DTMP

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... 

All NRTIs, as exemplified for AZT (Fig. 7), act in a similar fashion following their uptake by the cells, they are phosphorylated successively to their 5 -monophosphate, 5 -diphosphate, and 5 -triphosphate form (De Clercq 2002). Unlike the first phosphorylation step in the metabolic pathway of the acyclic guanosine analogues (see above), which is carried out by a virus-encoded enzyme (thymidine kinase), the first as well as the subsequent phosphorylations of the 2, 3 -dideoxynucleosides are carried out by cellular enzymes, that is, a 2 -deoxynucleoside (e.g., dThd) kinase, a 2 -deoxynucleotide (e.g., dTMP) kinase, and a (2 -deoxy)nucleoside 5 -diphosphate (NDP) kinase. 

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

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... 

Deoxytetracyclines, 24 595, 596 Deoxythymidylic acid (dTMP), folic acid and, 25 801... 

Folic acid derivatives are important in the production of purines and deoxythy-midine monophosphate (dTMP, a pyrimidine) required for nucleic acid synthesis in... 

Thymidylate synthase from E. coli hydride transfer from H4folate to form the methyl group of dTMP... 

The nomenclature for the commonly found bases, nucleosides, and nucleotides is shown in Tables I-l-2a and -2b. Note that the deoxy part of the names deoxythymidine, dTMP, etc., is sometimes understood, and not expressly stated, because thymine is almost always found attached to deoxyribose. 

Thymine Deoxythymidme Deoxythymidylic acid Deoxythymidine monophosphate (dTMP) Deoxythymidine diphosphate (dTDP) Deoxythymidine triphosphate (dTTP)... 

The primary end product is uridine monophosphate (UMP). In the conversion of UMP to dTMP, three important enzymes are ribonucleotide reductase, thymidylate synthase, and dihy-drofoiate reductase. All three enzymes are targets of antineoplastic drugs and are summarized in Table I-18-1. 

Thymidylate synthase Methylates dUMP to dTMP Requires THF 5-Fluorouiadl (S phase)... 

Ribonucleotide reductase is required for the formation of the deoxyribonucleotides for DNA synthesis. Figure 1-18-2 shows its role in dTMP synthesis, and Figure 1-18-3 shows all four nucleotide substrates ... 

Fig. 14.1 Cellular pathway of methotrexate. ABCBl, ABCCl-4, ABC transporters ADA, adenosine deaminase ADP, adenosine diphosphate AICAR, aminoimidazole carboxamide ribonucleotide AMP, adenosine monophosphate ATIC, AICAR transformylase ATP, adenosine triphosphate SjlO-CH -THF, 5,10-methylene tetrahydrofolate 5-CHj-THF, 5-methyl tetrahydro-folate DHFR, dihydrofolate reductase dTMP, deoxythymidine monophosphate dUMP, deoxy-uridine monophosphate FAICAR, 10-formyl AICAR FH, dihydrofolate FPGS, folylpolyglutamyl synthase GGH, y-glutamyl hydrolase IMP, inosine monophosphate MTHFR, methylene tetrahydrofolate reductase MTR, methyl tetrahydrofolate reductase MTX-PG, methotrexate polyglutamate RFCl, reduced folate carrier 1 TYMS, thymidylate synthase. Italicized genes have been targets of pharmacogenetic analyses in studies published so far. (Reproduced from ref. 73 by permission of John Wiley and Sons Inc.)...

The pyrimidine nucleotides (predominantly UMP, CMP, and dTMP) are hydrolysed to their respective bases (uracil, cytosine, and thymine) by reactions similar to those for metabolism of purine nucleotides. The pathways of... 

The enzyme tetrahydrofolate reductase, which is essential for the synthesises deoxythymidine monophosphate (dTMP) from deoxyuridine monophosphate, a process essential for DNA synthesis. This enzyme catalyses formation of methylene tetrahydrofate (CH3-FH4) a necessary co-substrate for synthesis of d-TMP catalysed by thymidylate synthase (See Figure 20.12(a) and p. 477). 

Antimetabolites (inhibition of purine and pyrimidine nucleotide synthesis) Methotrexate Folic acid antagonist, inhibits tetrahydrofolate reductase and therefore dTMP synthesis 6-Mercaptopurine Interferes with purine synthesis 5-Fluorouracil Inhibits dTMP synthesis ... 

We first applied Tethering to thymidylate synthase (TS). This enzyme converts de-oxyuridine monophosphate (dUMP) to thymidine monophosphate (dTMP), an activity essential for DNA synthesis. The cancer drug 5-fluorouracil irreversibly inhibits TS, and a selective inhibitor of a non-human form of the enzyme could yield a new antibiotic or antifungal drug [23]. 

Rapidly dividing cells need an abundant supply of dTMP for DNA synthesis, and this creates a need for dihydrofolate reductase activity. Specific dihydrofolate reductase inhibitors have become especially useful as antibacterials, e.g. trimethoprim, and antimalarial drugs, e.g. pyrimethamine. 

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