Project Summary The long-term objective of the proposed research is to develop a new drug for Chagas disease, an infectious disease caused by the parasite Trypanosoma cruzi. Chagas disease is estimated to effect 6-8 million people, mainly in Latin America. The work is motivated by the inadequacy of current therapies with respect to their poor efficacy and tolerability. In the previous grant period, we made excellent progress advancing an early lead series (benzothiazoles) to the late preclinical-candidate stage while defining the mechanism of action of mitosis inhibition in the trypanosome. This novel mechanism of action is uniquely associated with parasite clearance in both in vitro (washout) assays and in the chronic mouse model of T. cruzi infection. In this proposal, we will focus on three new compounds scaffolds discovered in high-throughput phenotypic screening assays against T. cruzi (EC50 values ~1 µM). The compounds were selected because of follow up experiments showing that they work by the same mechanism of action as the aforementioned benzothiazoles. The goal of the project will be to bring forward these compounds as backup series in lieu of the benzothiazole compounds which were associated with unexpected genotoxicity in Ames testing. The new compounds series (oxazoles, thienopyrimidinones, and diazines) will go into medicinal chemistry lead optimization campaigns for Chagas disease, an area in which we have over two decades of experience. With over 100 analogs of each scaffold already synthesized, we are developing a detailed understanding of structure activity relationships. Compound testing will include in vitro assays for anti- trypanosomal activity, mammalian cell cytotoxicity, solubility, mouse pharmacokinetics, and murine efficacy models following a screening cascade with defined go/no-go criteria. At the end of this three-year project, one to two drug candidates will be selected for late-stage preclinical development for Chagas disease.