Thymidylate synthase is the target of a number of drugs with real or potential use in cancer chemotherapy. We intend to elucidate details of the mechanism of this enzyme, as well as the mechanism of action of analogs of 2 feet deoxyuridylate and the folate cofactor which are potent inhibitors of this enzyme. Where applicable, we will atempt to apply our findings to study and optimize the efficacy of such inhibitors in tissue culture cells, with the anticipation that such studies will be relevant to in vivo systems. We shall attempt to obtain in crystals of the L.casei enzyme which are suitable for X-ray crystallography, and to obtain human tissue culture cell lines which overproduce this enzyme. Should the latter be successful, it would be possible to ascertain whether the human enzyme shares important mechanistic, inhibitory and structural properties with the better understood bacterial thymidylate synthases. We have formulated a hypothesis that the mechanism of thymidylate synthase may serve as a paradigm for many enzymes involved in modifications of the pyrimidine heterocycle. We believe that nucleophilic attack at the 6-position of the pyrimidine heterocycle to form transient 5, 6-dihydro-pyrimidine intermediates is a common feature of many enzymes which modify the 4- and 5-positions of the heterocycle and cleave the glycosidic bond of pyrimidine nucleosides/nucleotides. Using methodologies resulting from studies of thymidylate synthase, we shall test this hypothesis in studies of dCMP hydroxymenthylase, DNA(cytosine) methylases, Cyd and dCMP deaminases, Urd and dThd phosphorylases and PsiUrd synthases. We shall determine the mechanisms of (a) inhibition of DNA(cytosine) methylases by DNA containing 5-azacytosine and 5-fluorocytosine, and (b) inhibition of tRNA PsiUrd synthase by tRNA containing 5-fluorouracil. We believe that when contained in nucleic acids, these analogs form covalent adducts with the aforementioned enzymes they inhibit and are, in fact, mechanism-based inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA014394-13
Application #
3163917
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1978-09-01
Project End
1989-02-28
Budget Start
1985-03-01
Budget End
1986-02-28
Support Year
13
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Meyskens Jr, Frank L; Mukhtar, Hasan; Rock, Cheryl L et al. (2016) Cancer Prevention: Obstacles, Challenges and the Road Ahead. J Natl Cancer Inst 108:
Gonzalez-Pacanowska, Dolores; Ruiz-Perez, Luis M; Carreras-Gomez, Maria Angeles et al. (2003) The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase. Protein Eng 16:607-14
Kawase, S; Cho, S W; Rozelle, J et al. (2000) Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase. Protein Eng 13:557-63
Variath, P; Liu, Y; Lee, T T et al. (2000) Effects of subunit occupancy on partitioning of an intermediate in thymidylate synthase mutants. Biochemistry 39:2429-35
Morse, R J; Kawase, S; Santi, D V et al. (2000) Energetic contributions of four arginines to phosphate-binding in thymidylate synthase are more than additive and depend on optimization of ""effective charge balance"". Biochemistry 39:1011-20
Liu, Y; Barrett, J E; Schultz, P G et al. (1999) Tyrosine 146 of thymidylate synthase assists proton abstraction from the 5-position of 2'-deoxyuridine 5'-monophosphate. Biochemistry 38:848-52
Finer-Moore, J S; Liu, L; Birdsall, D L et al. (1998) Contributions of orientation and hydrogen bonding to catalysis in Asn229 mutants of thymidylate synthase. J Mol Biol 276:113-29
Liu, Y; Santi, D V (1998) A continuous spectrophotometric assay for thymidine and deoxycytidine kinases. Anal Biochem 264:259-62
Huang, L; Pookanjanatavip, M; Gu, X et al. (1998) A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst. Biochemistry 37:344-51
Chiericatti, G; Santi, D V (1998) Aspartate 221 of thymidylate synthase is involved in folate cofactor binding and in catalysis. Biochemistry 37:9038-42

Showing the most recent 10 out of 71 publications