Thymidylate Synthetase and Dihydrofolate Reductase

The methyl donor is methylene-tetrahydrofolate. The reaction involves formation of a methylene bridge between N-5 of the coenzyme and C-5 of dUMI) followed by transfer of hydrogen from the pyrazine ring of tetrahydrofolate, 

5-Fluorouracil is widely used in cancer chemotherapy. It is a precursor of 

5-fluoro-dUMP, which is a mechemism-dependent inhibitor of thymidylate synthetase. It forms a stable methylene-bridged complex with methyiene-tetrahydrofolate on the enzyme catalytic site that cemnot undergo reductive cleavage. 

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Thymidylate Synthetase and Dihydrofolate Reductase Methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP see Figure 10.8) is essential for the synthesis of DNA, although preformed TMP can be reutUized by salvage from the catabolism of DNA. 

The substituted perhydro-imidazo[l, 5-uJpyridine (113) was prepared in order to test for inhibition of enzymes thymidylate synthetase and dihydrofolate reductase. Pyridine-2-carboxaldehyde was condensed with ethyl-p-aminobenzoate to give the Schiff base (111) which, on sodium borohydride reduction followed by catalytic hydrogenation, resulted in 112. Condensation of 112 with 5-formyluracil gave 113 (70JMC276). 

Wu CL, Qasson M, Dyson N, Harlow E (1996) Expression of dominant-negative mutant DP-1 blocks cell cycle progression in GL Mol Cell Biol 16 3698-3706 Yasumasu I, Saitoh M, Fujimoto N, Kusunoki S (1979) Changes in activities of thymidylate synthetase and dihydrofolate reductase in sea urchin eggs after fertilization. Dev Growth Differ 21 237-243... 

Figure 11.21 The reaction of thymidylate synthetase and dihydrofolate reductase.

Several perhydro derivatives (74) of the ring system were synthesized as analogues of tetrahydrofolic acid. " They were not as effective as aminopterin as inhibitors of either thymidylate synthetase or dihydrofolate reductase. They are therefore presumably of little interest as anticancer agents and were found to have no inhibitory effect on the growth of various bacteria. ... 

The answer is E. Methotrexate is an analog of folic acid that binds with very high affinity to the substrate-binding site of dihydrofolate reductase, the enzyme that catalyzes conversion of DHF to THE, which is used in various forms by enzymes of both the purine and pyrimidine de novo synthetic pathways. Thus, synthesis of dTMP from dUMP catalyzed by thymidylate synthetase and several steps in purine synthesis catalyzed by formyltransferase are indirectly blocked by the action of methotrexate because both those enzymes require THE coenzymes. 

Methotrexate is a potent inhibitor of dihydrofolate reductase, with an affinity 1,000-fold greater than that of dUiydrofolate. Chemotherapy consists of alternating periods of administration of methotrexate and folate (normally as 5-formyl-tetrahydrofolate, leucovorin) to replete the normal tissues and avoid induction of folate deficiency- so-called leucovorin rescue. As well as depleting tissue pools of tetrahydrofolate, methotrexate leads to the accumulation of relatively large amounts of 10-formyl-dihydrofolate, which is apotentinhibitor of both thymidylate synthetase and glycinamide ribotide transformylase, an intermediate step in purine nucleotide synthesis. It is likely that this, rather than simple depletion of tetrahydrofolate, is the basis of the cytotoxic action of methotrexate (Barametal., 1988). 

Figure 10.8. Synthesis of thymidine monophosphate. Thymidylate synthetase, EC 2.1.1.45 and dihydrofolate reductase, EC 1.5.1.3. THE, tetrahydrofolate.

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are synthesized in the cytoplasm. At this stage the antifolate drugs (sulphonamides and dihydrofolate reductase inhibitors) act by interfering with the synthesis and recycling of the co-factor dihydrofolic acid (DHF). Thymidylic acid (2-deoxy-thymidine monophosphate, dTMP) is an essential nucleotide precursor of DNA synthesis. It is produced by the enzyme thymidylate synthetase by transfer of a methyl group from tetrahydrofolic acid (THF) to the uracil base on uridylic acid (2-deoxyuridine monophosphate, dUMP) (Fig. 12.5). THF is converted to DHF in this process and must be reverted to THF by the enzyme dihydrofolate reductase (DHFR) before... 

Methotrexate is a folic acid analogue. Its mechanism of action is based on the inhibition of dihydrofolate reductase. Inhibition of dihydrofolate reductase leads to depletion of the tetrahydrofolate cofactors that are required for the synthesis of purines and thymidylate (see Fig. 2). Enzymes that are required for purine and thymidylate synthesis are also directly inhibited by the polyglutamates of methotrexate which accumulate with dihydrofolate reductase inhibition. The mechanisms that can cause resistance include decreased transport of methotrexate into the tumor cells, a decreased affinity of the antifolate for dihydrofolate reductase, increased concentrations of intracellular dihydrofolate reductase and decreased thymidylate synthetase activity. 

Pemetrexed is chemically similar to folic acid. It inhibits three enzymes used in purine and pyrimidine synthesis - thymidylate synthetase, dihydrofolate reductase, and glycinamide ribonucleotide formyl transferase. By inhibiting the formation of precursor purine and pyrimidine nucleotides, pemetrexed prevents the formation of DNA and RNA. In 2004 it was approved for treatment of malignant pleural mesothelioma and as a second-line agent for the treatment of non-small cell lung cancer. Adverse effects include gastrointestinal complaints, bone marrow suppression, alopecia, allergic and neurotoxic reactions. 

Suramin (Germanin) is a derivative of a nonmetallic dye whose antiparasitic mechanism of action is not clear. It appears to act on parasite specific a-glyc-erophosphate oxidase, thymidylate synthetase, dihydrofolate reductase, and protein kinase but not on host enzymes. 

Methotrexate s principal mechanism of action at the low doses used in the rheumatic diseases probably relates to inhibition of aminoimidazolecarboxamide ribonucleotide (AICAR) transformylase and thymidylate synthetase, with secondary effects on polymorphonuclear chemotaxis. There is some effect on dihydrofolate reductase and this affects lymphocyte and macrophage function, but this is not its principal mechanism of action. Methotrexate has direct inhibitory effects on proliferation and stimulates apoptosis in immune-inflammatory cells. Additionally, inhibition of proinflammatory cytokines linked to rheumatoid synovitis has been shown, leading to decreased inflammation seen with rheumatoid arthritis. 

The active-site-directed inhibition of enzymes has been an important research topic in pharmaceutical drug design (Sandler, 1980). An early development of anti-cancer agents involved inhibitions of dihydrofolate reductase and thymidylate synthetase. Search enzyme resource sites for kinetic data (turnover number, Km and Kt) of these two enzymes. 

In the folate coenzymes, the pteridine ring is fully reduced to tetrahydro-folate, although the oxidized form, dihydrofolate, is an important metabolic intermediate. In the reactions of thymidylate synthetase (Section 10.3.3) and methylene tetrahydrofolate reductase (Section 10.3.2.1), the pteridine ring has a redox role in the reaction. The folate coenzymes are conjugated with up to six additional glutamate residues, finked by y-glutamyl peptide bonds. 

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