This proposal involves a multi-disciplinary research effort directed at studies of folate-cofactor utilizing enzymes in Leishmania, and, to some extent, other protozoa. Our objectives are to obtain fundamental scientific information on the biochemistry/biology of these important human pathogens; from such knowledge, we hope to develop approaches necessary for the design of selective chemotherapeutic agents for treatment of the diseases caused by such organisms. In particular, studies of the enzyme involved in the """"""""dTMP synthesis cycle"""""""" will be investigated with primary emphasis on the dihydrofolate reductase (DHFR)-thymidylate synthetase (TS) bifunctional protein which is apparently unique to protozoa; secondary emphasis will be placed on studies of serine transhydroxymethylase (STH). With DHFR-TS experiments are designed to: (1) ascertain whether structural features are common to all genera of protozoa; (2) develop methotrexate resistant Leishmania which overproduce TS-DHFR, and study aspects of this phenomenon; (3) develop effective methods for purification of the DHFR-TS from Leishmania which will be generally applicable to all such bifunctional enzymes; (4) determine important structural features of the bifunctional protein in Leishmania; (5) perform a complete kinetic analysis of the enzyme, with emphasis on cooperative activation/inhibition and substrate channeling; and (6) develop potent and selective inhibitors of Leishmania TS and DHFR with the hope that with STH are designed to: (1) determine the importance of this enzyme in thymidylate synthesis in Leishmania; (2) ascertain whether the enzyme is functionally or structurally associated with TS-DHFR and whether there is coordinate control of expression of these enzyme activities; and (3) map the cofactor binding site of the enzyme with the hope that leads will be uncovered which will permit development of potent inhibitors of this enzyme.
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