The enzyme, thymidylate synthase (TS) catalyzes the reaction that produces thymidylate (TMP). Depletion of TMP by inhibition of TS results in death of rapidly dividing cells, such as cancer cells. TS has been the focus of extensive research in cancer experimental therapeutics and is a well-validated target. TS inhibitors in current clinical use (e.g. 5-fluorouracil and raltitrexed) are limited in their utility by drug resistance. A main factor that is thought to underlie clinical drug resistance is an elevation of TS levels, which may be an intrinsic property of the tumor or may be induced by treatment with the current TS inhibitors. Human TS (hTS) exists in 2 conformations: 1 active and 1 inactive, which are in apparent equilibrium. We propose to test a hypothesis that TS inhibition via stabilization of the inactive conformer will not only prevent the induction in TS levels that is observed with classical active site directed inhibitors but also reduce TS levels in tumors with elevated expression of TS. To achieve this goal, mutant hTSs that are stabilized in either the active or inactive conformation will be created and characterized. These mutants will be utilized to analyze the effect of stabilization in an inactive conformation on the intracellular stability of the protein. Also, small molecule ligands stabilizing the inactive conformation will be developed. If our hypothesis is correct, then there is a strong rationale for developing inhibitors that would work by stabilization of the inactive conformation as opposed to current inhibitors that stabilize the active conformation. Such inhibitors should not only inhibit the catalytic activity of TS but also work by reducing the levels of TS protein, eliminating a major mechanism of resistance. We also propose to utilize hTS as a model for studies of synergistic inhibition by a mixture of inhibitors. This novel approach, which we term hetero-inhibition, would capitalize on the asymmetry of the hTS homodimer and may have general utility for the inhibition of proteins that are functional and/or structural heterodimers.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA076560-06
Application #
7147447
Study Section
Drug Discovery and Molecular Pharmacology Study Section (DMP)
Program Officer
Forry, Suzanne L
Project Start
1998-06-01
Project End
2009-11-30
Budget Start
2006-12-01
Budget End
2007-11-30
Support Year
6
Fiscal Year
2007
Total Cost
$163,827
Indirect Cost
Name
University of South Carolina at Columbia
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041387846
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

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