Project Summary Trichomonas vaginalis is the causative agent of trichomoniasis, the most common, non-viral sexually- transmitted disease, with 5-7 million cases in the U.S. and >200 million in the world each year. In addition to infections of the urogenital tract, trichomoniasis increases the risk of adverse pregnancy outcomes, HIV transmission, and cervical and prostate cancer. Only two drugs of the same class are FDA-approved for treatment, the nitro drugs metronidazole and tinidazole. Although generally effective, treatment failures occur in a substantial fraction of patients and the drugs have significant liabilities, with moderate to severe adverse effects and poor compliance due to seemingly benign but common side effects such as metallic taste. Given its prevalence, its association with multiple disease outcomes, and an increase in nitro drug-resistant strains, new antimicrobials against T. vaginalis are urgently needed, particularly in women where infection can persist for months or even years compared to generally less than ten days in men. In extensive preliminary studies, we determined that inhibitors of the proteasome, an essential cellular machinery for the degradation and recycling of cell proteins, kill T. vaginalis at sub-micromolar levels. Importantly, the inhibitors overcome nitro drug resistance and are efficacious in a murine model of trichomonad infection. We have also isolated and biochemically characterized proteasomes from T. vaginalis and human HeLa cells and found that they display significant differences in their peptide substrate specificity, providing the rationale for designing new potent and parasite-selective proteasome inhibitors. Based on these promising findings, the project has the overall objective to develop novel proteasome inhibitors with greatly improved potency and selectivity for the treatment of trichomoniasis. Using a hit compound with 50-fold selectivity, we will systematically develop T. vaginalis- specific proteasome inhibitors using a comprehensive combination of medicinal chemistry efforts, functional testing with multiple clinical strains of T. vaginalis, biochemical and structural investigations of the parasite proteasomes, and efficacy and toxicity testing in murine infection models. We have assembled a superb team of investigators with complementary expertise in parasitology, protease biology, antimicrobial drug development, and medicinal and peptide chemistry. The team has the experience and track record to conduct the critical pre-clinical studies to establish proteasome inhibitors as a new class of agents in the therapeutic armamentarium against trichomoniasis.