represent a significant knowledge gap. To fill this gap, our state-of-the-art T. brucei Gain-of-Function library was applied to the question of fexinidazole resistance and preliminary studies identified 11 survival associated ORFs including oxidoreductases, a peroxidase, dithiol glutaredoxin, and a glutathione S-transferase. Collectively, the identified genes support the central hypothesis that novel fexinidazole survival genes constitute a redox-based pathway able to promote nitroaromatic drug resistance with implications for fexinidazole’s MoA. This exploratory proposal will take the modest risk of investigating genes that promote survival during fexinidazole treatment, toward the high reward of identifying the drugs molecular targets and drug resistance mechanisms. The Specific Aims are targeted to answer two significant questions: Are fexinidazole survival ORFs specific to nitroaromatic compounds? and Do fexinidazole GoF survival genes constitute a redox-based drug resistance pathway? Aim 1 will employ a set of innovative T. brucei Gain-of-Function genetic screens to determine if the identified fexinidazole survival genes are (i) specific to fexinidazole resistance, (ii) general to all nitroaromatics, or (iii) broadly associated ROS stress management. AIM 2 will test the working hypothesis that fexinidazole resistance can arise from a novel redox- based resistance pathway through unbiased phenotypic analysis of drug resistance and cross-resistance, protein localization, mitochondrial functionality, cell cycle and DNA synthesis, and ROS and redox. The proposed research is significant because it will identify genes associated with fexinidazole’s MoA and resistance mechanisms, filling crit...