DTMP inhibitors

DHF to 5,10-methylenetetrahydrofolate for use by thymidylate synthase in de novo synthesis of dTMP. Inhibitors of dihydrofolate reductase, such as methotrexate and trimethoprim, ultimately inhibit production of dTTP. 

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... 

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. 

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. 

Correct answer = A. Methotrexate interferes with folate metabolism by acting as a competitive inhibitor of the enzyme dihydrofolate reductase. This starves cells for tetrahydrofolate, and makes them unable to synthesize purines and dTMP This is especially toxic to rapidly-growing cancer cells. Overproduction of dihydrofolate reductase, usually caused by amplification of its gene, can overcome the inhibition of the enzyme at the methotrexate concentrations used for chemotherapy, and can result in resistance of the tumor to treatment by this drug. 

Nucleotidase present in 48,000 X Q supernatant fractions of rat and guinea pig skeletal muscle extracts has been examined briefly (7-4). 5 -UMP seems to be the preferred substrate. The enzyme from fish skeletal muscle has also been studied (75). This enzyme hydrolyzes all ribo-and deoxyribonucleoside 5 -phosphates (except dCMP and dTMP) with preference for 5 -IMP and 5 -UMP. The enzyme is strongly activated by Mn2+ Mg2+ is a less powerful activator, and Zn2+ and EDTA are inhibitors. This enzyme thus appears similar to the soluble activity from mammalian liver (88, 86). 5 -Nucleotidase in mammary gland hydrolyzes all 5 -ribonucleotides and shows a decrease from pregnancy to early lactation (76). Rats injected with glucagon show increased 5 -nucleotidase in pancreatic islet tissue (77). The enzyme in mouse kidney has been examined histochemically and electrophoretically and found to exist as isozymes (75). Electrophoretic techniques have also provided evidence that the enzyme exists as isozymes in many other tissues of the mouse such as liver, spleen, intestine, testes, and heart (79). 

The folic acid-dependent conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) carried out by thymidylate synthase is an absolute requirement for DNA synthesis. An unusually high demand for uracil (ura) by certain tumor cells suggested that such an inhibitor of this process could have tumor cell selectivity. 5-Fluorouracil (fl5ura) (80), along with 5-fluorocytosine (fl5cyt) (81) and 5-fluoroorotic acid (fl5oro) (82), were synthesized by Heidelberger in 1957 as part of a... 

Several explanations have been repotted to account for hictciial resistance to the dihydrofolale reducia.se inhibitor trimethoprim, including intrinsic resistance at the cn/.ymaliu level, the development of the ability by the bacteria to use the host s S-dcoxythymidinc monophosphate (dTMP). and R-factor conjugation. 

Purines, pyrimidines and their nucleosides and nucleoside triphosphates are synthesized in the cytoplasm. At this stage the antifolate drugs (sulphonamides and dihydrofolate reductase inhibitors) act by interfering with the synthesis and recycling of the co-factor dihydrofolic acid (DHF). Thymidylic acid (2-deoxy-thymidine monophosphate, dTMP) is an essential nucleotide precursor of DNA synthesis. It is produced by the enzyme thymidylate synthetase by transfer of a methyl group from tetrahydrofolic acid (THF) to the uracil base on uridylic acid (2-deoxyuridine monophosphate, dUMP) (Fig. 12.5). THF is converted to DHF in this process and must be reverted to THF by the enzyme dihydrofolate reductase (DHFR) before... 

The dihydrofolate reductase inhibitor, methotrexate (Fig. 8.47), was developed as an anticancer drug, whose inhibition of formation of folic acid coenzymes would block purine synthesis. In other words, it was designed to induce a folic acid deficiency. Notice in Figs. 8.50 and 8.51 that formation of dTMP, FGAR, and AICAR also causes the oxidation of tetra-hydrofolate to dihydrofolate. The latter must be reduced by dihydrofolate reductase to tet-rahydrofolate before active coenzyme can form again. Thus, not only does methotrexate inhibit the initial formation of the tetrahydro-folate moiety, it blocks regeneration of the coenzyme form. 

The answer is c. (Murray, pp 48-73. Scriver, pp 4571-4636. Sack, pp 3-17. Wilson, pp 287-317.) Since rapidly multiplying cancer cells are dependent upon the synthesis of deoxythymidilate (dTMP) from deoxy-uridylate (dUMP), a prime target in cancer therapy has been inhibition of dTMP synthesis. The anticancer drug fluorouracil is converted in vivo to fluorodeoxyuridylate (FdUMP), which is an analogue of dlJMP FdUMP irreversibly forms a covalent complex with the enzyme thymidylate synthase and its substrate N5,N10-methylene-tetrahydrofolate. This is a case of suicide inhibition, where an enzyme actually participates in the change of a substrate into a covalently linked inhibitor that irreversibly inhibits its catalytic activity. 

The unique aspects of thymine synthesis allow the design of antimetabolites that may have greater specificity in this process. Aminopterin and methotrexate have been used with some success. These are inhibitors of tetrahydrofolate formation where, at the correct dosage, the conversion of dUMP to dTMP appears to be more sensitive than many other reactions and is preferentially inhibited. This inhibits DNA formation and cell proliferation. Other... 

A key enzyme in pyrimidine biosynthesis, thymidylate synthetase, catalyzes the reductive methylation of 2 -deoxyuridylate (dUMP) to thymidylate (dTMP) with the concomitant conversion of 5,10-methylene-H4-folate to 7,8-dihydrofolate. The cofactor serves both as a 1-carbon carrier and a reductant. There is substantive evidence based on direct and indirect studies with the inhibitor 5 -fluoro-2 -deoxyuridylate, which will not be reviewed here, that the dUMP is covalently bound to the enzyme, possibly through a thiol group [59]. The intermediate ternary complex is hypothesized to have the following structure (10), where the CH2 unit is attached through the N-5 of H4-folate. The ring opening of the 5,10-methylene-H4-folate is... 

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. 

Looked at simply, dTMP is produced via Cs-methylation of deoxyuridine monophosphate (dUMP). The rate-limiting enzyme of the dTMP synthetic pathway is the sulfhydryl-containing thymidylate synthase, with 5,10-methylenetetrahydrofolate (5,10-methylene-THF) serving as the methyl-donating cofactor. All dTMP synthesis inhibitors will inhibit thymidylate synthase either directly or indirectly, and this will result in a thymineless death in actively dividing cells. Without dTMP and its deoxythmidine triphosphate metabolite, DNAwill fragment, and the cell will die. 

Methotrexate and aminopterin, a similar compound, are analogs of dihydro folate (DHF) and inhibitors of dihydrofolate reductase, an enzyme that converts DHF to tetrahydro-folate (THF). The thymidylate synthase reaction converts N, N °-methylenetetrahydro-folate to DHF in the process of methylating dUMP to form dTMP In the presence of one of the inhibitors, this reaction functions as a sink that reduces the THF level of the cell by converting THF to DHF. Since THF derivatives are substrates in two reactions of purine metabolism and one of pyrimidine metabolism, both pathways are affected by the inhibitor. 

The primary mechanism of action of FU may be inhibition of thymidylate synthase (dUMP —> dTMP) by 5-fluorodeoxy UMP (FdUMP). FdUMP binds to thymidylate synthase with the other substrate 5,10-CHj-THF forming a tight ternary complex. A cysteine residue at the active site of the enzyme attacks the natural substrate, dUMP, forming a transient covalent bond. In FdUMP this bond caimot be broken due to the presence of the 5-fluoro group on the pyrimidine ring. Thymidylate synthase is thus permanently inactivated by this suicide inhibitor, with a consequent depletion of dTMP and thus dTTP in the cells. In addition,... 

Cell proliferation may be decreased by using various structural analogues to inhibit nucleic acid synthesis. Amethopterin (methotrexate) closely resembles folic acid (page 165) in structure and prevents the formation of the purine precursors of DNA, RNA and ATP. 6-Mercaptopurine is a purine analogue which interferes with the pathway in which the phosphoribosyl precursors of the purine nucleotides are formed. 5-Fluorouracilis a competitive inhibitor of the conversion of uridylic acid (dUMP) into thymidylic acid (dTMP). 

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