DUMP 2*-deoxyuridine

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). 

The de novo pathway to 2 -deoxythymidine monophosphate (dTMP) synthesis first requires the use of dUMP (2 -deoxyuridine-5 -monophosphate) from the metabolism of either UDP or CDP (cytidine diphosphate). The hydrolysis of dUTP (2 -deoxyuridine-5 -triphosphate) to dUMP and subsequent methylation at C-5 by the action of thymidylate synthase, using A, A i°-methylenetetrahydrofolate (THF) as the methyl donor, generate dTMP (Figure 6.54). The latter is subsequently phosphorylated to deoxy-thymidine triphosphate (dTTP) used in DNA synthesis and repair. 

The Ts used for the biosynthesis of DNA are synthesized from Us by thymidylate synthase, an enzyme that requires N, lV -methylene-THF as a coenzyme. The actual substrate is dUMP (2 -deoxyuridine 5 -monophosphate) and the product is dTMP (2 -deoxy thymidine 5 -monophosphate). 

Fig. 24.4 Thymidylate synthase (TS) biochemical pathway. dUMP-deoxyuridine monophosphate, dTMP-deoxythymine monophosphate, dTTP-deoxythymine triphosphate.

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.

Fig. 2. Target enzymes for methotrexate and 5FU. 5-FU = 5-Fluorouracil THF = tetrahydrofolic acid DHF = dihydrofolic acid dUMP = deoxyuridine-monophosphate dTMP = deoxythymidine-monophosphate.

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

Fig. 6 Methyl trap hypothesis 5,10-Methylenetetrahydrofolate is reduced to 5-methyltetiahy-drofolate in an irreversible reaction. When vitamin Bn is deficient, methyl groups are trapped as 5-methyltetrahydrofolate, resulting in decreased substrates for DNA synthesis and neural lipid methylation. MTHFR, methylenetetrahydrofolate reductase DHFR, dihydrofolate reductase MS, Methionine synthase TS, thymidylate synthase SAM, S-adenosyl-methionine dUMP, deoxyuridine 5 -monophosphate dTTP, deoxythymidine 5 -monophosphate...

Fig. 2.1. Examples from The Energy Hall of Fame. These molecules not only deliver energy, but transfer special groups in the process. Acyl, RCO— ADP, adenosine diphosphate ATP, adenosine triphosphate dUMP deoxyuridine monophosphate FAD, flavin adenine dinucleotide GTP, guanosine triphosphate NADH, nicotinamide adenine dinucleotide NADP, nicotinamide adenine dinucleotide phosphate P, phosphate TMP, thymidine monophosphate UDP, uridine diphosphate UTP, uridine triphosphate.

FIGURE 48-2 Action of flucytosine in fungi. 5-Flucytosine is transported by cytosine permease into the fungal cell, where it is deaminated to 5-fluorouracil (5-FU). The 5-FU is then converted to 5-fluorouracil-ribose monophosphate (5-FUMP) and then is either converted to 5-fluorouridine triphosphate (5-FUTP) and incorporated into RNA or converted by ribonucleotide reductase to 5-fluoro-2 -deoxyuridine-5 -monophosphate (5-FdUMP), which is a potent inhibitor of thymidylate synthase. 5-FUDP, 5-fluorouridine-5 -diphosphate dUMP, deoxyuridine-5 -monophosphate dTMP, deoxyuridine-5 -monophosphate UPRTase, uracil phosphoribosyl transferase. 

FIGURE 51—4 Sites of action of methotrexate and its pofyglutamates. AICAR, aminoimidazole carboxamide TMR thymidine monophosphate dUMP, deoxyuridine monophosphate FH Glu, dihydrofolate polyglutamate FH Glu, tetrahydrofolate polyglutamate GAR, glycinamide ribonucleotide IMP, inosine monophosphate PRPP, 5-phosphoribosyl-1 -pyrophosphate. 

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... 

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. 

In 1995, Horie et al. described a polymorphic tandem repeat found in the 5 -un-translated region of the thymidylate synthase gene [70]. Thymidylate synthase (TS TYMS) catalyzes the intracellular transfer of a methyl group to deoxyuridine-5-monophosphate (dUMP) to form deoxythymidine-5-monophosphate (dTMP), which is anabolized in cells to the triphosphate (dTTP). This pathway is the only de- novo source of thymidine, an essential precursor for DNA synthesis and repair. The methyl donor for this reaction is the folate cofactor 5,10-methylenetetrahydro-folate (CH2-THF) (Figure 24.4). 

Ribonucleotide reductase works on ribo-A, -U, -G, -C diphosphates to give the deoxynucleotide. The deoxyuridine, which is useless for RNA synthesis, is converted to deoxythymidine by the enzyme thymidylate synthase, which uses methylene tetrahydrofolate as a one-carbon donor. The odd thing here is that ribonucleotide reductase uses the UDP as a substrate to give the dUDP. This must then be hydrolyzed to the dUMP before thymidylate synthase will use it to make dTMP. Then the dTMP has to be kinased (phosphorylated) up to dTTP before DNA can be made. 

Thymidylate Synthase (TS) is a 70 kDa dimeric protein that catalyzes the conversion of 2 -deoxyuridine 5 -monophosphate (dUMP) into 2 -deoxythymidine 5 -monophosphate (dTMP) using 5,10-methylene-5,6,7,8-tetrahydrofolate as cofactor. Inhibitors of TS represent potential... 

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. 

Thymidylate synthase [EC 2.1.1.45] reductively methylates 2 -deoxyuridine-5 -monophosphate to form 2 -deoxythymidine-5 -monophosphate in the following folate-dependent reaction dUMP + A, A -methylene-tetrahydrofolate dTMP + dihydrofolate. 

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. 

Another route to dUMP is the reduction of UDP to dUDP, followed by phosphorylation of dUDP to dUTP (or direct reduction of UTP to dUTP in some microorganisms). The dUTP is then hydrolyzed to dUMP. This circuitous route to dUMP is dictated by two considerations. First, the ribonucleotide reductase in most cells acts only on ribonu-cleoside diphosphates, probably because this permits better regulation of its activity. Second, cells contain a highly active deoxyuridine triphosphate diphosphohydrolase (dUT-Pase). It prevents the incorporation of dUTP into DNA by keeping intracellular levels of dUTP low by means of the reaction... 

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). 

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... 

Many drugs have been developed to intervene in the synthesis of TMP (Figure 6.24). For example, methotrexate (6.66) inhibits DHFR by blocking the binding site of DHF. Another antimetabolite, 5-fluorouracil (5-FU, 6.67), is converted in the body to 5-fluoro-2 -deoxyuridine 5 -monophosphate (F-dUMP, 6.68), a potent inhibitor of TS.26... 

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