Deoxyuridine monophosphate

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... 

An interesting dinically useful prodrug is 5-fluorouracil, which is converted in vivo to 5-fluoro-2 -deoxyuridine 5 -monophosphate, a potent irreversible inactivator of thymidylate synthase It is sometimes charaderized as a dead end inactivator rather than a suicide substrate since no electrophile is unmasked during attempted catalytic turnover. Rathei since a fluorine atom replaces the proton found on the normal substrate enzyme-catalyzed deprotonation at the 5 -position of uracil cannot occur. The enzyme-inactivator covalent addud (analogous to the normal enzyme-substrate covalent intermediate) therefore cannot break down and has reached a dead end (R. R. Rando, Mechanism-Based Enzyme Inadivators , Pharm. Rev. 1984,36,111-142). 

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. 

Fig. lA. Anabolic and catabolic pathways of 5-FU. DPD dihydropyrimidine dehydrogenase, DP di-hydropyrimidinase, pUP beta-ureidopropionase, UP uridine phosphorylase, OPRT orotate phospho-ribosyl transferase, UK uridine kinase, TP thymidine phosphorylase, TK thymidine kinase, RNR ribonucleotide reductase. The three active metabolites (shown in rectangles) are FdUMP (5-fluoro-2 -deoxyuridine 5 -monophosphate) inhibiting TS (thymidylate synthase), and FUTP (5-fluorouridine 5 -triphosphate) and FdUTP (5-fluoro 2 -deoxyuridine 5 -triphosphate) interfering with RNA and DNA, respectively. 

Fluorouracil (5-FU) is inactive in its parent form and requires activation via a complex series of enzymatic reactions to ribosyl and deoxyribosyl nucleotide metabolites. One of these metabolites, 5-fluoro-2 -deoxyuridine-5 -monophosphate (FdUMP), forms a covalently ternary complex with the enzyme thymidylate synthase and the reduced folate 5,10-methylenetetrahydrofolate, a reaction critical for the de novo synthesis of thymidylate. This results in inhibition of DNA synthesis through "thymineless death." 5-FU is converted to 5-fluorouridine-5 -triphosphate (FUTP), which is then incorporated into RNA, where it interferes with RNA processing and mRNA translation. 5-FU is also converted to 5-fluorodeoxyuridine-5 -triphosphate (FdUTP), which can be incorporated into cellular DNA, resulting in inhibition of DNA synthesis and function. Thus, the cytotoxicity of 5-FU is thought to be the result of combined effects on both DNA- and RNA-mediated events. 

In 1967 Santi prepared (313) as an analogue of (305), a proposed intermediate in the TS-mediated conversion of 2 -deoxyuridine-5 -monophosphate... 

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... 

FdUMP[10], a 10-mer of 5-fluoro-2 -deoxyuridine 5 -monophosphate (FdUMP), was found to be 338-fold more potent than 5-fluorouracil at inhibiting cell proliferation in the National Cancer Institute 60 cell-line screen. The 5-fluoro-2 -deoxyuridine derivative 75 (Figure 11) showed a Pearson correlation coefficient of 0.781 with regard to the other compounds with the highest correlation which ranked 237th among all the compounds/extracts deposited in the DTP database <2005MI4844>. 

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). 

Fig. 5. (continued) Thymidylate synthase with bound 5-hydroxymethylene-6-hydrofolic acid and 5-fluoro-2 -deoxyuridine-5 -monophosphate (103) (b), dihydrofolate reductase with bound folate (104) (c). 

Thymidylate Synthase Thymidylate synthase catalyzes the reductive methylation of 2 -deoxyuridine-5 -monophosphate (dUMP) to 2 -deoxythymidine-5 -monophosphate (dTMP). The cofactor N, N -methylene-5,6,7,8-tetrahydrofolate (CH2H4fo-late) serves as a donor of both methylene and hydride [47]. We recently studied the hydride transfer step using competitive H/T and D/T KIEs [48], and the observed KIEs were used to calculate intrinsic KIEs. The observed and intrinsic KIEs were used to calculate the commitment to catalysis (Eq. (12.2)), and it was found that between 20 and 30 °C the hydride transfer is fully rate determining, while at elevated and reduced temperatures the commitment increases. At 20 °C, competitive KIE experiments resulted in 1° H/T KIE on kcat/kM and D/T KIE on k at/Ku KIEs of 6.91 + 0.05 and 1.78 + 0.02, respectively. The Swain-Schaad exponent for these KIEs is 3.35 + 0.07, suggesting that the hydride transfer is rate determining... 

Susceptible fungi deaminate flucytosine to 5-fluorouracil, a potent antimetabolite (Figure 48—2). Fluorouracil is metabolized to 5-fluorouracil-ribose monophosphate (5-FUMP) by the enzyme uracil phosphoribosyl transferase (UPRTase). 5-FUMP then is either incorporated into RNA (via synthesis of 5-fluorouridine triphosphate) or metabolized to 5-fluoro-2 -deoxyuridine-5 -monophosphate (5-FdUMP), a potent inhibitor of thymidylate synthetase that thus inhibits DNA synthesis. The selective action of flucytosine is due to the relatively low level of cytosine deaminase in mammalian cells, which prevents metabolism to fluorouracil. 

Mukherjee KL, Heidelberger C (1962) Studies of fluorinated pyrimidines. XV. Inhibition of the incorporation of formate-C14 into DNA thymine of ehrlich ascites carcinoma cells by 5-fluoro-2 -deoxyuridine-5 -monophosphate and related compounds. Cancer Res 22 815-822... 

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. 

Fluoro-2 -deoxyuridine 5 -monophosphate has been considered to be the cytotoxic anabolite of 5-fluorouracil, but the phosphates of 5-fluorouridine are also anabolites and are incorporated into RNA. The importance of this incorporation to the action of 5-fluorouracil is under... 

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). 

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