2 -Deoxyuridine 5 -monophosphate dUMP

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. 

The foundation for further study of combination regimens with hydroxyurea and 5-FU is based on in vitro pharmacokinetics denoting modulation of 5-FU by depleting deoxyuridine monophosphate (dUMP), ametabolic product of 5-FU. In turn, this will allow increased binding of 5-FU to thymidylate synthase and augment the properties of 5-FU. 

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. 

The TS mediates the conversion of 2-deoxyuridine monophosphate (dUMP) into deoxythymidine monophosphate (dTMP). This enzymatic methylation reaction is a key step in the synthesis of DNA and involves a ternary complex between the substrate, the enzyme and the co-factor [methylene tetrahydrofolic acid (CH2FAH4)] (Fig. 24) [8,80,81], This transformation represents the sole de novo source of dTMP, a building block for DNA synthesis and repair [82]. 

The other major class of antimalarials are the folate synthesis antagonists. There is a considerable difference in the drug sensitivity and affinity of dihydrofolate reductase enzyme (DHFR) between humans and the Plasmodium parasite. The parasite can therefore be eliminated successfully without excessive toxic effects to the human host. DHFR inhibitors block the reaction that transforms deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) at the end of the pyrimidine-synthetic pathway. This reaction, a methylation, requires N °-methylene-tetrahydrofolate as a carbon carrier, which is oxidized to dihydrofolate. If the dihydrofolate cannot then be reduced back to tetrahydrofolate (THF), this essential step in DNA synthesis will come to a standstill. 

Tetrahydrofolate cofactors participate in one-carbon transfer reactions. As described above in the section on vitamin B12, one of these essential reactions produces the dTMP needed for DNA synthesis. In this reaction, the enzyme thymidylate synthase catalyzes the transfer of the one-carbon unit of N 5,N 10-methylenetetrahydrofolate to deoxyuridine monophosphate (dUMP) to form dTMP (Figure 33-2, reaction 2). Unlike all of the other enzymatic reactions that utilize folate cofactors, in this reaction the cofactor is oxidized to dihydrofolate, and for each mole of dTMP produced, one mole of tetrahydrofolate is consumed. In rapidly proliferating tissues, considerable amounts of tetrahydrofolate can be consumed in this reaction, and continued DNA synthesis requires continued regeneration of tetrahydrofolate by reduction of dihydrofolate, catalyzed by the enzyme dihydrofolate reductase. The tetrahydrofolate thus produced can then reform the cofactor N 5,N 10-methylenetetrahydrofolate by the action of serine transhydroxy- methylase and thus allow for the continued synthesis of dTMP. The combined catalytic activities of dTMP synthase, dihydrofolate reductase, and serine transhydroxymethylase are often referred to as the dTMP synthesis cycle. Enzymes in the dTMP cycle are the targets of two anticancer drugs methotrexate inhibits dihydrofolate reductase, and a metabolite of 5-fluorouracil inhibits thymidylate synthase (see Chapter 55 Cancer Chemotherapy). 

The folic acid-dependent conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) carried out by thymidylate synthase is an absolute requirement for DNA synthesis. An unusually high demand for uracil (ura) by certain tumor cells suggested that such an inhibitor of this process could have tumor cell selectivity. 5-Fluorouracil (fl5ura) (80), along with 5-fluorocytosine (fl5cyt) (81) and 5-fluoroorotic acid (fl5oro) (82), were synthesized by Heidelberger in 1957 as part of a... 

Deoxyribose Deoxyuridine Deoxyuridylic acid deoxyuridine monophosphate (dUMP) Deoxyuridine diphosphate (dUDP) Deoxyuridine triphosphate (dUTP)... 

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. 

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. 

Figure 7-16. The transfer of a one-carbon unit from serine to deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP). FH4 is oxidized to FH2 (dihydrofolate) in this reaction. FH2 is reduced to FH4 by dihydrofolate reductase. Hi indicate the steps at which the antimetabolites methotrexate and 5-fluo-rouracil (5-FU) act.

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... 

Several analytes are known to be indicative of folate metabolism. Plasma total homocysteine increases when there is a deficiency of 5-MTHF, such that the methylation of homocysteine to methionine is compromised. However, though plasma homocysteine is considered to be a sensitive functional indicator, it is not specific because its concentration can be influenced by deficiency of other vitamins (Bg and B12) involved in the metabolism of homocysteine. Similarly the methylation of DNA is dependent upon adequate 5-MTHF. A sensitive new method for the rapid detection of abnormal methylation patterns in global DNA patterns has been reported and may have promise as a functional marker, as may the measurement of the degree of uracil incorporation into DNA, 5,10-metliylene THF being required for die conversion of deoxyuridine monophosphate (dUMP) to dTMP by thymidylate synthetase. ... 

The best-known example of this type of inhibition is 5 -fluorouracil, a rather old cytostatic agent. Fluorouracil is first converted metabolically into the corresponding phosphodeoxyriboside. This, in turn, blocks DNA biosynthesis by inhibiting thy-midylate synthase, an enzyme which methylates deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), one of the four building blocks of DNA [77]. 

Of the two objectives (Section I) of investigations on the models of this cofactor, the one translating its functional capability of carbon transfers in the evolution of chemical synthesis has been amply illustrated. With respect to the second objective of providing support to the molecular mechanism of the enzymatic process in which this cofactor plays an essential role, Pandit chose to mimic in totality the thymidylate synthase (TS) catalyzed conversion of deoxyuridine monophosphate (dUMP, 129) to deoxythymine monophosphate (dTMP, 131) that is reported to proceed by transfer of a methylene group of an imidazolidine component of 1 to C-5 of dUMP 129, the nucleophilicity of which is enhanced by the addition of a thiol of apoenzyme at C-6, generating the methylene intermediate 130. The subsequent delivery of the C-6H of 1 as an hydride equivalent to the methylene carbon is followed by elimination of a thiol and formation of dTMP 131 along with 7,8-dihydrofolate 132. 

Colin Tuma is being treated with 5-fluorouracil (5-FU), a pyrimidine base similar to uracil and thymine. 5-FU inhibits the synthesis of the thymine nucleotides required for DNA replication. Thymine is normally produced by a reaction catalyzed by thymidylate synthase, an enzyme that converts deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). 5-FU is converted in the body to F-dUMP, which binds tightly to thymidylate synthase in a transition state complex and inhibits the reaction (recall that thymine is 5-methyl uracil). Thus, thymine nucleotides cannot be generated for DNA synthesis, and the rate of cell proliferation decreases. 

The one-carbon groups carried by FH4 are used for many biosynthetic reactions. For example, one-carbon units are transferred to the pyrimidine base of deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP), to the amino acid glycine to form serine, to precursors of the purine bases to produce carbons C2 and C8 of the purine ring, and to vitamin B12. 

The nucleotide deoxythymidine monophosphate (dTMP) is produced from deoxyuridine monophosphate (dUMP) by a reaction in which dUMP is methylated to form dTMP (Fig. 40.5). The source of carbon is N, N °-methylene FH4. Two hydrogen atoms from FH4 are used to reduce the donated carbon to the methyl level. Consequently, dihydrofolate (FH2) is produced. Reduction of FH2 by NADPH in a... 

Decreased methylation of deoxyuridine monophosphate (dUMP) to form deoxythymidine monophosphate (dTMP), a reaction that requires N, N °-methyl-ene tetrahydrofolate as a coenzyme (see Fig. 40.5), leads to an increase in the intracellular dUTP/dTTP ratio. This change causes a significant increase in the incorporation of uracil into DNA. Although much of this uracil can be removed by DNA repair enzymes, the lack of available dTTP blocks the step of DNA repair catalyzed by DNA polymerase. The result is fragmentation of DNA as well as blockade of normal DNA rephcation. 

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. 

Thymidine monophosphate (deoxythymidine monophosphate [dTMP]) is generated from deoxyuridine monophosphate (dUMP). The methyl group added to the ring is derived from the CIO methylene unit of tetrahydrofolate as described in Chapter 12 (Scheme 12.9), and reduction is brought about by nicotine adenine dinucleotide (NADH, NAD+). A representation of the process is provided in Scheme 14.12. 

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