Validation of N-myristoyltransferase as Potential Chemotherapeutic Target in Chagas Disease

The protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas disease, a problem endemic to Central and South America. In recent years, this neglected infectious disease has become a global health concern. The only clinically available drugs for the chemotherapy of Chagas disease have important disadvantages such as, high toxicity, strain resistance and variable efficacy. Therefore, there is an urgent need for the development of new antichagasic agents. The enzyme N-myristoyltransferase (NMT) has been characterized in a range of eukaryotes, from Saccharomyces cerevisae to Homo sapiens. Moreover, NMT has been shown to be essential in protozoan parasites, including Leishmania major and Trypanosoma brucei. Here, we report the validation of T. cruzi NMT as a target and the discovery of lead compounds that specifically inhibit this enzyme. The results from the first part of this dissertation indicate that in T. cruzi NMT is constitutively expressed in all stages of the parasite and show at least partial endoplasmic reticulum association. Moreover, we have standardized the heterologous expression and purification of TcNMT for future enzyme kinetics analysis and in vitro inhibition studies. In addition, we report for the first time in trypanosomes, an alternative and rapid, non-radioactive method to study protein myristoylation. The second part of this work focuses on the evaluation of NMT inhibitors as anti-T. cruzi agents. These compounds were originally designed to target T. brucei NMT; here we show their effects on the intracellular parasite T. cruzi. Compounds DDD86481, DDD100097 and DDD100144 showed anti-proliferative characteristics in submicromolar concentrations. Moreover, metabolic labeling with myristic acid azide showed decreased myristoylation of proteins in treated parasite, providing evidence of the on target activity of the inhibitors. These inhibitors hold great potential for further exploration as urgently needed new therapeutic agents for Chagas disease