Sunday, December 22, 2024
12/22/2024
PROJECT SUMMARY Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, is endemic in the Americas, but has also globalized due to human migration. Despite being one of the major causes of infection-induced heart disease worldwide, current therapies for Chagas disease have inconsistent efficacy and frequent side effects. A major contributor to treatment failure is thought to be the transiently dormant intracellular forms of T. cruzi that are resistant to otherwise highly effective trypanocidal compounds. The newly discovered benzoxaborole AN15368 represents the first extensively validated and safe clinical candidate for the treatment of Chagas disease. With the mRNA processing pathway as the apparent target, AN15368 acts as a prodrug that requires cleavage by parasite serine carboxypeptidases (TcCBPs) to yield the active metabolite. This proposal aims to gain additional understanding of this processing step for AN15368 as a prodrug, as well as to assess the potential for acquired resistance as this compound proceeds toward human clinical trials. Aim 1 will take advantage of our well-established quantitative mass spectrometry platform to extensively study the kinetics of AN15368 metabolism in amastigotes and epimastigotes, the two proliferative life stages of T. cruzi. A concentration-gradient model will be constructed based on this study to test the hypothesis that only the prodrug, but not the processed form, can enter T. cruzi efficiently and once processed, accumulates therein. Furthermore, these kinetic studies will address the differential susceptibility of epimastigotes and amastigotes to AN15368 to further understand the process of prodrug metabolism. Aim 2 will focus on the breadth of susceptibility to the prodrug in the amastigote populations. A fluorogenic substrate will be developed to allow for the quantitation of prodrug activation in individual amastigotes, including the transient dormant ones. We will test the hypothesis that the susceptibility to this prodrug is broad in amastigotes, including those in dormancy. Lastly, since the prodrug feature brings AN15368 the liability of acquiring selected resistance via loss of the processing pathway, we propose to carry out Aim 3 to comprehensively monitor the outcome of infection when TcCBPs are disrupted, with and without AN15368 treatment. This will provide an assessment for the risk of this resistance if developed. The proposed work will enhance our understanding of how prodrug AN15368 achieves sterile cure of T. cruzi infection, and will be the first study attempting to quantify a metabolic process in dormant parasites to address drug- mediated cure. The results of these studies are expected to provide insights into the high therapeutic potential of benzoxaboroles in this and other parasitic infection, as well as one of the risks for induction of drug resistance to this class of compounds and thus inform on the safety and efficacy of this clinical candidate.
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