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 #
5R01CA014394-14
Application #
3163918
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1978-09-01
Project End
1989-02-28
Budget Start
1986-03-01
Budget End
1987-02-28
Support Year
14
Fiscal Year
1986
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
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