Enzyme inhibitors thymidylate synthase

Drugs that can be used to control tumour cell proliferation inhibit a variety of enzymes, including thymidylate synthase and topoisomerase (Chapter 20). The enzyme aromatase converts a ring in a steroid to an aromatic ring. It converts, for example, adrenal steroid hormones into female sex hormones, which bind to oestrogenic receptors in the ovary or breast and increase the risk of ovarian or breast cancer. Aromatase inhibitors are used to treat patients with breast or ovarian cancers that are sensitive to oestrogen. Unfortunately, none of the inhibitors is specific for enzymes in tumour cells and they can therefore have severe side-effects (Chapter 21). 

Dihydrofolate reductase acts as an auxiliary enzyme for thymidylate synthase. It is involved in the regeneration of the coenzyme N, N -methylene-THF, initially reducing DHF to THF with NADPH as the reductant (see p. 418). The folic acid analogue methotrexate, a frequently used cytostatic agent, is an extremely effective competitive inhibitor of dihydrofolate reductase. It leads to the depletion of N, N -methylene-THF in the cells and thus to cessation of DNA synthesis. 

The methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA. The one-carbon fragment of methy-lene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division. Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anticancer drug. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme inhibitors of these enzymes can be used as antibacterial drugs, eg, trimethoprim, and anti-malarial drugs, eg, pyrimethamine. 

Fig. 1.4 The increase in thymidylate synthase inhibition activity obtained from an initial hit identified as a disulphide-bound enzyme adduct and optimised to a reversible potent 330nM inhibitor using crystal structure-guided design.

Freer, Calculation of solvation and binding free energy differences for folate-based inhibitors of the enzyme thymidylate synthase, J. Am. Chem. Soc. 114 10117 (1992). 

Reddy et al29 studied the molecule N6,N6-dimethyl-2,6-diaminobenz[cd]-indole, which is shown in Figure 6. The aim was to assess its suitability as an inhibitor of thymidylate synthase, an enzyme which is important in DNA biosynthesis. Three structures were investigated N1H, which is the amine form depicted, and N2H, the imine form, in anti- and syn-conformations, i.e. with the proton on Ni away from or towards the N2 proton, respectively. In the gas phase it was found that the order of stability is N1H > anti-N2H > syn-N2H, in a ratio of 73.1 20.3 6.6. Solvation then further favours the N1H form, and the ratios in solution are calculated to be 98.5 0.5 1.0. Unfortunately, the syn-N2H form is likely to be the conformation that binds most strongly to the enzyme, and so the calculations indicate that this molecule is unlikely to be a suitable inhibitor. 

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]. 

Other useful targets for pharmaceutical agents are thymidylate synthase and dihydrofolate reductase, enzymes that provide the only cellular pathway for thymine synthesis (Fig. 22-49). One inhibitor that acts on thymidylate synthase, fluorouracil, is an important chemotherapeutic agent. Fluorouracil itself is not the enzyme inhibitor. In the cell, salvage pathways convert it to the deoxynucleoside monophosphate FdUMP, which binds to and inactivates the enzyme. Inhibition by FdUMP (Fig. 22-50) is a classic example of mechanism-based enzyme inactivation. Another prominent chemotherapeutic agent, methotrexate, is an inhibitor of dihydrofolate reductase. This folate analog acts as a competitive inhibitor the enzyme binds methotrexate with about 100 times higher affinity than dihydrofolate. Aminopterin is a related compound that acts similarly. 

In a process similar to the inhibition of thymidylate synthase by ffdUMP, fl5dC blocks (cytosine-5)-methyltransferase- (DCMtase)-catalyzed methylation of dC residues in DNA. In this case, a catalytic SH group on the enzyme adds to the 6-position of the pyrimidine. After transfer of a methyl group from AdoMet (( S)-adenosyl metionine) to the 5-position of this -enzyme-inhibitor complex, the presence of fluorine blocks the elimination of enzyme-SH (Figure 7)121. 

The chemical structures of four commonly used anticancer drugs are shown in Fig. 15-17. Methotrexate was the first "true anticancer drug, synthesized in 1949, and has been in clinical use for treatment of a variety of cancers since the early 1950s. Methotrexate is a potent inhibitor of dihydrofolate reductase with an inhibition constant (Kt) for interaction with the enzyme of 10 9Af. Inhibition of this enzyme in a cell leads to major accumulation of DHF to concentrations of 2.5 fiM, and minor decreases in THF. Marked decreases in THF may not be seen due to the release of bound THF in methotrexate-treated cells. The high levels of DHF are toxic to the cell, inhibiting the reaction catalyzed by thymidylate synthase,... 

The inhibition of thymidylate synthase is recognized as a viable tqiproach to the control of cancer and DNA viral infections. Mertes and coworkers proposed S-quinone derivatives of 2 deoxyuridine S -I iosphate as potential irreversible inhibitors of this enzyme and prepared the quinones (32a) by oxidative demethylation of the appropriate dimethoxyarenes using CAN. Interestingly, the quinones (32b) were also prepared from the corresponding dimethoxyarenes but using silveifU) oxide and nitric acid in aqueous dioxane to effect this transformation. Cerium(IV) ammonium nitrate could not be used, due to precipitation. 

Suicide inhibitors generally look like the substrate, but attack the enzyme when activated. 5-fluorouracil (which is converted in the body to 5F-dUMP) is a suicide inhibitor of thymidylate synthase, and prevents DNA synthesis in cancerous cells. 

Fluoropropynyl-dUMP (23), a novel mechanism based inhibitor of the A A -methylenetetrahydrofolate-dependent enzyme thymidylate synthase, has been synthesised. This 5-fluoropropynyl-dUMP caused rapid, irreversible inactivation of thymidylate synthase both in the presence and in the absence of the cofactor. 

Several novel 5-substituted A -hydroxy-2 -deoxycytidine 5 -phosphates (24) with substituents of different electronic, hydrophobic and steric properties at the 5-position were synthesised chemoenzymatically with the aid of the wheat shoot phosphotransferase system and evaluated as putative inhibitors of thymidylate synthase. All A -hydroxy-dCMP (24a) and dUMP analogues (24b) were competitive inhibitors of the enzyme-catalysed dUMP methylation. The inhibitory activity was attributed to the rare ram-rotamer (A/ -OH pointing towards C5), and therefore weaker slow-binding inhibitors were detected when unfavourable 04-C5-substituent steric interactions were present. 

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