Thymidylate synthase

Fig. 8. Thymidylate synthase inhibitors designed to fit into the A/, AJ -methylenetetrahydrofolate binding site. The best inhibitors from each class are the classical antifolate TS inhibitor (31), the naphthostyril-based lead compound (32), and the tetrahydroquinoline-based lead compound (33), iC values (in nAI)...

Fig. 9. Thymidylate synthase inhibitors found after analysis with the computet program DOCK (see Table 2).

Mn-+ K+ Ni " Arginase Pyruvate kinase (also requires Mg ) U rease Tetrahydrofolate (THF) Other one-carbon groups Thymidylate synthase... 

The 6-methylacetylamino-l,2,3,4-tetrahydroquinoline, after nitration and separation of isomers, following reduction and deprotection, gave the 7-amino-6-methylamino derivative, which cyclized with cyanogen bromide. Alkylation of the cyclization products afforded inhibitors of thymidylate synthase, 5-substituted 2-amino-l//-l-methyl-5,6,7,8-tetrahydroimidazo[4,5-g]quinolines 136, designed for use in iterative protein crystal analysis (Scheme 42) (92JMC847). 

Heterocyclic templates and nonpolyglutamatable inhibitors of thymidylate synthase as potential antitumor agents 99JHC827. 

Ralitrexed is a folate analog with greater selectivity. It easily crosses the cell membrane and undergoes polyglutamation. Within tissues, ralitrexed may be stored up to 29 days. It directly inhibits thymidylate synthase, the key enzyme for synthesizing thymidine triphosphate (TTP). The drug has been described to induce apoptosis in tumor cells. Ralitrexed is used for the treatment of colon carcinomas. 

Proguanil appears to have a dual activity. Part of it is metabolized to cycloguanil, which subsequently inhibits the protozaon dihydrofolate reduc-tase/thymidylate synthase (DHFR/TS) (Fig. 4). In addition, the native form, proguanil itself, exerts a potent antimalarial activity, especially in combination with other antimalarial drugs. The target of proguanil is unknown. 

Toyoda T, Brobey RKB, Sano G, Horii T, Tomioka N, Itai Akiko. Lead discovery of inhibitors of the dihydrofolate reductase domain of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase. Biochem Biophys Res Commun 1997 235 515-19. 

The methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA. The one-carbon fragment of methy-lene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division. Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anticancer drug. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme inhibitors of these enzymes can be used as antibacterial drugs, eg, trimethoprim, and anti-malarial drugs, eg, pyrimethamine. 

Fluoro-2 -deoxyuridine has been extensively used in studies of the mechanism of action of thymidylate synthase, and 5-fluorouracil is an anticancer drug that has provided a lead to the development of others. The metabolism of 5-fluorouracil by the ascomycete fungus Nectria haematococca has been studied using F NMR (Parisot et al. 1991). a-Fluoro-P-alanine (2-fluoro-3-aminopropionate) was produced (Figure 10.27), while 5-fluorouridine-5 -mono-, di-, and triphosphate were found in acid extracts of the mycelia, and the 2 - and 3 -monophosphates were recovered from RNA. 

Fig. 1.4 The increase in thymidylate synthase inhibition activity obtained from an initial hit identified as a disulphide-bound enzyme adduct and optimised to a reversible potent 330nM inhibitor using crystal structure-guided design.

G. F. Maley, P. Van Roey 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(5), 988. 

Fluorouracil (5-FU) acts as a false pyrimidine, inhibiting the formation of the DNA base thymidine.26,35 The main mechanism by which it accomplishes this is by inhibiting the enzyme thymidylate synthase, the rate-limiting step in thymidine formation. 5-FU first must be metabolized to its active metabolite (F-dUMP). Additionally, metabolites of 5-FU may incorporate into RNA, inhibiting its synthesis. 

Xanthine oxidase (1-Lactamase Ornithine decarboxylase Steroid 5a-reductase Thymidylate synthase Aromatase... 

Monoamine oxidase B Monoamine oxidase Thymidylate synthase GABA transaminase... 

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.

TK), 5-FU is activated to 5-fluorodeoxyuridine monophosphate (5-FdUMP). Potent inhibition of thymidylate synthase (TS) by 5-FdUMP is considered critical for 5-FU cytotoxicity. TS catalyzes the rate-limiting step of DNA synthesis, such as the conversion of dUMP into dTMP. Optimal TS function requires the formation of a covalent ternary complex consisting of TS, the folate cofactor 5,10-methylenetetra-hydrofolate (CH2THF), and 5-FdUMP. Inadequate cellular levels of 5,10-methyle-netetrahydrofolate reduce the stability of the ternary complex and consequently the inhibition of TS by 5-FdUMP. For this reason, 5-FU is administered in association with folinic acid, a precursor of 5,10-methylenetetrahydrofolate [40]. 

Fig. 14.10 Folate metabolism and role of MTHFR. Genetically reduced MTHFR activity affects the distribution between folate species required for protein and DNA synthesis. Higher availabil ity of 5,10-methylenetetrahydrofolate (CH2THF) potentiates the TS inhibition by 5-FdUMP, the active metabolite of 5-FU. Hey, homocysteine Met, methionine CH3HF, 5-methyltetrahydrofolate TS, thymidylate synthase 5-FdUMP, fluorodeoxyuridine monophosphate.

Recent studies focused on the importance of pharmacogenetic determinants of response in cancer patients. Screening of patients for polymorphic mutants of glutathione-S-transferase and thymidylate synthase has the potentiality to predict response and hence outcome of chemotherapy. 

Thymidylate Synthase Gene Promoter Polymorphism and Response to 5-Flurouracil-Based Chemotherapy... 

Thymidylate synthase (TS) is the rate-limiting enzyme in the DNA synthetic pathway and the target for 5-FU and folate analogs (Figure 14.3). Compared to normal tissues, TS is often overexpressed in tumor cells, probably as a result of tumor suppression loss of function, gene amplification or other mechanisms. Acute induction of TS protein as well as stable amplification of TS-specific genes may be associated with resistance to fluoropyrimidine derivatives [118, 119], and an inverse correlation between tumor TS expression and clinical response was found [120-122]. 

Berger SH, Jenh CH, Johnson LF et al. Thymidylate synthase overproduction and gene amplification in fluorodeoxyuri-dine-resistant human cells. Mol Pharmacol 1985 28 461—467. 

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