Folate reductase inhibition

Methotrexate has been measured in biological specimens using a wide variety of techniques. RIA and the folate reductase inhibition techniques have been used, but nonisotopic immunoassays are now the method of choice. Liquid chromatographic procedures have also been developed to allow for co-analysis of the drug and its metabolites. ... 

A. J. Hopfinger, /. Am. Chem. Soc., 102, 7196 (1980). A QSAR Investigation of Dihydro-folate Reductase Inhibition by Baker Triazines Based upon Molecular Shape Analysis. 

Folate metabolism Sulphonamides (also ) Trimethoprim Pyrimethamine Trimetrexate / Inhibit folate synthesis Inhibits dihydrofolate reductase Inhibits dihydrofolate reductase Inhibits dihydrofolate reductase Not present in mammalian cells Mammalian enzyme not inhibited Mammalian enzyme not inhibited Toxicity overcome with leucovorin... 

The active form of folate is the tetrahydro-derivative that is formed through reduction by dihydrofolate reductase. This enzymatic reaction (Figure 29.5) is inhibited by trimethoprim, leading to a decrease in the folate coenzymes for purine, pyrimidine, and amino acid synthesis. Bacterial reductase has a much stronger affinity for trimethoprim than does the mammalian enzyme, which accounts for the drug s selective toxicity. [Note Examples of other folate reductase inhibitors include pyrimethamine, which is used with sulfonamides in parasitic infections (see p. 353), and methotrexate, which is used in cancer chemotherapy (see p. 378).]... 

FH4 is carried out by the crucial enzyme thimidylate synthetase (Fig. 4-13). It should be noted that this is a reductive methylation where the tetrahydrofolate coenzyme serves both as a one-carbon donor and reductant. The resultant FH2 must be reduced back to FH4 by folate reductase, thus offering further opportunity for DNA synthesis inhibition at this point. Thymidylate synthetase is the sole de novo path to thymidylate there are no alternate routes. The interest of this point as a target for anticancer agents is obvious. 

Methotrexate inhibits the enzyme folate reductase at two steps (Fig. 4-12). Even though the inhibition is technically a competitive one, the enzyme binds MTX much more strongly than the natural substrate, FH2 (Table 4-5). In practical terms there is no dissociation of the enzyme-drug complex. For thymidylate synthetase to continue to produce thymidylic acid (and therefore DNA), perpetual reduction of FH2 is essential for cellular survival. The efficiency of MTX inhibition of folate reductase thus indirectly becomes the mechanism by which this drug is so cytotoxic in the S phase. In addition, there is evidence that MTX may, by binding to the tetrahydrofolate coenzyme, also inhibit the thymidylate synthetase... 

Methotrexate is a close strnctnral analogne of folate and inhibits dihydrofolate reductase. This prevents the rednction of folate and dihydrofolate (DHF) to tetrahydrofolate (THF), which is the precursor of A ,lV -methylene THF. This is essential for dTTP and DNA synthesis. Unfortnnately, normal cells are also attacked by methotrexate. Folinic acid (A -formyl tetrahydrofolate) is an active form of folate that can be given after the start of methotrexate treatment to rescue normal cells from this drug toxicity. 

The folate analogues are powerful inhibitors of tetrahydrofolate dehydrogenase (or folate reductase), the enzyme which converts folate to the active tetrahydro derivative. Reduced forms of aminopterin also inhibit tetrahydrofolate dehydrogenase, dihydroaminopterin being about as... 

Several of the enzymes which catalyze the interconversion of the 114-folate coenzymes are inhibited or repressed by purine nucleotides. Thus, 5,10-methylene H4-folate dehydrogenase is inhibited by ATP, GTP, and ITP (66), and 5,10-methylene H4-folate reductase is inhibited by S-adenosylmethionine (67). 10-Formyl H4-folate synthetase is repressed in cells grown in the presence of purines (68). 

Folic Acid Antagonists - Interest in antimetabolites that interfere with the synthesis of nucleic acids continues. Studies on the transport and uptake of methotrgxate ° and 2,4-diamino-5-(3,4-dichlorophenyl)-6-methylpyrimidine indicate that clinical response is related to cellular uptake of drugl and the resistance of certain cells to methotrexate appears to be due to lack of transport into these cells. Work on other resistant cell lines indicates that resistance can also be due to an increase in cellular content of folate reductase, but no correlation was observed between resistance and the level of other enzymes involved in folate metabolism. Despite these results there is evidence that the ability of methotrexate to kill cells cannot be entirely explained by its inhibition of folate reductase. Leucovorin at appropriately timed intervals improved the therapeutic index of methotrexate in the treatment of head and neck cancer, and lymphosarcoma and reticulum cell sarcoma. The use of methotrexate in the treatment of hormone-refractory metastatic breast carcinoma " and the use of intrathecal methotrexate also appear promising. Oxidation of methotrexate to 7-hydroxymethotrexate by liver aldehyde oxidase is probably a detoxification mechanism. Material previously reported to be tetrahydromethotrexate has now been found to be a mixture of di- and tetrahydromethotrexate, both of which are less effective than methotrexate in the inhibition of folate reductase, but more effective in the inhibition of thymidylate synthetase. ... 

Inhibition of folate reductase accounts for the beneficial effects of methotrexate in certain leukemias and in choriocarcinoma. Unfortunately, the differential effect of methotrexate on tumor cells is not nearly as great as the selective inhibition of folate metabolism in protozoa by trimethoprim. Baker has reported compounds that inhibit dihydrofolic reductase obtained from leukemic cells but do not affect the same enzyme isolated from normal cells. 

Proguanil (Fig. 3.16) was a relatively early antimalarial drug and it is still useful as a first line of defence against the erythrocytic stage of malaria. It is metabolised in the human body to an active metabolite which mimics the pteridine ring in folic acid and acts as a selective inhibitor of folate reductase in the parasite, thus inhibiting its DNA synthesis. 

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). 

Methotrexate (MTX, chemical structure shown in Fig. 1.) competitively inhibits the dehyrofolate reductase, an enzyme that plays an essential role in purine synthesis. The dehydrofolate reductase regenerates reduced folates when thymidine monophosphate is formed from deoxyuridine monophosphate. Without reduced folates cells are unable to synthesize thymine. Administration of N-5 tetrahydrofolate or N-5 formyl-tetrahydrofolate (folinic acid) can bypass this block and rescue cells from methotrexate activity by serving as antidote. 

Folic acid antagonist inhibits dihydrofolate reductase (DHFR) blocks reduction of folate to tetrahydrofolate inhibits de novo purine synthesis results in arrest of DNA, RNA, and protein synthesis... 

Methotrexate acid produced Antineoplastic (Cancer) Inhibits dihydrofolate reductase, enzyme that converts folate... 

Inhibit Enzymes Many drugs are competitive inhibitors of key enzymes in pathways. The statin drugs (lovastatin, simvastatin), used to control blood cholesterol levels, competitively inhibit 3-hvdroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in cholesterol biosynthesis. Methotrexate, an antineoplastic drug, competitively inhibits dihydrofolate reductase, depriving the cell of active folate needed for purine and deoxythymidine synthesis, thus interfering with DNA replication during S phase. 

Megaloblastic anemia results from insufficient active THF to support cell division in the bone marrow. Methotrexate inhibits DHF reductase, making it a useful antineoplastic drug. Folate deficiencies may be seen during pregnancy and in alcoholism. 

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. 

Folate coenzyme concentrations may also decline as a result of treatment with drugs that inhibit dihydrofolate reductase, eg, methotrexate. 

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