Inhibition/inhibitors reductase

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. 

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. 

All of these compounds are inhibitors of dihydrofolate reductase in bacteria, plasmodia, and humans. Fortunately, they have a significantly higher affinity to bacterial and protozoal dihydrofolate reductase. Pyrimethamine, for example, inhibits dihydrofolate reductase in parasites in concentrations that are a several hundred times lower than that required to inhibit dihydrofolate reductase in humans. This is the basis of their selective toxicity. Selective toxicity can be elevated upon the host organism s production of folic acid, which parasites are not able to use. 

Folate deficiency can be dietary, especially in the eiderly, due to increased demand like in pregnancy, or due to maiabsorption syndromes. Agents which can cause folic acid deficiency with long-term use include phenytoin, oral contraceptives, isoniazid and glucocorticosteroids. In rare instances the use of dihydrofolate reductase inhibitors like trimethoprim, methotrexate or pyrimethamine can contribute to the occurrence of folate deficiency. Folinic acid can circumvent the need for the inhibited dihydrofolate reductase. 

Inhibitors. Aside from its role in providing reduced folate coenzymes for cells, this enzyme has attracted a great deal of attention because it appears to be a site of action of the important anticancer drugs methotrexate (amethopterin) and aminopterin.293 364 365 These compounds inhibit dihydrofolate reductase in concentrations as low as 10 8 to 10 9 M. Methotrexate is also widely used as an immunosuppresant drug and in the treatment of parasitic infections. 

What are the criteria for regarding a compound as a TS analog The observation that the binding affinity of an inhibitor is greater than that of a substrate, i.e., X, < XM, is insufficient as many potent inhibitors bind differently to an enzyme than the substrate examples are methotraxate, inhibiting dihydrofolate reductase (DHFR) X] = 0.15 pM (Werkheiser, 1961), and sulfonyl urea herbicides, inhibiting acetolactate synthase (ALS) at picomolar levels. 

In Ayurveda and folklore medicines, cinnamon is used in the treatment of diabetes. Cinnamon is reported to reduce the blood glucose level in non-insulin-dependent diabetics. Therapeutic studies have proved the potential of cinnamaldehyde as an antidiabetic agent. Cinnamaldehyde inhibits aldose reductase, a key enzyme involved in the polyol pathway. This enzyme catalyses the conversion of glucose to sorbitol in insulin-insensitive tissues in diabetic patients. This leads to accumulation of sorbitol in chronic complications of diabetes, such as cataract, neuropathy and retinopathy. Aldose-reductase inhibitors prevent conversion of glucose to sorbitol, thereby preventing several diabetic complications (Lee, 2002). 

Several general comments about NOS inhibitors can be made. An inhibitor that inhibits electron transfer from the reductase or directly inhibits the reductase domain might... 

Vitamin deficiency can result from treatment with certain drags. Thus, destruction of intestinal microorganisms by antibiotic therapy can produce symptoms of vitamin K deficiency. Isoniazid, used to treat tuberculosis, is a competitive inhibitor of pyridoxal kinase, which is needed to produce pyridoxal phosphate. Isoniazid can produce symptoms of pyridoxine deficiency. To prevent this, pyridoxine is often incorporated into isoniazid tablets. Methotrexate and related folate antagonists act by competitively inhibiting dihydrofolate reductase (Chapter 27). 

Inhibitors of sterol biosynthesis provide many, useful, broad spectrum, fungicides, and it is essential that their effectiveness is retained as long as possible. Of the two major groups of SBI fungicides, only.the morpholines, which inhibit A reductase and/or A -A isomerase depending on their... 

Today we can envisage at least five important and potentially useful types of inhibitors that could inhibit ribonucleotide reductase in different ways ... 

Svoboda and co-workers (Svoboda and Robbins, 1967 1968 Svoboda et al., 1967 1969) found that triparanol, 2-(4-chlorophenyl)-l-[4-(2-diethylamino-ethoxy)phenyl]-l-(4-tolyl) ethanol and 22,25-diazacholesterol (99) inhibit sterol reductase and also disrupt the normal growth and development of the larvae of tobacco homworm, Manduca sexta. Both compounds are known as inhibitors of steroid biosynthesis in vertebrates. 22,25-DiazacholesteroI is a hypocholeste-rolaemic agent acting as a competitive antagonist of cholesterol. A similar activity was found later for 25-azacholesterol (100). For the determination of the minimum structural requirements of activity, Svoboda and Robbins synthesised and tested... 

Vidarabine is an inhibitor of viral DNA synthesis. Cellular enzymes phosphorylate vidarabine to the triphosphate, which inhibits viral DNA polymerase activity in a manner that is competitive with deoxyadenosine triphosphate. Vidarabine triphosphate is incorporated into both cellular and viral DNA, where it may act as a chain terminator. Vidarabine triphosphate also inhibits ribonucleoside reductase, RNA polyadenylation, and 5 -adenosylhomocysteine hydrolase, an enzyme involved in transmethylation reactions. Resistant variants due to mutations in viral DNA polymerase can be selected in vitro. 

The antimicrobial activity of the trimethoprim/sulfamethoxazole combination results from actions on two steps of the biosynthetic pathway for tetrahydrofolic acid. Trimethoprim prevents the reduction of dihydrofolate to tetrahydrofolate (Figure 43-2). Mammalian cells use preformed folates from the diet and do not synthesize the compound. Trimethoprim is a highly selective inhibitor of dihydrofolate reductase of lower organisms -100,000 times more drug is required to inhibit human reductase than the bacterial enzyme. This relative selectivity is vital because the enzyme is essential to all species. 

All of these effects, fluorescence artifacts, edge effects, insolubility, etc., have been understood for some time. But even taking them all into account still wasn t enough to explain a mysterious problem that had probably been observed in many labs. Dr Brian Shoichet of UCSF elaborates We were looking for inhibitors of 3-lactamase. We found all these hits and they were all noncompetitive, which was weird, and they were all time-dependent, which was weird. None of those things were impossible, but to have them all We had twenty of these molecules and so we said, Well, let s just make sure they re not inhibiting chymotrypsin. And they did. Then we said, Let s make sure they don t inhibit dihydrofolate reductase. And they did. We said, Let s make sure they don t inhibit P-galactosidase. And they did. We knew we were in trouble. Usually, when that happens in pharma, people drop the project. ... 

The former can be prepared by reductive alkylation of 2-acetamido-5-amino-4(3H)pyrimidone with dimethyl iV-(p-(A-(formylmethyl)formamido)ben-zoyl -L-glutamate, followed by reformylation, partial deblocking, and acid hydrolysis to (LXIII). Both (LXIII) and (LXIV) were reported to have inhibitory activity against S. faecalis equal to that of aminopterin. They both inhibited dihydrofolate reductase (source not named) at 4 x 10" m, for 50 per cent inhibition. Compound (LXIII) is a better inhibitor of thymidylate synthetase than (LXIV) (3 x 10 vj. 7 x I0 M for 50 per cent inhibition). 

Competitive enzyme inhibitors are used as drugs. They have structures similar to the natural substrate and can therefore compete with it for access to the same binding site on the enzyme. For example, methotrexate (an anticancer drug) structurally resembles folate, which is the natural substrate for dihydrofolate reductase (Chapter 58). Methotrexate is used to inhibit dihydrofolate reductase in cancer chemotherapy. 

Two of the derivatives (i.e., those containing 5-methylamino and 5-ethyl-amino groups) demonstrated impressive antitumour activity against Sarcoma 180 ascites cells and several others were potent inhibitors of ribonucleoside diphosphate reductase, requiring concentrations in the range of 10" to 10" M for 50% inhibition [43], 5-Methylamino-l-formylisoquinoline thiosemicarbazone, which was the most effective of the newly synthesized compounds required a concentration of 3 10" M for 50% inhibition of reductase activity and increased the life span of tumour-bearing mice over untreated animals by a factor of 2.5 at the optimal daily dose. 

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