Dihydrofolate reductase, effect

Fig. 7.15 The variation in torsion angles can be effectively represented as a series of dials, where the time corresponds to the distance from the centre of the dial. Data from a molecular dynamics simulation of an intermolecular complex between the enzyme dihydrofolate reductase and a triazine inhibitor [Leach and Klein 1995].

The sulfa dmgs are stiH important as antimicrobials, although they have been replaced in many systemic infections by the natural and semisynthetic antibiotics. They are of great value in third world countries where problems of storage and lack of medical personnel make appropriate use of antibiotics difficult. They are especially useful in urinary tract infections, particularly the combination of sulfamethoxazole with trimethoprim. Their effectiveness has been enhanced by co-adniinistration with dihydrofolate reductase inhibitors, and the combination of sulfamethoxazole with trimethoprim is of value in treatment of a number of specific microbial infections. The introduction of this combination (cotrimoxazole) in the late 1960s (1973 in the United States) resulted in increased use of sulfonamides. 

In view of the well-documented inhibition of dihydrofolate reductase by aminopterin (325), methotrexate (326) and related compounds it is generally accepted that this inhibitory effect constitutes the primary metabolic action of folate analogues and results in a block in the conversion of folate and dihydrofolate (DHF) to THF and its derivatives. As a consequence of this block, tissues become deficient in the THF derivatives, and this deficiency has many consequences similar to those resulting from nutritional folate deficiency. The crucial effect, however, is a depression of thymidylate synthesis with a consequent failure in DNA synthesis and arrest of cell division that has lethal results in rapidly proliferating tissues such as intestinal mucosa and bone marrow (B-69MI21604, B-69MI21605). 

Overproduction of the chromosomal genes for the dihydrofolate reductase (DHFR) and the dihydroptero-ate synthase (DHPS) leads to a decreased susceptibility to trimethoprim and sulfamethoxazol, respectively. This is thought to be the effect of titrating out the antibiotics. However, clinically significant resistance is always associated with amino acid changes within the target enzymes leading to a decreased affinity of the antibiotics. 

Chromosomal mutations in E. coli result in overproduction of dihydrofolate reductase (DHFR). Higher concentrations of trimethoprim, which may not be therapeutically achievable, are therefore required to inhibit nucleotide metabolism. Other mutations lower the affinity of DHFR for trimethoprim. These two mechanisms of resistance may coexist in a single strain, effectively increasing the level of resistance to the antibiotic. 

In the Kohn-Sham Hamiltonian, the SVWN exchange-correlation functional was used. Equation 4.12 was applied to calculate the electron density of folate, dihydrofolate, and NADPH (reduced nicotinamide adenine dinucleotide phosphate) bound to the enzyme— dihydrofolate reductase. For each investigated molecule, the electron density was compared with that of the isolated molecule (i.e., with VcKt = 0). A very strong polarizing effect of the enzyme electric field was seen. The largest deformations of the bound molecule s electron density were localized. The calculations for folate and dihydrofolate helped to rationalize the role of some ionizable groups in the catalytic activity of this enzyme. The results are,... 

Drug efficacy is directly related to its intracellular concentration level, so it is necessary to evaluate the MTX concentration in cells. In particular, MTX is a folate antagonist, thus it binds to dihydrofolate reductase in competition with folate [71-77]. A low intracellular level of MTX caused by high efflux and low uptake in resistant cells is also the main disadvantage of MTX medication [78,79]. This leads to a high dosage of MTX for cancer treatment, which is also directly associated with adverse effects. 

III. The answer is d, (Hardman, pp 1247, L135.) Leucovorin prevents methotrexate from inhibiting dihydrofolate reductase and reverses all of its adverse effects except neurotoxicity... 

Classical methodology was used to prepare the dibenz[b,f]azepine derivative 21 (R = substituted pyrido[2,3-d]pyrimidine) utilising amide ion formation from dibenz[b,f]azepine itself with sodium hydride and then iV-alkylation with 2,4-diamino-6-bromomethylpyrido[2,3-d]pyrimidine. The bulky bis-fused azepine moiety was required to introduce steric bulk in the system and to study the effect of this on inhibition of the enzyme dihydrofolate reductase <00JHC921>. 

Although affinity chromatography has not been used directly as an analytical method, it may be modified in the future to produce a viable technique. Leucovorin has been used as an effective spacer in obtaining active samples of dihydrofolate reductase.79 If the enzyme could be immobilized without losing its activity, perhaps it could be used to separate folates. 

Urlaub, G., Mitchell, PJ., Kas, E., Chasin, L.A., Funanage, V.L., Myoda, T.T., and Hamlin, Effect of gamma rays at the dihydrofolate reductase locus deletions and inversions, Somat. Cell Mol Genet., 12, 555-556, 1986. 

Methotrexate is an antimetabolite of folic acid and has immunosuppressant properties. It inhibits the enzyme dihydrofolate reductase that is required for the synthesis of purines and pyrimidines. It is used in malignant disease, Crohn s disease, rheumatic disease and psoriasis. Folic acid is given with methotrexate to reduce the occurrence of side-effects particularly the risk of mucositis. 

These three compounds exert many similar effects in nucleotide metabolism of chicks and rats [167]. They cause an increase of the liver RNA content and of the nucleotide content of the acid-soluble fraction in chicks [168], as well as an increase in rate of turnover of these polynucleotide structures [169,170]. Further experiments in chicks indicate that orotic acid, vitamin B12 and methionine exert a certain action on the activity of liver deoxyribonuclease, but have no effect on ribonuclease. Their effect is believed to be on the biosynthetic process rather than on catabolism [171]. Both orotic acid and vitamin Bu increase the levels of dihydrofolate reductase (EC 1.5.1.4), formyltetrahydrofolate synthetase and serine hydroxymethyl transferase in the chicken liver when added in diet. It is believed that orotic acid may act directly on the enzymes involved in the synthesis and interconversion of one-carbon folic acid derivatives [172]. The protein incorporation of serine, but not of leucine or methionine, is increased in the presence of either orotic acid or vitamin B12 [173]. In addition, these two compounds also exert a similar effect on the increased formate incorporation into the RNA of liver cell fractions in chicks [174—176]. It is therefore postulated that there may be a common role of orotic acid and vitamin Bj2 at the level of the transcription process in m-RNA biosynthesis [174—176]. 

These are pyrimidine derivatives and are effective because of differences in susceptibility between the enzymes in humans and in the infective organism. Anticancer agents based on folic acid, e.g. methotrexate, inhibit dihydrofolate reductase, but they are less selective than the antimicrobial agents and rely on a stronger binding to the enzyme than the natural substrate has. They also block pyrimidine biosynthesis. Methotrexate treatment is potentially lethal to the patient, and is usually followed by rescue with folinic acid (A -formyl-tetrahydrofolic acid) to counteract the folate-antagonist action. The rationale is that folinic acid rescues normal cells more effectively than it does tumour cells. 

Inhibition of nucleobase synthesis (2). Tetrahydrofolic acid (THF) is required for the synthesis of both purine bases and thymidine. Formation of THF from folic acid involves dihydrofolate reductase (p. 272). The folate analogues aminopterin and methotrexate (ame-thopterin) inhibit enzyme activity as false substrates. As cellular stores of THF are depleted, synthesis of DNA and RNA building blocks ceases. The effect of these antimetabolites can be reversed Ltillmann, Color Atlas of Pharmacology 2000 Thieme All rights reserved. Usage subject to terms and conditions of iicense. 

Successful fusion (2) is a rare event, but the frequency can be improved by adding polyethylene glycol (PEG). To obtain only successfully fused cells, incubation is required for an extended period in a primary culture with HAT medium (3), which contains hypoxan-thine, aminopterin, and thymidine. Amino-pterin, an analogue of dihydrofolic acid, competitively inhibits dihydrofolate reductase and thus inhibits the synthesis of dTMP (see p. 402). As dTMP is essential for DNA synthesis, myeloma cells cannot survive in the presence of aminopterin. Although spleen cells are able to circumvent the inhibitory effect of aminopterin by using hypoxanthine and thymidine, they have a limited lifespan and die. Only hybridomas survive culture in HAT medium, because they possess both the immortality of the myeloma cells and the spleen cells metabolic side pathway. 

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. 

Timelines can also be portrayed in charts or figures, as illustrated in excerpt 14D. In fact, charts and figures represent excellent ways to illustrate how smaller, individual projects contribute to larger research goals and how smaller projects complement one another and overlap in time. (Note In excerpt 14D, Kohen uses the following abbreviations in his chart, each defined previously in the proposal hydrogen (H), tritium (T), deuterium (D), kinetic isotope effect (KIE), dihydrofolate reductase (a relatively small protein) (DHFR), and wild type (WT).)... 

Trimethoprim acts in the body by interfering with the action of hydrofolate reductase, an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid. This process is necessary for purine biosynthesis of live organisms and DNA, respectively. Reducing the dihydrofolic acid to tetrahydrofolic acid is also catalyzed in humans by dihydrofolate reductase. However, trimethoprim has thousands of more inhibitory effects with respect to bacterial enzymes than with respect of analogons enzymes of mammals, which is the main benefit of trimethoprim. 

Pharmacology Leucovorin is one of several active, chemically reduced derivatives of folic acid. It is useful as an antidote to drugs that act as folic acid antagonists. Administration of leucovorin can counteract the therapeutic and toxic effects of folic acid antagonists such as methotrexate, which act by inhibiting dihydrofolate reductase. 

Dolnick BJ, Pink JJ. Effects of 5-fluorouracil in dihydrofolate reductase and dihydrofolate reductase mRNA from methotrexate-resistant KB cells. J Biol Chem 1985 260 3006-3014. 

Trimethoprim is a competitive inhibitor of the enzyme dihydrofolate reductase and can thus prevent the formation of tetrahydrofolate thereby blocking the synthesis of purines. The affinity of trimethoprim for the enzyme in microorganisms is 10,000 times higher than for the human enzyme which explains the selective toxicity. Used alone its main indication is acute uncomplicated urinary tract infections. It is then as effective as co-trimoxazole but has the advantage of fewer adverse reactions. 

Adverse effects include skin rashes, pruritus, nausea, epigastric pain and glossitis. Megaloblastic anemia, leukopenia, granulocytopenia can occur due to the inhibition of the human dihydrofolate reductase. Folinic acid, the reduced form of tetrahydrofolate is sometimes used to prevent these effects. 

VI.a.2.2. Biguanides. Proguanil is a dihydrofolate reductase inhibitor. It is a slow acting blood schizonticide and not effective on its own. It has also a marked effect on the primary tissue stages of Plasmodium falciparum. It is used in combination with chloroquine for the prophylaxis of chloroquine-resistant Plasmodium falciparum. 

Used for malaria chemoprophylaxis and treatment the dihydrofolate reductase inhibitors do not cause pharmacological side-effects in the host. In the higher dose used for toxoplasmosis macrocytic anaemia and other adverse effects may occur. 

Pemetrexed is chemically similar to folic acid. It inhibits three enzymes used in purine and pyrimidine synthesis - thymidylate synthetase, dihydrofolate reductase, and glycinamide ribonucleotide formyl transferase. By inhibiting the formation of precursor purine and pyrimidine nucleotides, pemetrexed prevents the formation of DNA and RNA. In 2004 it was approved for treatment of malignant pleural mesothelioma and as a second-line agent for the treatment of non-small cell lung cancer. Adverse effects include gastrointestinal complaints, bone marrow suppression, alopecia, allergic and neurotoxic reactions. 

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