Protein Synthesis Inhibitors as anti-T. Cruzi agents

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Project Abstract Trypanosoma cruzi is the causative agent of the Chagas’ disease. Despite the devastating effects of chronic T. cruzi infections on human health, there are no satisfactory options for their treatment. The goal of this proposal is to develop effective, affordable, and safe therapies against chronic T. cruzi infections. Such therapies must target a biological process essential for T. cruzi viability without disrupting it in humans. This concept has successfully been used for the development of antibiotics, many of which target bacterial protein synthesis with minimal toxicity to humans. T. cruzi protein synthesis differs substantially from that of humans at the levels of mRNA structure, translation initiation and elongation factors, ribosomal RNA and proteins and ribosome structure. These differences render every stage of T. Cruzi protein synthesis potentially suitable for the development of T,Cruzi specific protein synthesis inhibitors for the treatment of chronic infections. We therefore propose to optimize and adapt a T. cruzi in vitro translation assay developed in our laboratory to high throughput screening (Specific Aim 1) and implement an HTS campaign to identify agents that specifically inhibit T. cruzi protein synthesis and selectively kill these parasites (Specific Aim 2). PI has developed and implemented several high throughput screening (HTS) assays, obtained three first-in- class compounds and optimized these by evaluating focused libraries and structure-activity-relationships studies. These probes have been widely adapted by the scientific community and fostered research that resulted in more than 100 peer-reviewed publications by PI, his collaborators and others. Two of these lead series have been licensed to industry for further development into drugs for cancer therapy. In this application we propose to utilize our expertise in biology of translation and HTS assay development and implementation to identify the specific/selective inhibitors T.Cruzi protein synthesis. In addition to primary screening assay we will employ a GFP reporter T. cruzi in vitro translation assay to eliminate false positives and a counter dual luciferase mammalian in vitro translation assay to eliminate hit compounds not specific for T. cruzi, i.e. those that also inhibit mammalian protein synthesis. We will test specific inhibitors of T. cruzi protein synthesis for anti-parasitic activity and selective toxicity to parasite vs. host cells. While what we propose here is a pilot screen, we possess all the expertise and facilities needed to conduct a large scale screen and/or hit-to-lead optimization for the hits we will identify. Effort towards the hit-to-lead optimization would require that one or more of our specific/selective T.cruzi protein synthesis inhibitor compounds will display toxicity towards T.cruzi but not the mammalian cells and will be chemically accessible for design of focused libraries and structure-activity-relationship studies. Identification of the direct target of hit compounds is another area of future research that will elucidate biological differences between translation machineries of human and T. cruzi, and all well within the PI’s capabilities.