FdUMP

Fig. 14.3 5 -FU catabolism, anabolism and mechanism of action. 5-FUH2, 5-fluoro-5,6-dihydrouracil 5-FdUMP, 5-fluorodeoxyuridine monophosphate TP, thymidine phosphorylase TK, thymidine kinase TS, thymidylate synthase CH2THF, 5,10-methylenetetrahydrofolate.

TK), 5-FU is activated to 5-fluorodeoxyuridine monophosphate (5-FdUMP). Potent inhibition of thymidylate synthase (TS) by 5-FdUMP is considered critical for 5-FU cytotoxicity. TS catalyzes the rate-limiting step of DNA synthesis, such as the conversion of dUMP into dTMP. Optimal TS function requires the formation of a covalent ternary complex consisting of TS, the folate cofactor 5,10-methylenetetra-hydrofolate (CH2THF), and 5-FdUMP. Inadequate cellular levels of 5,10-methyle-netetrahydrofolate reduce the stability of the ternary complex and consequently the inhibition of TS by 5-FdUMP. For this reason, 5-FU is administered in association with folinic acid, a precursor of 5,10-methylenetetrahydrofolate [40]. 

Fig. 14.10 Folate metabolism and role of MTHFR. Genetically reduced MTHFR activity affects the distribution between folate species required for protein and DNA synthesis. Higher availabil ity of 5,10-methylenetetrahydrofolate (CH2THF) potentiates the TS inhibition by 5-FdUMP, the active metabolite of 5-FU. Hey, homocysteine Met, methionine CH3HF, 5-methyltetrahydrofolate TS, thymidylate synthase 5-FdUMP, fluorodeoxyuridine monophosphate.

The carbon-donating cofactor for this reaction is N, N methylenetetrahydrofolate, which is converted to dihydrofolate. The reduced folate cofactor occupies an allosteric site on thymidylate synthetase, which allows for the covalent binding of 5-FdUMP to the active site of the enzyme. 

Fluorouracil and fluorodeoxyuridine (floxuridine) inhibit pyrimidine nucleotide biosynthesis and interfere with the synthesis and actions of nucleic acids. To exert its effect, fluorouracil (5-FU) must first be converted to nucleotide derivatives such as 5-fluorodeoxyuridylate (5-FdUMP). Similarly, floxuridine (FUdR) is also converted to FdUMP by the following reactions ... 

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. 

The most widely known chemotherapeutic agent directed against TS is 5-fluorour-acil (5-FU). 5-FU was first used clinically almost 50 years ago, yet still remains a mainstay for the treatment of carcinoma of the breast and gastrointestinal tract. In cells, 5-FU is metabolized to 5-FdUMP, which forms a stable inhibitory ternary complex with the co-substrate N5N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4-folate) and TS. In this complex, a covalent bond links the thiol of cysteine 195 of human TS to C6 of deoxyuracil monophosphate (dUMP) and the methylene carbon of the co-substrate is joined to C5 of the nucleotide [55]. The fluorine at C5, unlike the proton, cannot... 

Cell cycle-selective agents comprise antimetabolites (of DNA synthesis) and inhibitors of mitosis (cell division see below). A widely used example of an antimetabolite is 5-fiuorouracil (5-FU Figure 13.11). Before this drug actually does something interesting, it needs to be converted to the nucleotide analog 5-fluoro-deoxyuridinemonophos-phate (5-FdUMP), which occurs in the same way as with normal uracil. 

Figure 13.12. The thymidylate synthase reaction, and its inhibition by 5-fluoro-deoxyuridinemonophosphate (5-FdUMP). a Overview of the reaction, b The catalytic mechanism. The enzyme forms an intermediate in which it is covalently linked to both the substrate (UMP) and, via the latter, to the coenzyme (bottom center). Resolution of this intermediate does not happen with 5-FdUMP because it requires abstractionof the hydrogen normally found in position 5 of the uracil ring.

Flucytosine (FQ and 5-fluorouraci (5-FU) are bioactivated to 5-fluorodeoxyuridine monophosphate (5-FdUMP), which inhibits thymidylate synthetase —> "thymineless death" of fungal cells (FC) or neoplastic cells (5-FU). 

Answer C. All of the drugs listed are antimetabolites used in cancer chemotherapy or as immunosuppressants. The 5-fluorouracil is bioactivated to 5-fluorodeoxyuridine monophosphate (5-FdUMP), a substrate for and inhibitor of thymidylate synthase. When used in drug regimens for treatment of cancer, 5-FU causes thymineless death of cells. 

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. 

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. 

Mechanisms Fluorouracil is biotransformed to 5-fluoro-2 -deoxyuridine-5 -monophos-phate (5-FdUMP). which inhibits thymidylate synthase and leads to thymineless death of cells. Tumor cell resistance mechanisms include decreased activation of 5-FU, increased thymidylate S)mthase activity, and reduced drug sensitivity of this enzyme. 

Fig. 40.11. Flucytosine, a pro-drug, is converted by fungal cytosine deaminase to 5-fluorouracil (5-FU). This reaction does not occur in mammalian cells. A further transformation of 5-FU to the actual cytotoxic agent, 5-fluorodeoxyuridine monophosphate (5-FdUMP), also occurs.

The synthetic pirimidine, 5-fluouracil (5-FU), is used in the treatment of a variety of epithelial tumors including, colorectal, breast cancer, ovarian, head, and neck cancers. 5-FU enters tumor cells by a facilitated nucleobase transporter and is converted into active metabolites (5 -fluoro-2 -deoxyuridine) by a complex metabolic pathway that interferes with both DNA and RNA syntheses. Of particular interest is the conversion of 5-FU to 5 -fluoro-2 -deoxyur-idine (5-FUdR) and subsequent phosphorylation by thymidine kinase to the active metabolite 5 -fluoro-2 -DUMP (5-FdUMP). Unfortunately, the response rate of patients receiving 5-FU-based chemotherapy is relatively poor, varying between 10 and 30%. To further evaluate, 5-FU labeled with F (5- FU) has been used in PET studies to understand the factors behind this strong interindividual variability. As 5- FU is biochemically identical to 5-FU, PET has been used for quantitative pharmacokinetic measurements of 5- FU in human and animal models. 

Fig. 14. Effect of 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FdUMP) on the synthesis of RNA and DNA. 5-FU is incorporated into RNA whereas 5-FdUMP inhibits thymidilate synthetase competitively and blocks DNA synthesis. The conversion of thymidine (dTr) into thymidilate (dTMP) by means of thymidine kinase is only a subsidary routine to thymidilate which can become important, however, under certain conditions such as in developing pollen (page 185). U = uridine triphosphate, Tr = thymidine, TMP = thymidine monophosphate = thymidilate, TDP = thymidine diphosphate, TTP = thymidine triphosphate, 5-F = 5-fluoro, d = deoxy, 1 = thymidine kinase, 2 = thymidilate synthetase.

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