Validation of Trypanosoma cruzi dihydroorotate dehydrogenase as a drug target for Chagas´disease.

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ABTRACT Trypanosoma cruzi is the etiological agent of Chagas disease, a pathogenesis that affects 6 to 7 million people worldwide, mainly in Latin America. There is no effective treatment for chronic Chagas disease and resistant strains to the current frontline treatment have already emerged. There is a growing need for new pharmacological alternatives to treat this disease. This proposal exploits a target-based approach to search for lead compounds against Chagas disease. Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) is a flavin mononucleotide containing enzyme, which catalyzes the oxidation of L-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. TcDHODH was also described as a soluble fumarate reductase playing a role in connecting succinate/fumarate metabolism to de novo pyrimidine biosynthesis. DHODH has already been extensively exploited as a drug target for proliferative and parasitic diseases. Genetic studies have shown that DHODH is essential for T. cruzi survival providing evidence that this enzyme is an attractive target for the development of antichagasic drugs. The goal of this project is to chemically validate TcDHODH as a new drug target for Chagas disease and to provide leads for drug development. Our approach combines multiple techniques including computational chemistry, enzymology, structural biology, parasitology, and medicinal chemistry to develop selective and covalent inhibitors of TcDHODH. Initially, we will seek to gain a greater understanding of the DHODH hot spots by using solvent mapping to characterize the binding pockets with the flexible active-site loop open and closed. This information will help strengthen the computational modeling, and subsequent compound design. Covalent inhibition of enzymes based on the identification of a set of cysteine targeting covalent warheads including acrylamides, vinyl sulfones and nitriles, has been shown to be a successful approach in drug discovery pipelines. In our proposal, an initial set of putative inhibitors that incorporate an acrylamide warhead have already been rationally designed to target the TcDHODH active-site cysteine. Predicted inhibitors will be evaluated for inhibitory potency and mechanism of inhibition against TcDHODH, cytotoxicity and its anti-parasitic effects in cell culture. X-ray crystallography, fragment screening and medicinal chemistry will be combined to provide the chemical basis for the synthesis of a new generation of potent, selective, and drug-like inhibitors. The top 3 inhibitors that meet lead criteria (including PK and tolerability) will have their efficacy evaluated by DNDi via an in-kind contribution to the project. This proposal represents an unprecedent initiative to significantly contribute to building capacity in the field of drug discovery in Brazil, a field of research still very incipient in developing countries.