This proposal involves a multi-disciplinary research effort directed at studies of enzymes in folate metabolism in protozoa, in particular the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR). The sources to be studied are Leishmania, Trypanosoma and Plasmodium, casual agents of diseases of worldwide importance (Leishmaniasis, Trypanosomiasis and Malaria, respectively). Our objectives are aimed at obtaining fundamental biochemical information, with the belief that such knowledge will provide insight into how to assist in controlling these organisms and the diseases they cause. We will continue studies on the structure, function and inhibition of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) which is uniquely found in protozoa. We intend to express large amounts of the enzymes and study their structures and functions in detail; studies on molecular modeling and drug design will be performed. In Plasmodium falciparum, we will also prepare TS-DHFR genes from antifolate drug-resistant mutants, express the gene products in E. coli or yeast, and characterize them in detail. We will study molecular aspects of drug resistance towards antifolates to prove the molecular mechanisms of antifolate-resistance, and hopefully obtain drugs which circumvent resistance.
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