Thymidylate synthetase

Thymidylate synthetase catalyzes the conversion of deoxyuridylate to deoxy-thymidylate in a folate-dependent reaction. This enzyme is a target for many agents used in chemotherapy of cancer and treatment of infectious diseases. 

Deoxythymidylate was separated from other reaction components by chromatography on a LiChrosorb RP-8 column (4.6 mm X 250 mm, 5 /Am). The mobile phase contained 2-propanol-85% phosphoric acid-triethylamine-water (3 3 10 984, v/v). The effluent was monitored at 254 nm. 

The enzyme assay contained in a total volume of 250 /xL 50 m M Tris-HCl buffer (pH 7.8), 50 mM 2-mercaptoethanol, 5 mil/ formaldehyde, and 1 mM methylenetetrahydrofolate. The reaction was initiated by adding 25 /xL of 1 mM deoxyuridylate. After 30 minutes at 37°C, the reaction was stopped by addition of 250 /xL of ice-cold 1 M perchloric acid. After 30 minutes in an ice bath, the reaction mixture was centrifuged and the resulting supernate was neutralized with 0.1 volume of 10 M KOH containing 1 M KH2PO4. The supernate obtained by centrifugation was injected onto the HPLC column. Formation of deoxythymidylate was linear with time up to 30 minutes, and with protein in the range of 80 to 720 /xg. 

The source of enzyme was the human malaria parasite Plasmodium falciparum, cultured in red blood cells. Intact parasites were obtained by lysis of the cells with saponin, and the enzyme extract was obtained by freezing and thawing. 

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With the aid of cytosine permease, flucytosine reaches the fungal cell where it is converted by cytosine deaminase into 5-fluorouracil [51-21-8]. Cytosine deaminase is not present in the host, which explains the low toxicity of 5-FC. 5-Fluorouracil is then phosphorylated and incorporated into RNA and may also be converted into 5-fluorodeoxyuridine monophosphate, which is a potent and specific inhibitor of thymidylate synthetase. As a result, no more thymidine nucleotides are formed, which in turn leads to a disturbance of the DNA-synthesis. These effects produce an inhibition of the protein synthesis and cell repHcation (1,23,24). 5-Fluorouracil caimot be used as an antimycotic. It is poorly absorbed by the fungus to begin with and is also toxic for mammalian cells. 

Wataya Y, DV Santi (1975) Thymidylate synthetase catalyzed dehalogenation of 5-bromo- and 5-iodo-2 -deoxyuridylate. Biochem Biophys Res Commun 61 818-823. 

The answer is b. (Hardman, p 1203.) Trifluridine inhibits viral activity in HSV types 1 and 2, CMV, vaccinia, and perhaps adenovirus. It acts as a viral DNA synthesis inhibitor by irreversibly blocking thymidylate synthetase. Trifluridine triphosphate is a competitive inhibitor of thymidine triphosphate accumulation into DNA It is used in the treatment of primary keratoconjunctivitis and recurrent epithelial keratitis caused by HSV 1 and 2. 

A second relevant enzyme to 5-FU metabolism is thymidylate synthetase (TYMS). If this enzyme is complexed with 5-FU metabolites along with 5,10-methylene-tetrahydrofolate, it cannot maintain a thymidine-5 -monophosphate pool required for DNA replication and repair. A tandem repeat polymorphism in the 5 -promoter region of the TYMS gene can increase enzyme expression (85,86). Tumors carrying the repeats have higher enzyme expression, resulting in lower response to chemotherapy compared to wild type (87). 

Szeto, D.W., Cheng, Y.C., Rosowsky, A., t al. (1979) Human thymidylate synthetase—III. Effects of methotrexate and folate analogs. Biochemical Pharmacology. 28, 2633-2637. 

Thymine derivatives - 5-[7V-(2-Amino-4-hydroxy-6-methyl-5-pyrimidinyl-propyl)-p-carboxyanilinomethyl] uracil (XXXIII) was synthesized for study as a possible intermediate in the enzymatic synthesis of thymidylate. It is active as an enzyme inhibitor against thymidylate synthetase isolated from E. coli [298]. Certain thymine derivatives containing a 2-thioimidazole moiety (XXXIV, R = alkyl) inhibit growth of Ehrlich ascites carcinoma (fluid form) in mice [299]. 

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. 

C. Deoxythymidylate (dTMP) is formed from 2 -deoxyuridylate (dUMP) in a one-carbon transfer by thymidylate synthetase (Figure 10-5). 

Figure 10-5. Conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) by thymidylate synthetase. The importance of folate coenzymes in this reaction is illustrated. NADPH + H provide the necessary reducing equivalents and serine is the source of one-carbon units present on N, N °-methylene tetrahydrofolate (THF).

Methotrexate is a folate analog that acts as a potent competitive inhibitor of dihydrofoiate reductase, causing a decreased supply of THF coenzymes needed by thymidylate synthetase. 

The thymine analog 5-fluorouracil (5-FU) is converted to 5-fluoro-dUMP, which acts as a suicide inhibitor of thymidylate synthetase. 

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. 

Flucytosine is an oral antifungal pro-drug. It has to be enzymatically deaminated by the fungi to the active metabolite, fluorouracil. Fluorouracil inhibits thymidylate synthetase and DNA synthesis. Its indications are treatment of cryptococcal meningitis and serious systemic candidiasis. Resistance develops rapidly, due to altered drug-permeability. For this reason Amphotericin B and flucytosine are often given in combination as they have synergistic effects. 

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. 

Raltitrexed is a folic acid analogue which inhibits thymidylate synthetase. Intracellularly formed raltitrexed polyglutamates are even stronger inhibitors of thymidylate synthetase than the parent compound. Similar to methotrexate polyglutamates... 

Trifluridine (Viroptic) is a fluorinated pyrimidine nucleoside that has in vitro activity against HSV-1 and HSV-2, vaccinia, and to a lesser extent, some adenoviruses. Activation of trifluridine requires its conversion to the 5 monophosphate form by cellular enzymes. Trifluridine monophosphate inhibits the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) by thymidylate synthetase. In addition, it competes with deoxythymidine triphosphate (dTTP) for incorporation by both viral and cellular DNA polymerases. Trifluridine-resistant mutants have been found to have alterations in thymidylate synthetase specificity. 

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. 

The carbon-donating cofactor for this reaction is N, N methylenetetrahydrofolate, which is converted to dihydrofolate. The reduced folate cofactor occupies an allosteric site on thymidylate synthetase, which allows for the covalent binding of 5-FdUMP to the active site of the enzyme. 

It is fluorinated analogue of pyrimidine, act by binding the enzyme thymidylate synthetase and preventing the production of basic component of DNA-th5miine. It... 

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