Thymidylate synthetase mechanism

Flucytosine is a fluorinated derivative of pyrimidine. Its spectrum of activity is narrower than that of amphotericin B. However, it exhibits a synergetic effect when used in combination with amphotericin B. In sensitive fungi, flucytosine is transformed into 5-fluorouracil, which in turn is turned into 5-fluorodeoxyuracilic acid, an inhibitor of thymidylate synthetase, and correspondingly, DNA synthesis. 5-Fluorouracil triphosphate, which causes the formation of defective RNA, may also be involved in this process. The mechanism is highly selective because mammahan cells are not able to turn a large amount of flucytosine into 5-fluorouracil. 

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. 

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. 

A considerable amount of work, both with thymidylate synthetase itself and model systems, indicates that in the mechanism of thymidylate synthetase a key step is the attack of an enzymatic nucleophile (believed to be cysteine) on C-6 of dUMP to give a 5,6-dihydro-dUMP intermediate (B-77MI11003). A number of observations are in accord with such a nucleophilic catalysis by the enzyme. Among these are the demonstration that thymidylate synthetase, in the absence of the cofactor, catalyzes the exchange of tritium from [5-3H]dUMP for protons of water (79B2794), and the recent isolation of a covalent adduct formed between 5-nitro-dUMP and thymidylate synthetase (80JBC(255)5538 see also 80MI11003). 

Mechanism of 5-fluorouracil s cytotoxic action. 5-Fluorouracil is converted to 5-FdUMP, which competes with deoxyuridine monophosphate (dUMP) for the enzyme thymidylate synthetase. 

Fluorouracil is widely used in cancer chemotherapy. It is a precursor of 5-fluoro-dUMP, which is a mechanism-dependent inhibitor of thymidylate synthetase. It forms a stable methylene-bridged complex with methylene-tetrahydrofolate on the enzyme catalytic site that cannot undergo reductive cleavage. 

Like idoxuridine. the antiviral mechanism of trifluridinc involves inhibition of viral DNA synthesis. Trifluridinc monophosphate is an irreversible inhibitor of thymidylate synthetase, and the biologically generated triphosphate competitively inhibits thymidine triphosphate incorporation into DNA by DNA polymera.se. In addition, trilluridine in its triphosphate form is incorporated into viral and cellular DNA. creating fragile, poorly functioning DNA. 

In 1968, my graduate student Charles Walsh and I addressed the following question What are the pyrimidine sources for nucleic acid synthesis by Plasmodium lophurae We found the parasite synthesized pyrimidines de novo (Walsh and Sherman, 1968b). The evidence for a de novo synthesis was the presence of the key enzymes, thymidylate synthetase and oroti-dine-5-monophosphate pyrophosphorylase, as well as the demonstration of the incorporation of 14C-bicarbonate into cytosine, uracil and thymine. Finding a de novo pathway for the synthesis of pyrimidines by the malaria parasite would, in the next three decades, provide a biochemical basis for the mechanism of action of anti-folate anti-malarials as well as contributing to an understanding of the unique properties of the malaria parasite mitochondrion. 

Methotrexate inhibits the enzyme folate reductase at two steps (Fig. 4-12). Even though the inhibition is technically a competitive one, the enzyme binds MTX much more strongly than the natural substrate, FH2 (Table 4-5). In practical terms there is no dissociation of the enzyme-drug complex. For thymidylate synthetase to continue to produce thymidylic acid (and therefore DNA), perpetual reduction of FH2 is essential for cellular survival. The efficiency of MTX inhibition of folate reductase thus indirectly becomes the mechanism by which this drug is so cytotoxic in the S phase. In addition, there is evidence that MTX may, by binding to the tetrahydrofolate coenzyme, also inhibit the thymidylate synthetase... 

As with thiopurines, 5-FU must first be activated by a series of steps to the active cytotoxic agent, 5-fluorodeoxyuridine monophosphate (FdUMP) (Fig. 4-17). Tumor cells deficient in any of the enzymes needed for these conversions (e.g., phosphoribosyltransferase) are likely to be resistant to 5-FU. Since the early discovery that thymidylate synthetase is very sensitive to 5-FU (actually FdUMP), the mechanism of action was believed to be impairment of the conversion of dUMP to dTMP (Fig. 4-13). A more detailed mechanism is now understood to be in the first step, thymidylate synthetase reacts covalently with C-5 of FdUMP (as if it were the normal substrate, dUMP) via nucleophilic attack of its reactive site s SH group. Methylene-FH4 cofactor then adds to C-5 of this initial complex. 

The antiviral mechanism of action of triflnridine involves inhibition of viral DNA synthesis. Triflnridine monophosphate irreversibly inhibits thymidylate synthetase, and trifluridine triphosphate is a competitive inhibitor of thymidine triphosphate incorporation into DNA by DNA polymerases. Trifluridine is incorporated into viral and ceUnlar DNA. Trifluridine-resistant HSV with altered thymidine kinase substrate specificity can be selected in vitro, and resistance in clinical isolates has been described. 

While no firm underlying mechanisms have been demonstrated, some authors have suggested that irreversible oxidation of the cobalt atom in vitamin B12 by nitrous oxide can lead to inactivation of enzymes dependent on this vitamin, with resultant metabolic aberrations. Such examples have included methionine synthetase and thymidylate synthetase, which are essential in the synthetic pathways leading to the production of myelin and thymidine, respectively. Should these enzymes be impaired during the sensitive periods of in utero development, the potential for malformations may unfortunately be realized. To date, no studies have been able to demonstrate conclusively that low-level exposure to nitrous oxide is associated with a meaningful disruption of crucial metabolic functions to produce the above-described toxicity however, measures including improved waste gas-scavenging systems should be taken to minimize exposure of personnel. 

Studies on the mechanism of the bacterial thymidylate synthetase reaction employing tritiated H4-folate showed that a hydrogen atom of 5,10-methylene H4-folate was transferred to the thymine methyl group and suggested the formation of a methylene bridge between folate and deoxyuridylate (9). 

Proposed mechanisms for the thymidylate synthetase reaction are discussed in more detail by Blakley (2). 

Folic Acid Antagonists - Interest in antimetabolites that interfere with the synthesis of nucleic acids continues. Studies on the transport and uptake of methotrgxate ° and 2,4-diamino-5-(3,4-dichlorophenyl)-6-methylpyrimidine indicate that clinical response is related to cellular uptake of drugl and the resistance of certain cells to methotrexate appears to be due to lack of transport into these cells. Work on other resistant cell lines indicates that resistance can also be due to an increase in cellular content of folate reductase, but no correlation was observed between resistance and the level of other enzymes involved in folate metabolism. Despite these results there is evidence that the ability of methotrexate to kill cells cannot be entirely explained by its inhibition of folate reductase. Leucovorin at appropriately timed intervals improved the therapeutic index of methotrexate in the treatment of head and neck cancer, and lymphosarcoma and reticulum cell sarcoma. The use of methotrexate in the treatment of hormone-refractory metastatic breast carcinoma " and the use of intrathecal methotrexate also appear promising. Oxidation of methotrexate to 7-hydroxymethotrexate by liver aldehyde oxidase is probably a detoxification mechanism. Material previously reported to be tetrahydromethotrexate has now been found to be a mixture of di- and tetrahydromethotrexate, both of which are less effective than methotrexate in the inhibition of folate reductase, but more effective in the inhibition of thymidylate synthetase. ... 

P. Reyes and C. Heidelberger, Fluorinated pyrimidines. XXVI. Mammalian thymidylate synthetase Its mechanism of action and inhibition by fluorinated nucleotides. Mol. Pharmacol. /, 14 (1965). 

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