application): The targets of antifolate drugs are the parasite dihydropteroate synthase (DHPS) and, in protozoa, the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS). Their activities are essential for folate biosynthesis and homeostasis and thymidylate biosynthesis. Inhibition of any of these enzymes will result in the cessation of thymidylate and DNA synthesis and death. A large number of substrate analogues have been tested for inhibition of these enzymes; many of them have been found efficacious in the treatment of infectious and neoplastic diseases. Although it is well known that the potency of these drugs varies among microbial DHFRs, there has been no attempt to assess their efficacy in inhibiting the target C. parvum enzyme. The applicants have previously expressed C. parvum DHFR-TS and human DHFR genes in E. coli, purified the active recombinant enzymes, and developed a microtiter plate-based assay to measure inhibitor I50s and Kis. The applicants have also homology-modeled the tertiary structure of the DHFR domain and applied a docking program to the model to identify candidate inhibitors. The investigators propose to: (1) determine the kinetic constants of distinct C. parvum DHFR-TS enzymes from bovine and human isolates; (2) identify lead inhibitors by screening rationally and empirically selecting candidates by using recombinant enzymes, and refining leads by molecular modeling and structure-activity relationship studies; (3) purify sufficient mass of the recombinant enzyme for crystallization and structure determination; (4) employ a short-term epithelial cell culture system to examine folate synthesis and thymidine salvage by intracellular parasites; and (5) investigate the permeability properties of the C. parvum dense band-feeder organelle by microinjection of fluorescent tracers into the host cell cytoplasm and microscopic observation of their intracellular trafficking.
