This proposal involves a multi-disciplinary research effort directed at studies of a) enzymes in folate metabolism in protozoa and b) mechanisms of drug resistance in these organisms. The organisms to be studied are Leishmania and Plasmodium, casual agents of diseases (Leishmaniasis and malaria, respectively) of worldwide importance. Our objectives are aimed at obtaining fundamental biochemical information, with the belief that such knowledge will provide insight into how to exploit what is found and assist in controlling these organisms. We will continue studies on the structure, function and inhibition of the bifunctional thymidylate synthase (TS)-dihydrofolate reductase (DHFR) which is uniquely found in protozoa. We intend to express the enzyme from Leishmania, and study its higher order structure in detail; studies on computer assisted structure modeling and drug design will be initiated. We will continue studies on the amplified DNA found in anti-folate resistant Leishmania. We will further characterize the structure of these DNAs and delineate differences observed in different cell lines. In Plasmodium falciparum, we will isolate the TS-DHFR gene, characterize it and express it in E. coli or yeast; we will study molecular aspects of drug resistance towards anti-folates and initiate other studies of enzymes in folate metabolism.
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