5-Fluorodeoxyuridine-5 monophosphate

With the aid of cytosine permease, flucytosine reaches the fungal cell where it is converted by cytosine deaminase into 5-fluorouracil [51-21-8]. Cytosine deaminase is not present in the host, which explains the low toxicity of 5-FC. 5-Fluorouracil is then phosphorylated and incorporated into RNA and may also be converted into 5-fluorodeoxyuridine monophosphate, which is a potent and specific inhibitor of thymidylate synthetase. As a result, no more thymidine nucleotides are formed, which in turn leads to a disturbance of the DNA-synthesis. These effects produce an inhibition of the protein synthesis and cell repHcation (1,23,24). 5-Fluorouracil caimot be used as an antimycotic. It is poorly absorbed by the fungus to begin with and is also toxic for mammalian cells. 

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]. 

Thymidylate synthase (E.C. 2.1.1.45) is the enzyme that methylates UMP to thymidine, using methylene tetrahydrofolate as the carbon carrier. The enzyme can be inhibited directly by analogues of uracil such as 5-fluorouracil (8.34, 5-FU). The antimetabolite must be in the 5-fluorodeoxyuridine monophosphate (FdUMP) form to become active, and the capability of cells to achieve this transformation is a major determinant of their sensitivity to such drugs. 

Flucytosine is taken up by fungal cells via the enzyme cytosine permease. It is converted intracellularly first to 5-FU and then to 5-fluorodeoxyuridine monophosphate (FdUMP) and fluorouridine triphosphate (FUTP), which inhibit DNA and RNA synthesis, respectively (Figure 48-1). Human cells are unable to convert the parent drug to its active metabolites, resulting in selective toxicity. 

FU also forms 5-fluorodeoxyuridine monophosphate (5-Fd-UMP), which inhibits thymidy-late synthase ->4- thymine. 

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. 

As with thiopurines, 5-FU must first be activated by a series of steps to the active cytotoxic agent, 5-fluorodeoxyuridine monophosphate (FdUMP) (Fig. 4-17). Tumor cells deficient in any of the enzymes needed for these conversions (e.g., phosphoribosyltransferase) are likely to be resistant to 5-FU. Since the early discovery that thymidylate synthetase is very sensitive to 5-FU (actually FdUMP), the mechanism of action was believed to be impairment of the conversion of dUMP to dTMP (Fig. 4-13). A more detailed mechanism is now understood to be in the first step, thymidylate synthetase reacts covalently with C-5 of FdUMP (as if it were the normal substrate, dUMP) via nucleophilic attack of its reactive site s SH group. Methylene-FH4 cofactor then adds to C-5 of this initial complex. 

Fluorodeoxyuridine monophosphate (FdUMP) is a molecule that is a mechanism-based inhibitor. Irreversible binding of FdUMP to thymidylate synthase occurs only in the presence of 5,10-methyl enetetrahydrofolate. Crystallographic analysis of thymidylate synthase with dUMP and an analog of 5,10-methylenetetrahydrofolate (that could not be acted on by the enzyme) reveals that thymidylate synthase normally makes a transient covalent bond in the process of catalysis of the reaction. Apparently FdUMP s structure traps the enzyme-substrate covalent bond and prevents it from breaking down. 

Flucytosine (5-FC) is an analogue of the natural pyrimidine cytosine that is converted to 5-fluorouracil (5-FU) in susceptible fungi. Formation of 5-FU is essential to the antimycotic effect of 5-FC 5-FU acts as a pyrimidine antimetabolite and is phosphorylated to the cytotoxic agent 5-fluorodeoxyuridine monophosphate. All of these facts are commonly emphasized in a medicinal chemistry sequence, and readers probably are aware that 5-FU is a chemotherapeutic agent that causes myelosuppression as its major toxicity. Therefore, it should not be surprising that the same side effect can be seen in patients receiving 5-FC. 

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.

Currently, the compound 217 is the only available antimetabolite drug having antifungal activity. It inhibits fungal protein synthesis by replacing 187 with 177 in fungal RNA. 217 also inhibits thymidylate synthetase via 5-fluorodeoxyuridine monophosphate and thus interferes with fungal DNA synthesis. 

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