vitamin-k-semiquinone-radical and tretazicar

A nitroreductase enzyme has been isolated from Escherichia coli that has the unusual property of being equally capable of using either NADH or NADPH as a cofactor for the reduction of its substrates which include menadione as well as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954). This property is shared with the mammalian enzyme, DT diaphorase. The nitroreductase can, like DT diaphorase, also use simple reduced pyridinium compounds as virtual cofactors. The intact NAD(P)H molecule is not required and the simplest quaternary (and therefore reducible) derivative of nicotinamide, 1-methylnicotinamide (reduced), is as effective as NAD(P)H in its ability to act as an electron donor for the nitroreductase. The structure-activity relationship is not identical to that of DT diaphorase and nicotinic acid riboside (reduced) is selective, being active only for the nitroreductase. Irrespective of the virtual cofactor used, the nitroreductase formed the same reduction products of CB 1954 (the 2- and 4-hydroxylamino derivatives in equal proportions). Nicotinic acid riboside (reduced), unlike NADH, was stable to metabolism by serum enzymes and had a plasma half-life of seven minutes in the mouse after an i.v. bolus administration. NADH had an unmeasurably short half-life. Nicotinic acid riboside (reduced) could also be produced in vivo by administration of nicotinic acid 5'-O-benzoyl riboside (reduced). These results demonstrate that the requirement for a cofactor need not be a limitation in the use of reductive enzymes in antibody directed enzyme prodrug therapy (ADEPT). It is proposed that the E. coli nitroreductase would be a suitable enzyme for ADEPT in combination with CB 1954 and a synthetic, enzyme-selective, virtual cofactor such as nicotinic acid riboside (reduced).

Topics: Animals; Antibodies; Aziridines; Escherichia coli; Mice; NAD; Nitroreductases; Oxidation-Reduction; Prodrugs; Pyridinium Compounds; Ribonucleosides; Vitamin K

The enzyme DT diaphorase (NAD(P)H dehydrogenase (quinone), EC 1.6.99.2) is unusual in that it can utilize either NADH or NADPH as a co-factor for the reduction of its substrates. We have shown that the intact NAD(P)H molecule is not required and that other reduced pyridinium compounds can also act as co-factors for DT diaphorase. The entire adenine dinucleotide portion of NAD(P)H can be dispensed with entirely and the simplest quaternary (and therefore reducible) derivative of nicotinamide, 1-methylnicotinamide, was as effective as NAD(P)H as a co-factor for the reduction of the quinone, menadione. Nicotinamide 5'-O-benzoyl riboside was also as effective a co-factor as NAD(P)H, whilst nicotinamide ribotide and riboside have a higher Km, and decreased the kcat of DT diaphorase. Nicotinic acid derivatives had little activity. Kinetic analysis indicated that both nicotinamide ribotide and riboside may be interacting with the menadione binding site rather than the NAD(P)H site. Irrespective of the differences between the various reduced pyridinium derivatives in their ability to act as co-factors for the reduction of menadione by DT diaphorase, all the compounds that showed activity in this assay were equally effective co-factors for the reduction of the nitrobenzamide, CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The apparent Km of DT diaphorase for all these co-factors approached zero. It was concluded that co-factor binding is not a rate-limiting step in the nitroreductase activity of DT diaphorase.

Topics: Animals; Aziridines; Carcinoma 256, Walker; Coenzymes; Kinetics; NAD(P)H Dehydrogenase (Quinone); NADP; Niacinamide; Nicotinamide Mononucleotide; Oxidation-Reduction; Pyridinium Compounds; Rats; Structure-Activity Relationship; Vitamin K

A form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, menadione reductase (NMOR), phylloquinone reductase, quinone reductase, EC 1.6.99.2) has been isolated from Walker 256 rat carcinoma cells. This enzyme can convert 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) to a cytotoxic DNA interstrand crosslinking agent by reduction of its 4-nitro group to the corresponding hydroxylamino species (Knox et al. Biochem Pharmacol, 37: 4661-4669 and 4671-4677, 1988). 2-Phenyl-5(4)-aminoimidazole-4(5)-carboxamide and AICA [5(4)-aminoimidazole-4(5)-carboxamide] have previously been reported to be antagonists of the anti-tumour effects of CB 1954. We have shown that both these compounds are inhibitors of the above enzyme and that AICA protects against both the cytotoxicity and the formation of DNA interstrand crosslinks, produced by CB 1954 in Walker cells. Similarly, known inhibitors of NAD(P)H dehydrogenase (quinone) such as dicoumarol, also reduced the cytotoxicity and DNA-interstrand crosslinking of CB 1954 in Walker cells. Caffeine was shown to be a novel inhibitor of NAD(P)H dehydrogenase (quinone) and also elicited the above protective effects. All of the above inhibitors were also shown to potentiate the toxic effects of menadione against the Walker cell. This quinone is known to be detoxified by NAD(P)H dehydrogenase (quinone) and thus emphasises the ability of these compounds to inhibit this enzyme within the cell.

Topics: Aminoimidazole Carboxamide; Animals; Antineoplastic Agents; Aziridines; Caffeine; Cross-Linking Reagents; Dicumarol; DNA; Drug Synergism; NAD(P)H Dehydrogenase (Quinone); Neoplasms, Experimental; Quinone Reductases; Rats; Tumor Cells, Cultured; Vitamin K