Deoxyuridine metabolism

Fluorouracil (5-fluorouracil, 5-FU, Fig. 5) represents an early example of rational drag design in that it originated from the observation that tumor cells, especially from gut, incorporate radiolabeled uracil more efficiently into DNA than normal cells. 5-FU is a fluorinated pyrimidine analog that must be activated metabolically. In the cells 5-FU is converted to 5-fluoro-2>deoxyuridine-monophosphate (FdUMP). This metabolite inhibits thymidilate synthase which catalyses the conversion of uridylate (dUMP) to thymidilate (dTMP) whereby methylenetetrahydrofo-late plays the role of the carbon-donating cofactor. The reduced folate cofactor occupies an allosteric site of... 

Fluoro-2 -deoxyuridine has been extensively used in studies of the mechanism of action of thymidylate synthase, and 5-fluorouracil is an anticancer drug that has provided a lead to the development of others. The metabolism of 5-fluorouracil by the ascomycete fungus Nectria haematococca has been studied using F NMR (Parisot et al. 1991). a-Fluoro-P-alanine (2-fluoro-3-aminopropionate) was produced (Figure 10.27), while 5-fluorouridine-5 -mono-, di-, and triphosphate were found in acid extracts of the mycelia, and the 2 - and 3 -monophosphates were recovered from RNA. 

D. J. Sweeney, S. Barnes, R. B. Diasio, Formation of Conjugates of 2-Fluoro-/3-alanine and Bile Acids during the Metabolism of 5-Fluorouracil and 5-Fluoro-2-deoxyuridine in the Isolated Perfused Liver , Cancer Res. 1988, 48, 2010-2014. 

Dideoxyuridine (ddU) is an antiviral agent that proved ineffective at controlling human immunodeficiency virus type 1 (HIV-1) infection in human T-cells. This ineffectiveness was ascribed to a lack of substrate affinity of ddU for cellular nucleoside kinases, which prevent it from being metabolized to the active 5 -triphosphate. To overcome this problem, bis[(pivaloyloxy)methyl] 2, 3 -dideoxyuridine 5 -monophosphate (9.41) was prepared and shown to be a membrane-permeable prodrug of 2, 3 -di-deoxyuridine 5 -monophosphate (ddUMP, 9.42) [93]. Indeed, human T-cell lines exposed to 9.41 rapidly formed the mono-, di-, and triphosphate of ddU, and antiviral activity was observed. This example again documents... 

The metabolic fate of trifluridine (11.17), an antiviral agent closely related to 5-fluoro-2 -deoxyuridine, was comparable. The main metabolite in the urine of mice administered the drug was the free 5-trifluorothymidine, i. e., the product of C-N bond cleavage [39]. All other metabolites were products of further biotransformation of trifluorothymidine. 

H. Foth, J. Hellkamp, E. M. Kunellis, G. E Kahl, Pulmonary Elimination and Metabolism of 5-Fluoro-2 -deoxyuridine in Isolated Perfused Rat Lung and Lung Slices , Drag Metab. Dispos. 1990, 18, 1011 - 1017. 

TMPase acts to dephosphorylate both TMP and its precursor dUMP, forming a mixture of TdR and 2 -deoxyuridine (UdR). As a starting material for zidovudine synthesis, TdR must be essentially free of this impurity, which would pass through the manufacturing process to form a demethylated analogue of zidovudine. Separation of TdR and UdR requires difficult and costly downstream processing hence, the key to a commercial process is metabolic engineering to minimize biosynthetic UdR. 

Fig. 3. Metabolism of the fluoropyrimidines dTMP = deoxythymidine monophosphate, dUMP = deoxyuridine monophosphate, FdUDP = fluorodeoxyuridine diphosphate, FdUMP - fluoro-deoxyuridine monophosphate, FdUTP = fluorodeoxyuridine triphosphate, FU-DNA= fluorouracil-deoxyribonucleic acid, FUDP = fluorouracil diphosphate, FUMP = fluorouracil monophosphate, FU-RNA = fluorouracil-ribonucleic acid, FUTP = fluorouracil triphosphate.

Fluorouracil is normally given intravenously (Table 55-3) and has a short metabolic half-life on the order of 15 minutes. It is not administered by the oral route because its bioavailability is erratic due to the high levels of the breakdown enzyme dihydropyrimidine dehydrogenase present in the gut mucosa. Floxuridine (5-fluoro-2 -deoxyuridine, FUDR) has an action similar to that of fluorouracil, and it is only used for hepatic artery infusions. A cream incorporating fluorouracil is used topically for treating basal cell cancers of the skin. 

A metabolic pathway that has received considerable attention is the conversion of 2 -deoxyuridine 5 -monophosphate (dUMP, 6.60) to thymidine 5 -monophosphate (TMP, 6.61) (Scheme 6.13). Without an adequate supply of TMP, a cell or bacterium cannot create DNA for cell division. Therefore, blocking TMP synthesis is an attractive method for slowing the advancement of certain cancers and bacterial infections. Important molecules in the methylation of dUMP are the various folic acid derivatives folic acid (FA, 6.62), dihydrofolic acid (DHF, 6.63), tetrahydrofolic acid (THF, 6.64), and N5, A1 "-methylene tetrahydrofolic acid (MTHF, 6.65) (Figure 6.23). These structures... 

In humans, the mean maximum plasma concentration (Cmax) is 79 ng/mL and the time to achieve Cmax was 2.67 h after a single 3-h infusion of decitabine (1) at 15 mg/m2. At this dose, the volume of distribution at steady state is 148 mL/kg, and the total plasma clearance is 122 L/kg/m2. The terminal half-life is approximately 35 min as decitabine (1) is primarily metabolized in the liver by cytidine deaminase to yield noncytotoxic 5-aza-2 -deoxyuridine. Urinary excretion of unchanged decitabine (1) is low (0.01-0.9% of total dose).13... 

In the 1990s, it was found that tylophorine alkaloids inhibit several key targets for clinically important anticancer drugs, including the metabolic enzymes thymidylate synthase (TS) and dihydrofolate reductase [8, 94], TS catalyzes the reductive methylation of the substrate dUMP (2 -deoxyuridine 5 -monophosphate) to dTMP (2 -deoxythymidine 5 -monophosphate thymidylate) with concomitant conversion of the cofactor CH2THF (5,10-methylenetetrahydrofolate) to DHF (7,8-dihydrofolate) (see Equation 1). 

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]. 

Maghni K, Nicolescu OM, Martin JG. Suitability of cell metabolic colorimetric assays for assessment of CD4+ T cell proliferation comparison to 5-bromo-2-deoxyuridine (BrdU) ELISA. J Immunol Methods 1999 223 185-194. 

Capecitabine is a pyrimidine analog. It is an oral systemic prodrug that is enzymatically converted to 5-fluorouracil (5-FU). Healthy and tumor cells metabolize 5-FU to 5-fluoro-2-deoxyuridine monophosphate (FdUMP) and 5-flu-orouridine triphosphate (FUTP). These metabolites cause cell injury by two different mechanisms. First, they inhibit the formation of thymidine triphosphate, which is essential for the synthesis of DNA. Second, nuclear transcriptional enzymes can mistakenly incorporate FUTP during the synthesis of RNA. This metabolic error can interfere with RNA processing and protein synthesis. Capecitabine is indicated in the treatment of resistant metastatic breast cancer alone or in combination with docetaxel, and colorectal cancer. 

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. 

See also Figure 4.3, Figure 4.2, De Novo Pyrimidine Nucleotide Metabolism, Deoxyuridine Nucleotide Metabolism, Nucleotide Salvage Synthesis... 

See also Ribonucleotide Reductase and Deoxyribonucleotide Biosynthesis, Deoxyuridine Nucleotide Metabolism, Salvage Routes to Deoxyribonucleotide Synthesis, Nucleotide Analogs in Selection. 

See also Biosynthesis of Thymine Deoxyribonucleotides (from Chapter 22), Deoxyuridine Nucleotide Metabolism (from Chapter 22), Replication Complexes... 

This drug is metabolized to 5-deoxyuridine monophosphate (5-dUMP), which binds to thymidylate synthase, inhibiting the interaction between the enzyme and its major cofactor. Thus, synthesis of thymidine is inhibited, causing cell death. 

Fluorouracil (5-FU) undergoes metabolism to form 5-fluoro-2 -deoxyuridine 5 -phosphate (5-dUMP). This metabolite forms a covalently bound ternary complex with thymidylate synthase and its coenzyme A-methylenetetrahydrofolate. The synthesis of thymine nucleotides is blocked, and a thymineless death of cells results. The answer is (E). 

Other halogenated uridine derivatives have been reported to exhibit antiviral activity. Fluorodeoxyuridine has in vitro antiviral activity but is not used in clinical practice. Bromodeoxyuridine is used in subacute sclerosing panencephalitis, a deadly, virus-induced CNS disease. This agent appears to interfere with DNA synthesis in the same way as idoxuridine. The 5 -amino analogue of idoxuridine (5-iodo-5 -amino-2, 5 -dideoxyuridine) is a better antiviral agent than idoxuridine, and it is less toxic. It is metabolized in herpesvirus-infected cells only by thymidine kinase to di- and triphosphoramidates. These metabolites inhibit HSV-specific late RNA transcription, causing reduction of less infective abnormal viral proteins. 5-Bromo-2 -deoxyuridine has an action similar to that of other iodinated compounds. None of these compounds are commercially available in the United States. 

The metabolic pathway of fluorouracil is presented schematically in Figure 8. In man, the major biochemical effect of fluorouracil is the inhibition of DNA synthesis, since concentrations which inhibit DNA synthesis may still permit RNA synthesis. Fluorouracil is converted to fluorouridine and then to the mono-,di-, and triphosphates of fluorouridine. This is then incorporated into the fraudulant RNA. Fluorouridine monophosphate is also reduced to fluoro-2 -deoxyuridine monophosphate. There is no further metabolism to the di- and triphosphate nucleotides... 

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