Thymidylate synthesis, inhibitor

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. 

The stereospecific preparation of E- and Z-a-fluorovinylstannanes via the radical reaction of the corresponding a-fluorovinylsulfones with tributyltin hydride has been reported by McCarthy et al. These reagents undergo a variety of destannylation reactions including protolysis, deuterolysis, acylation, iodina-tion, electrophilic fluorination and the Stille coupling reaction [186-189] (Scheme 66). The last reaction has been employed successfully in the synthesis of a fluorinated thymidylate synthetase inhibitor [189] (Scheme 67). 

Another group of inhibitors prevents nucleotide biosynthesis indirectly by depleting the level of intracellular tetrahydrofolate derivatives. Sulfonamides are structural analogs of p-aminobenzoic acid (fig. 23.19), and they competitively inhibit the bacterial biosynthesis of folic acid at a step in which p-aminobenzoic acid is incorporated into folic acid. Sulfonamides are widely used in medicine because they inhibit growth of many bacteria. When cultures of susceptible bacteria are treated with sulfonamides, they accumulate 4-carboxamide-5-aminoimidazole in the medium, because of a lack of 10-formyltetrahydrofolate for the penultimate step in the pathway to IMP (see fig. 23.10). Methotrexate, and a number of related compounds inhibit the reduction of dihydrofolate to tetrahydrofolate, a reaction catalyzed by dihydrofolate reductase. These inhibitors are structural analogs of folic acid (see fig. 23.19) and bind at the catalytic site of dihydrofolate reductase, an enzyme catalyzing one of the steps in the cycle of reactions involved in thymidylate synthesis (see fig. 23.16). These inhibitors therefore prevent synthesis of thymidylate in replicating... 

A large number of compounds in category 3 act at different sites in the pathways for purine and pyrimidine biosynthesis. These compounds are very toxic for rapidly growing tumors and bacteria, making them useful in cancer chemotherapy and treatment of bacterial infections. 6-Mercaptopurine is a potent inhibitor of purine biosynthesis, and 5-fluorouracil inhibits thymidylate synthesis. Some compounds, such as hydroxyurea and sulfonamides, inhibit the synthesis of both purine and pyrimidine nucleotides. These are only a few of the many compounds useful in treating cancer and infectious diseases (see Chapter 10). 

Phenanthroindolizidine and phenanthroquinolizidine alkaloids involve a phenanthrene (Phe ] Phe Phe (angular)) fused with an indolizidine or quinolizidine, respectively. The phenanthroindolizidines tylophorine (phenanthrene G5N G4N ) and tylocrebrine (phenanthrene G5N C4N ) and the phenanthroquinolizidine crypto-pleurine (phenanthrene G5N C5N ) are toxic, cytotoxic protein synthesis inhibitors. The phenanthroindolizidines tylophorine and pergularinine are thymidylate synthase inhibitors. 

There are several others that inhibit DNA replication. Fagaronine and nitidine have ben mentioned previously as intercalating agents, and this intercalation evidently inhibits the action of DNA polymerase [334]. Several benzophenanthridines and protoberberines inhibit reverse transcriptase, which catalyzes the synthesis of DNA with an RNA template [356, 357]. Mimosine at the cellular level, rather than directly on the process, is an inhibitor of DNA synthesis because it chelates iron [358] and because it blocks the synthesis of thymidylate, an essential precursor of DNA [359]. Vincristine may also act as an inhibitor of thymidylate synthesis, perhaps because it alkylates an essential thiol group of an enzyme [360]. 

Looked at simply, dTMP is produced via Cs-methylation of deoxyuridine monophosphate (dUMP). The rate-limiting enzyme of the dTMP synthetic pathway is the sulfhydryl-containing thymidylate synthase, with 5,10-methylenetetrahydrofolate (5,10-methylene-THF) serving as the methyl-donating cofactor. All dTMP synthesis inhibitors will inhibit thymidylate synthase either directly or indirectly, and this will result in a thymineless death in actively dividing cells. Without dTMP and its deoxythmidine triphosphate metabolite, DNAwill fragment, and the cell will die. 

To date no drugs preferentially target leishmanial components of the pyrimidine biosynthetic pathway. This is likely due to similarities in enzymatic activities shared between Leishmania and humans. For example, acivicin and PALA inhibit both human and leishmanial pyrimidine biosynthesis. Similarly, classical inhibitors such as methotrexate, which target leishmanial DHFR-TS, an essential component of thymidylate synthesis, are poor anti-leishmanial drugs because they also inhibit the human enzyme. ... 

In conclusion, an inhibitor of thymidylate synthesis and uptake and thus of DNA synthesis has been found. It consists of two components, methotrexate and uridine. It inhibits DNA synthesis to the extent and in ways which lead to blockage of the next cell division. This would be expected if DNA replication itself or events coupled to progression in the S phase are required to take the cell on to division. Inhibition by methotrexate + uridine can be discontinued by addition of excess thymidine, or just by washing of the cells with fresh growth medium, with or without added folate. These agents shall be used as tools in attempts to control DNA replication independently of cell division in populations with temperature-synchronized cell division. 

Thus, the formation of thymidylate involves the formation of a carbon-carbon bond and a reduction 5,10-methyIene H4-folate is both donor of the one-carbon unit and reductant. It is apparent that a catalytic amount of H4-folate will serve in the cycle. This is consistent with the observation that in S. faecalis extracts the synthesis of thymidylate in the presence of excess H4-folate was unaffected by aminopterin, a potent inhibitor of tetrahydrofolate dehydrogenase when this reaction was conducted with catalytic amounts of H4-folate and excess NADPH, thymidylate synthesis was blocked by aminopterin (5). 

It should be emphasized that fluorodeoxyuridylate is a very potent inhibitor of the synthetase as a competitive inhibitor of deoxyuridylate in thymidylate synthesis by the enzyme from Ehrlich ascites tumor cells, the inhibition constant for fluorodeoxyuridylate is in the order of 10 M (8). 

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. 

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. 

We first applied Tethering to thymidylate synthase (TS). This enzyme converts de-oxyuridine monophosphate (dUMP) to thymidine monophosphate (dTMP), an activity essential for DNA synthesis. The cancer drug 5-fluorouracil irreversibly inhibits TS, and a selective inhibitor of a non-human form of the enzyme could yield a new antibiotic or antifungal drug [23]. 

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]. 

Dihydrofolate reductase acts as an auxiliary enzyme for thymidylate synthase. It is involved in the regeneration of the coenzyme N, N -methylene-THF, initially reducing DHF to THF with NADPH as the reductant (see p. 418). The folic acid analogue methotrexate, a frequently used cytostatic agent, is an extremely effective competitive inhibitor of dihydrofolate reductase. It leads to the depletion of N, N -methylene-THF in the cells and thus to cessation of DNA synthesis. 

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. 

The pyrimidine antagonists inhibit the biosynthesis of pyrimidine nucleotides or interfere with vital cellular functions, such as the synthesis or function of nucleic acids. The analogues of deoxycytidine and thymidine that are used are inhibitors of DNA synthesis while 5-fluorouracil (5-FU) an analogue of uracil, is an inhibitor of both RNA function and of the synthesis of thymidylate (see Fig. 2). PALA (N-phosphonoacetyl-L-aspartate), an inhibitor of as-... 

Other useful targets for pharmaceutical agents are thymidylate synthase and dihydrofolate reductase, enzymes that provide the only cellular pathway for thymine synthesis (Fig. 22-49). One inhibitor that acts on thymidylate synthase, fluorouracil, is an important chemotherapeutic agent. Fluorouracil itself is not the enzyme inhibitor. In the cell, salvage pathways convert it to the deoxynucleoside monophosphate FdUMP, which binds to and inactivates the enzyme. Inhibition by FdUMP (Fig. 22-50) is a classic example of mechanism-based enzyme inactivation. Another prominent chemotherapeutic agent, methotrexate, is an inhibitor of dihydrofolate reductase. This folate analog acts as a competitive inhibitor the enzyme binds methotrexate with about 100 times higher affinity than dihydrofolate. Aminopterin is a related compound that acts similarly. 

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