Thymidylate synthetase, action

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 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. 

Fluorouracil/ Topical [5-FU] (Efudex) [Anrineoplasric/ Antimetabolite] Uses Basal cell carcinoma acdnic/solar keratosis Action Inhibits thymidylate synthetase (X DNA synth, S-phase specific) Dose 5% cream bid x 2-6 wk Caution [D, ] Irritant chemo Contra Component sensitivity Disp Cream, soln SE Rash, dry skin, photosens EMS See Fluorouracil OD See Fluorouracil... 

Site of action 5-FU per se is devoid of antineoplastic activity and must be converted to the corresponding deoxynucleotide (5-FdUMP, Figure 38.9), which competes with deoxyuridine monophosphate (dUMP) for thymidylate synthetase. 5-FdUMP acts as a pseudosubstrate and is entrapped with the enzyme and its N5,N10-methylene tetrahydrofolic acid coenzyme in a ternary complex that cannot proceed to products. DNA synthesis decreases due to lack of thymidine, leading to imbalanced cell growth and cell death. [Note Leucovorin is given with 5-FU because the reduced folate coenzyme is required in the thymidylate synthetase reaction. Lack of sufficient coenzyme reduces the effectiveness of the antipyrimidine.] 5-FU is also incorporated into RNA and low levels have been detected in DNA. 

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. 

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). 

B. Replacement of hydrogen Exchange of a hydrogen atom with any of the Class I isosteres often leads to antimetabolite activity. Particularly, replacement of H with the spatially similar F results in analogues that are readily utilized by the various enzymes and incorporated into macromolecules (e.g., 18a 29), except, when the F substitutes for an H which would undergo displacement in the enzymic reaction (cp. action of FUDR-P (18c) as inhibitor of thymidylate synthetase Section 2.3.). 

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. 

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. 

All susceptible fungi are capable of deaminating flucytosine to 5-fluorouracil, a potent antimetabolite that is used in cancer chemotherapy. Fluorouracil is metabolized fast to 5-fluorouracil-ribose monophosphate (5-FUMP) by the enzyme uracil phosphodbosyi transferase (UPRTase, also called uridine monophosphate pyrophosphorylase). As in mammalian cells, 5-FUMP then is either incorporated into RNA (via synthesis of 5-fluorouridine triphosphate) or metabolized to 5-fluoro-2 -5 deoxyuridine-5-monophos-phate (5-FdUMP), a potent inhibitor of thymidylate synthetase. DNA synthesis is impaired as the ultimate inhibition of this latter reaction. The selective action of flucytosine is due to the lack or low levels of cytosine deaminase in mammalian cells, which prevents metabolism to fluorouracil. 

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. 

That the folate analogues do not interfere directly with the action of thymidylate synthetase, or with the action of the H4-folate coenzyme, was... 

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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