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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA076560-03
Application #
6173127
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Forry, Suzanne L
Project Start
1998-06-01
Project End
2001-05-31
Budget Start
2000-06-01
Budget End
2001-05-31
Support Year
3
Fiscal Year
2000
Total Cost
$122,186
Indirect Cost
Name
University of South Carolina at Columbia
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
111310249
City
Columbia
State
SC
Country
United States
Zip Code
29208
Celeste, Lesa R; Chai, Geqing; Bielak, Magdalena et al. (2012) Mechanism of N10-formyltetrahydrofolate synthetase derived from complexes with intermediates and inhibitors. Protein Sci 21:219-28
Qin, Jie; Chai, Geqing; Brewer, John M et al. (2012) Structures of asymmetric complexes of human neuron specific enolase with resolved substrate and product and an analogous complex with two inhibitors indicate subunit interaction and inhibitor cooperativity. J Inorg Biochem 111:187-94
Gibson, Lydia M; Celeste, Lesa R; Lovelace, Leslie L et al. (2011) Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding. Acta Crystallogr D Biol Crystallogr 67:60-6
Luo, BeiBei; Repalli, Jayanthi; Yousef, Al-Motassem et al. (2011) Human thymidylate synthase with loop 181-197 stabilized in an inactive conformation: ligand interactions, phosphorylation, and inhibition profiles. Protein Sci 20:87-94
Luo, BeiBei; Johnson, Saphronia R; Lebioda, Lukasz et al. (2011) Evolution of metamorphism in thymidylate synthases within the primate lineages. J Mol Evol 72:306-14
Huang, Xiao; Gibson, Lydia M; Bell, Brittnaie J et al. (2010) Replacement of Val3 in human thymidylate synthase affects its kinetic properties and intracellular stability . Biochemistry 49:2475-82
Lovelace, Leslie L; Johnson, Saphronia R; Gibson, Lydia M et al. (2009) Variants of human thymidylate synthase with loop 181-197 stabilized in the inactive conformation. Protein Sci 18:1628-36
Gibson, Lydia M; Dingra, Nin N; Outten, Caryn E et al. (2008) Structure of the thioredoxin-like domain of yeast glutaredoxin 3. Acta Crystallogr D Biol Crystallogr 64:927-32
Gibson, Lydia M; Lovelace, Leslie L; Lebioda, Lukasz (2008) The R163K mutant of human thymidylate synthase is stabilized in an active conformation: structural asymmetry and reactivity of cysteine 195. Biochemistry 47:4636-43
Lovelace, Leslie L; Gibson, Lydia M; Lebioda, Lukasz (2007) Cooperative inhibition of human thymidylate synthase by mixtures of active site binding and allosteric inhibitors. Biochemistry 46:2823-30

Showing the most recent 10 out of 22 publications