PROJECT SUMMARY/ABSTRACT Human African trypanosomiasis (HAT) is a neglected tropical disease that is endemic to sub-Saharan Africa, where millions are at risk for infection. The disease, which is caused by the eukaryotic pathogen Trypanosoma brucei, is typically fatal if untreated. Several therapeutic strategies are available, but outside of fexinidazole, these drugs are marred by relatively high toxicity, serious side-effects, and emerging resistance. Thus, there is a need for novel therapeutic strategies to treat this disease, and those caused by related organisms such as T. cruzi (American trypanosomiasis) and Leishmania spp. (leishmaniasis). Another protozoan target of our efforts, Trichomonas vaginalis, causes the most prevalent non-viral sexually-transmitted infection in the United States (ca. 3 million cases) with in excess of 120 million cases worldwide. Recently, we discovered a straightforward (one-step) route to synthesize a novel class of compounds, the diazacyclobutenes (DCBs), and we determined that some of them have potent anti-trypanosomal activity. The central goals of this proposal are to further explore the structure- activity relationship of these compounds as anti-trypanosomal agents, to uncover their mode of action in trypanosomes, and to explore their utility against another common parasite, Trichomonas vaginalis. The efforts of this proposal are subdivided into three Aims. Specific Aim 1: To rapidly expand the library of diazacyclobutenes and explore the structure-activity relationships that govern their anti-trypanosomal activity. Specific Aim 2: To uncover the mechanism of action responsible for the observed anti-trypanosomal activity of the diazacyclobutenes. Specific Aim 3: To explore the utility of diazacyclobutenes against the Parabasalid protozoan, Trichomonas vaginalis. Overall, this study will represent the first characterization of diazacyclobutenes as anti-parasitic drugs, while developing the synthetic routes necessary for expansion of this interesting class of compounds. Successful completion of these studies will provide the framework for a future R01 submission that will focus on the development of much needed drugs for parasite infections.