Opportunistic infections are the primary cause of suffering and death in individuals with AIDS. Many of these infections are produced by parasites which rarely affect individuals who are not immunocompromised. Unfortunately, successful combination therapies against the HIV-1 virus still leave most patients susceptible to opportunistic parasitic infections. The drugs which are currently available for the treatment of these parasitic infections suffer from a lack of selectivity resulting in host toxicity and untoward side effects. Thus there is a need for novel therapeutic strategies which may be more selective and less toxic. This proposal outlines mechanistic and structural studies on a unique bifunctional enzyme which will serve as basis for the design of novel antiparasitic drugs. Two enzymes crucial for DNA synthesis and one-carbon transfers are thymidylate synthase and dihydrofolate reductase. In many protozoan parasites, these two catalytic activities are located on a single polypeptide chain to form a bifunctional thymidylate synthase (TS)/dihydrofolate reductase (DHFR) enzyme. In mammalian species, the thymidylate synthase and dihydrofolate activities occur as separate catalytic activities on mono functional enzymes. A considerable amount of mechanistic information is available for the human monofunctional thymidylate synthase and dihydrofolate reductase since each enzyme has been successfully targeted with the anticancer drugs, 5-fluorouracil and methotrexate, respectively. Earlier work as well as preliminary transient kinetic studies from the Pl's lab indicate substantial mechanistic differences in the bifunctional parasitic and mono functional human enzymes. The three dimensional structure of the bifunctional TS-DHFR enzyme is available for computer modeling studies to identify inhibitors through docking programs. The central theme of this proposal is that an in-depth kinetic and structural evaluation of the bifunctional TS-enzyme at a molecular level will provide a crucial mechanistic understanding that can be exploited as a novel therapeutic approach. ? ? ?
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