Thymidylate synthase (TS) plays a critical role in DNA biosynthesis by providing the only de novo source of thymidylate (dTMP). Although the TS inhibitor FdUMP has been utilized for several decades, clinical trials have recently been established to determine the efficacy of novel TS targeted anticancer agents. Included among these are ZD1694, LY21514, ZD9331, 1843489, and AG337. These novel inhibitors of TS have demonstrated anticancer activity against several cancer types and their cytotoxic mechanism of action has been attributed to a decrease in the de novo synthesis of dTMP. TS, therefore, continues to be a prime target for the development of novel chemotherapeutic agents. The applicants propose to utilize steady state and transient state kinetics to determine the mode of binding for TS targeted anticancer agents that are currently in clinical trials. They believe these inhibitors can also be used as probes to reveal insights into the mechanism by which TS catalyzes the conversion of dUMP to dTMP. Because much is known about the physical characteristics of TS and the sequence of reactions catalyzed by TS, but little about the catalytic mechanism of action of this key enzyme, we plan to define the molecular mechanism of catalysis for Escherichia coli and human TS by determining rate constants which govern the individual reactions catalyzed by TS. This fundamental information will be used to analyze the effects certain amino acid substitutions have on specific rate constants as well as their effects on inhibitor binding. They will also determine the three dimensional structure of TS and variants of TS complexed with various inhibitors to target residues that stabilize inhibitor binding but are not required for substrate binding or catalysis.
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