Thymidine kinase Thymidylate synthase

Fig. 14.3 5 -FU catabolism, anabolism and mechanism of action. 5-FUH2, 5-fluoro-5,6-dihydrouracil 5-FdUMP, 5-fluorodeoxyuridine monophosphate TP, thymidine phosphorylase TK, thymidine kinase TS, thymidylate synthase CH2THF, 5,10-methylenetetrahydrofolate.

Fig. lA. Anabolic and catabolic pathways of 5-FU. DPD dihydropyrimidine dehydrogenase, DP di-hydropyrimidinase, pUP beta-ureidopropionase, UP uridine phosphorylase, OPRT orotate phospho-ribosyl transferase, UK uridine kinase, TP thymidine phosphorylase, TK thymidine kinase, RNR ribonucleotide reductase. The three active metabolites (shown in rectangles) are FdUMP (5-fluoro-2 -deoxyuridine 5 -monophosphate) inhibiting TS (thymidylate synthase), and FUTP (5-fluorouridine 5 -triphosphate) and FdUTP (5-fluoro 2 -deoxyuridine 5 -triphosphate) interfering with RNA and DNA, respectively. 

In order to determine the relationship between protein structure and function and to create mutant enzymes with altered properties useful for biotechnology and cancer therapy, a directed evolution approach has been explored and novel proteins developed for Pol I DNA polymerase enzymes thymidylate synthase, thymidine kinase and 06-alkylguanine-DNA alkyltransferase. In every case the creation of a large variety of altered proteins has been achieved, and the emerging picture is that even highly conserved proteins can tolerate wide-spread amino acid changes at the active site with-... 

We describe the various approaches that one might use to generate novel enzymes and the application of one such technique, Random Oligonucleotide Mutagenesis, to the DNA synthesis and repair enzymes DNA polymerase I from E. coli and Thermus aquaticus, as well as human thymidylate synthase, thymidine kinase and 06-alkylgua-nine-DNA alkyltransferase. 

Random oligonucleotide mutagenesis was first applied to promotor sequences that regulate the production of enzymes in cells [21] and was the first method used to alter systematically the functions of enzymes by directed evolution [22], Based on our experience, we will focus on this approach and emphasize recent applications of this methodology to enzymes involved in DNA repair and synthesis, including DNA polymerase enzymes, thymidylate synthase, thymidine kinase, and 06-alkylguanine-DNA alkyltransferase. 

The deoxyuridine analogue 5-azidomethyl-2 -deoxyuridine (159) was one of a large number of compounds investigated as inhibitors of thymidylate synthase and thymidine kinase from L1210 cells, but the compound was... 

Thymidylate (dTMP) is formed intracellularly either de novo, in a process of the C(5) methylation of 2 -deoxyuridylate (dUMP), catalyzed by the enzyme thymidylate synthase (TS), or as a product of thymidine salvage via phosphorylation, catalyzed by the enzyme thymidine kinase. The dUMP methylation reaction involves a concerted transfer and reduction of the one-carbon group of... 

Enzyme Assays Activities of thymidylate synthase (Rode et al. 1990), thymidine kinase (Tsukamoto et al. 1991) and dihydrofolate reductase (Mathews et al. 1963) were assayed according to previously published procedures. dUTP-ase activity was determined by coupling with the thymidylate synthase-catalyzed reaction and measuring tritium released from [ H]dUMP (Golos and Rode 1999). The activity unit was defined as the enzyme amount required to convert 1 pmol of substrate per 1 min at 37°C. 

FIG. 6.13 Mammalian pyrimidine salvage and interconversion pathways. Enzymes listed in Figs 6.13-6.17 are as follows (1) deoxyCMP deaminase (2) thymidylate synthase (3) ribonucleotide reductase (4) deoxyuridine triphosphatase (5) CTP synthetase (6) nucleotide kinase (7) deoxyTMP kinase (8) nucleotide diphosphokinase (9) non-specific phosphatase or nucleotidase (10) cytidine kinase (11) pyrimidine phos-phorylase or hydrolase (12) uracil PRTase (13) cytidine deaminase (14) thymidine kinase (15) cytidine phosphotransferase (16) uridine phosphotransferase (17) thymidine phosphotransferase (18) deoxyribo-nucleotide phosphotransferase (19) cytosine PRTase. 

In HeLa ceils hydroxyurea is an efficient inhibitor of histone synthesis. This action requires protein synthesis and leads to rapid disappearance of cytoplasmatic histone mRNA The effect is not specific for hydroxyurea since suppression of DNA synthesis by arabino-cytosine or temperature-sensitive mutations yields analogous results. Similarly, the synthesis of some enzymes necessary for DNA replication and active in S-phase is altered by hydroxyurea. Increased activity of ribonucleotide reductase in HeLa and in hamster cells and of the salvage enzyme thymidine kinase in HeLa cells and KB cells has been observed, probably as a consequence of the increased fraction of cells in S-phase. Repression occurs for thymidine kinase in human lymphocytes and for ornithine decarboxylase in Chinese hamster fibroblasts whereas no or only slight effects were seen on ribonucleotide reductase in hamster fibroblasts , on thymidylate synthase in extracts from synchronous mouse cells " , and on DNA polymerase in rabbit kidney cells or HeLa cells . ... 

Chinese hamster fibroblasts co-sedimentation on sucrose gradients kinetic coupling nascent and template DNA, ribonucleotide reductase, thymidylate synthase, dihydrofolate reductase, thymidine kinase, NDP kinase, DNA polymerase 83, 273, 353... 

Thymidylate residues required for cellular DNA synthesis are obtained exogenously via the salvage pathway and/or are synthesised de novo via dUMP in a reaction catalysed by thymidylate synthase (dUMP dXMP). The thymidine monophosphate can then be phosphorylated by kinases to dTDP and dTTP before being incorporated into DNA. 

The active principle of both TAS-102 and FTC-092 with anti-cancer effect is Trifluridine (7). As in the case of Fluorouracil, one of the mechanisms by which compound 7 exhibits its antitumor activity is inhibition of thymidylate synthase [100], More precisely, Trifluridine is transformed into a,a,a-trifluorothymidine monophosphate (76) by thymidine kinase (Scheme 21) similarly to the Fluorouracil derivatives discussed in the previous sections, compound 76 is true inhibitor of thymidylate synthase. However, compound 7 exhibits an anticaneer effect on colorectal cancer cells that have acquired Fluorouracil resistance as a result of the overexpression of thymidylate synthase. 

Nucleoside and nucleotide analogues have been widely used in the treatment of various diseases. Koegler et al. reported the synthesis of 6-aza-2 -deo3qruridine monophosphate analogues (6). These compounds can inhibit thymidylate synthases, and also act as substrates or inhibitors of thymidine monophosphate kinase in mycobacterium tuberculosis. 

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