Because of the role that thymidylate synthase (TS) and deoxycytidylate deaminase (DD) play in providing, as well as regulating the supply of nucleotide products for DNA synthesis, these enzymes are targets for chemotherapy. We have studied the two enzymes extensively over a period of 27 years and have reached a point where we can provide significant information on the topography of both enzymes which might be exploited for the purpose of developing even more effective chemotherapeutic agents. Thus, in the case of TS, the binding sites of the folate substrate and its analogues methotrexate and 10-propargyl-5,8- dideazafolate will be determined, as well as that of photofixed deoxyuridine 5'-monophosphate and its 5-azido derivative. In addition site-directed mutagenesis will be employed to evaluate those regions of the protein which influence enzyme activity. This technique will also be used to study the mechanism of splicing associated with the intron-containing td gene encoding T4-phage TS. Methods will be developed to isolate and to amplify the expression of the human TS gene for purposes of x-ray crystallography. Cell culture studies will be conducted to examine the mechanism by which CB3717 affects its inhibitory potential. In the case of DD, similar studies on infrastructure probing will be conducted particularly with reference to its substrate and allosteric binding sites. These studies should be greatly aided by site-directed mutagenesis. Isolation and sequencing of the human DD gene is planned, as well as its amplification by a high expression vector, which will provide sufficient amount of protein for x-ray crystallographic studies. Location of the intracellular compartment of the enzyme will be established by immuno-electron microscopy. Eventually, it is hoped to examine the possibility that TS and DD are elevated in the sera of tumor containing animals, which would portend their use as diagnostic tools.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Unknown (R35)
Project #
5R35CA044355-07
Application #
3479559
Study Section
Special Emphasis Panel (SRC (88))
Project Start
1987-07-01
Project End
1994-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
7
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Wadsworth Center
Department
Type
DUNS #
110521739
City
Menands
State
NY
Country
United States
Zip Code
12204
Arvizu-Flores, Aldo A; Sugich-Miranda, Rocio; Arreola, Rodrigo et al. (2008) Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant. Int J Biochem Cell Biol 40:2206-17
Saxl, Ruth L; Reston, James; Nie, Zhe et al. (2003) Modification of Escherichia coli thymidylate synthase at tyrosine-94 by 5-imidazolylpropynyl-2'-deoxyuridine 5'-monophosphate. Biochemistry 42:4544-51
Gribaudo, Giorgio; Riera, Ludovica; Caposio, Patrizia et al. (2003) Human cytomegalovirus requires cellular deoxycytidylate deaminase for replication in quiescent cells. J Gen Virol 84:1437-41
Johnson, Eric F; Hinz, Wolfgang; Atreya, Chloe E et al. (2002) Mechanistic characterization of Toxoplasma gondii thymidylate synthase (TS-DHFR)-dihydrofolate reductase. Evidence for a TS intermediate and TS half-sites reactivity. J Biol Chem 277:43126-36
Aschele, C; Debernardis, D; Bandelloni, R et al. (2002) Thymidylate synthase protein expression in colorectal cancer metastases predicts for clinical outcome to leucovorin-modulated bolus or infusional 5-fluorouracil but not methotrexate-modulated bolus 5-fluorouracil. Ann Oncol 13:1882-92
Saxl, R L; Changchien, L M; Hardy, L W et al. (2001) Parameters affecting the restoration of activity to inactive mutants of thymidylate synthase via subunit exchange: further evidence that thymidylate synthase is a half-of-the-sites activity enzyme. Biochemistry 40:5275-82
Almog, R; Waddling, C A; Maley, F et al. (2001) Crystal structure of a deletion mutant of human thymidylate synthase Delta (7-29) and its ternary complex with Tomudex and dUMP. Protein Sci 10:988-96
Changchien, L M; Garibian, A; Frasca, V et al. (2000) High-level expression of Escherichia coli and Bacillus subtilis thymidylate synthases. Protein Expr Purif 19:265-70
Keefe, R G; Maley, G F; Saxl, R L et al. (2000) A T4-phage deoxycytidylate deaminase mutant that no longer requires deoxycytidine 5'-triphosphate for activation. J Biol Chem 275:12598-602
Aschele, C; Debernardis, D; Tunesi, G et al. (2000) Thymidylate synthase protein expression in primary colorectal cancer compared with the corresponding distant metastases and relationship with the clinical response to 5-fluorouracil. Clin Cancer Res 6:4797-802

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