Reduction of misonidazole and its derivatives by xanthine oxidase

The nitroimidazole drug misonidazole, now undergoing clinical trials as a radiosensitizer of hypoxic cells, is selectively toxic to hypoxic mammalian cells; this toxicity may be due to metabolic reduction of the drug. Zinc reduction of misonidazole yields its azo and azoxy derivatives [P. D. Josephy, B. Palcic and L. D. Skarsgard, in Radiation Sensitizers (Ed. L. W. Brady), p. 61. Masson, New York (1980)]. We have shown in the present work that misonidazole and its azo and azoxy derivatives were reduced by xanthine oxidase, under hypoxic conditions. The nature of the products of misonidazole reduction was examined; hydroxylamino-misonidazole appeared to be the main product.     

Inhibition of monoamine oxidase by analogues of amiloride

Amiloride analogues with nonaromatic substituents on the 5-amino group or different substituents on carbon-6 of the pyrazine ring were tested as inhibitors of monoamine oxidase A and B in rat brain homogenate. The inhibition was competitive and reversible. 5-(N,N-Tetramethylene)amiloride protected the A type in the homogenate against irreversible inhibition by clorgyline. A reciprocal relation was found to exist between inhibitory constants of 5-N-substituted amiloride analogues for monoamine oxidase A and the ratio of overflows of endogenous noradrenaline and 3,4-dihydroxyphenylethylene glycol from the isolated rat tail artery incubated in the presence of a 50 microM concentration of the analogue, when the tissue was exposed to 10 microM tyramine. The 5-amino group appeared to be essential for inhibition of the A but not of the B type. Bell-shaped relations between inhibitory constants of 5-(N-alkyl)- and 5-(N,N-dialkyl)-substituted analogues and lengths of alkyl chains were different for each type. The presence of a methyl group in the alpha-position of the chain increased substantially the inhibitory constant for the A type. Halogen atoms as substituents on carbon-6 increased inhibitory constants for both types of the enzyme in the sequence: I less than Br less than Cl less than F. These findings are consistent with the existence of hydrophobic binding sites of restricted dimensions in both types of the enzyme.     

Oxidation of selected pteridine derivatives by mamalian liver xanthine oxidase and aldehyde oxidase.

Considerable information is available concerning the oxidation of pteridine derivatives by bovine milk xanthine oxidase, but few investigations have been carried out on the oxidation of such compounds by mammalian liver xanthine oxidase and the related aldehyde oxidase. Xanthine oxidase, obtained from rat liver, oxidizes a variety of substituted amino- and hydroxypteridines in a manner identical to that previously observed for milk xanthine oxidase. For example, 2-aminopteridine and its 4- and 7-hydroxy derivatives were oxidized efficiently to 2-amino-4,7-dihydroxypteridine (isoxanthopterin) by the rat liver enzyme, and 4-aminopteridine and its 2- and 7-hydroxy derivatives were oxidized to 4-amino-2,7-dihydroxypteridine.4-Hydroxypteridine and the isomeric 2- and 7-hydroxypteridines were oxidized by rat liver xanthine oxidase to 2,4,7-trihydroxypteridine. Rabbit liver aldehyde oxidase, but not rat liver xanthine oxidase, was able to catalyze the oxidation in position 7 of 2,4-diaminopteridine and its 6-methyl and 6-hydroxymethyl derivatives. 2-Aminopteridine and 4-aminopteridine were both oxidized to the corresponding 7-hydroxy derivatives in the aldehyde oxidase system; 2-amino-4-hydroxypteridine appeared to be a minor product in the oxidation of 2-aminopteridine by rabbit liver aldehyde oxidase. Both aldehyde oxidase and xanthine oxidase were able to catalyze the oxidation of 2-amino-6,7-disubstituted pteridines to the corresponding 4-hydroxy derivatives; 4-hydroxy-6,7-disubstituted pteridines were oxidized in position 2 by both enzymes. 4-Amino-6,7-disubstituted pteridines were not oxidized by either enzyme. 2-Amino-4-methylpteridine was oxidized in position 7 by aldehyde oxidase but was not an effective substrate for xanthine oxidase; 2-hydroxypteridine and 7-hydroxypteridine were not oxidized to a detectably extent by aldehyde oxidase. All oxidations mediated by xanthine oxidase were strongly inhibited by allopurinol (4-hydroxypyrazolo[3,4-d]pyrimidine), and all oxidations mediated by aldehyde oxidase were inhibited by menadione (2-methyl-1,4-naphthoquinone). Rat liver xanthine oxidase and, to a lesser extent, rabbit liver aldehyde oxidase were inhibited by 4-chloro-6,7-dimethylpteridine; 2-amino-3-pyrazinecarboxylic acid inhibited xanthine oxidase but not aldehyde oxidase. The oxidations of 2- and 4-aminopteridines by aldehyde oxidase resulted in concomitant reduction of cytochrome c.     

Inhibition of xanthine oxidase by phenolic conjugates of methylated quinic acid

The caffeoyl conjugates of prenylhydroquinone glucoside and of quinic acid, either in the carboxyl-free or carboxymethyl forms, isolated from Phagnalon rupestre (Asteraceae), showed inhibitory activity on lipid peroxidation induced by Fe 2+/ascorbate and by CCl4/NADPH in rat liver microsomes, with IC50 values ranging from 3 to 11 microM. After having demonstrated their effect on the xanthine oxidase-regulated superoxide production, the active compounds were tested for the direct inhibition of this enzyme. Methylated dicaffeoylquinic conjugates competitively inhibited the enzyme and the highest potency was obtained for the 4,5-diester, with an IC50 value of 3.6 microM, nearly ten times lower than that of the 3,5-analogue. In conclusion, the presence of the caffeoyl moiety is essential for both the antiperoxidative and radical scavenging activities, and the methylation of the quinic carboxyl group enhances the potency on xanthine oxidase inhibitory activity.     

Metabolism of azoxy derivatives of procarbazine by aldehyde dehydrogenase and xanthine oxidase.

Procarbazine, a 1,2-disubstituted hydrazine, is employed therapeutically in the treatment of Hodgkin's disease and a limited number of other neoplasias. The isomeric azoxy metabolites of procarbazine have recently been identified as the precursors of species responsible for both the anti-cancer efficacy and toxic effects mediated by this drug. This study demonstrates that cytosolic enzymes are involved in the metabolism of the azoxy metabolites of procarbazine. Two azoxy procarbazine oxidase activities were resolved by diethylaminoethyl (DEAE)-cellulose chromatography. The activity which did not bind to this column was purified to homogeneity and was identified as a phenobarbital-inducible form of cytosolic aldehyde dehydrogenase. This protein fraction was shown to metabolize only the azoxy 2 procarbazine isomer to yield N-isopropy-p-formylbenzamide (ALD) in a reaction which did not require NAD+ as cofactor. The ALD product formed was also a substrate for a subsequent NAD(+)-dependent reduction reaction catalyzed by that purified protein. The azoxy 2 procarbazine isomer and ALD were shown to be potent inhibitors of both the dehydrogenase and esterase activities of aldehyde dehydrogenase. The second azoxy procarbazine oxidase activity which was retained by the DEAE-cellulose column co-eluted with xanthine oxidase activity. Both the xanthine dehydrogenase/oxidase and azoxy procarbazine oxidase activities of this protein fraction were inhibited by allopurinol, a specific inhibitor of xanthine dehydrogenase. Xanthine dehydrogenase/oxidase was partially purified by an alternative procedure and was shown to metabolize both the azoxy 2 procarbazine isomer and ALD, ultimately producing N-isopropylterephthalamic acid. The ability of xanthine oxidase to metabolize azoxy 2 procarbazine and ALD was confirmed using commercial, purified milk xanthine oxidase.     

Inhibition of xanthine oxidase by 4-hydroxy-6-mercaptopyrazolo[3,4-d]pyrimidine

Compound B103U, 4-hydroxy-6-mercaptopyrazolo[3,4-d]pyrimidine, was investigated as an inhibitor of human xanthine oxidase. Studies in vitro demonstrated that it was significantly more potent than oxypurinol, 4,6-dihydroxypyrazolo[3,4-d]pyrimidine. It formed an initial complex with electron-rich (reduced) human xanthine oxidase that was tighter than the corresponding complex formed by oxypurinol. The initial complexes with each inhibitor and reduced enzyme were internally rearranged into more stable complexes with first-order rate constants of 2.5 to 3 per min. However, the half-life of the isomerized (stable) complex with B103U was three to four times longer than the half-life of the analogous complex with oxypurinol. This stability was previously noted by Massey et al. (J. Biol Chem 254: 2837-2844, 1970) with B103U and bovine xanthine oxidase. The overall Ki values accounting for the initial and isomerized complexes were 5 nM for B103U and 100 nM for oxypurinol. B103U was also more potent as an inhibitor of bovine xanthine oxidase-catalyzed generation of superoxide radicals. Studies in mice revealed that the relative in vitro potency of B103U was not sustained in vivo. Compared to the inhibition of xanthine oxidase by oxypurinol, inhibition by B103U was neither more potent nor longer lasting. This shortcoming was not caused by weaker inhibition of mouse xanthine oxidase. Instead, it was the result of poor bioavailability. Plasma levels of available B103U rapidly decreased from samples of mouse and human blood because of reversible binding to serum proteins. B103U was also susceptible to oxidation. Two equivalents of H2O2 stoichiometrically oxidized the 6-thiol substituent to a sulfinic acid. This oxidized product was three orders of magnitude weaker as an inhibitor of xanthine oxidase than was B103U.     

Superoxide radical production by allopurinol and xanthine oxidase

Oxypurinol, an inhibitor of xanthine oxidase (XO), is being studied to block XO-catalyzed superoxide radical formation and thereby treat and protect failing heart tissue. Allopurinol, a prodrug that is converted to oxypurinol by xanthine oxidase, is also being studied for similar purposes. Because allopurinol, itself, may be generating superoxide radicals, we currently studied the reaction of allopurinol with xanthine oxidase and confirmed that allopurinol does produce superoxide radicals during its conversion to oxypurinol. At pH 6.8 and 25 degrees C in the presence of 0.02 U/ml of XO, 10 and 20 microM allopurinol both produced 10 microM oxypurinol and 2.8 microM superoxide radical (determined by cytochrome C reduction). The 10 microM allopurinol was completely converted to oxypurinol, while the 20 microM allopurinol required a second addition of xanthine oxidase to complete the conversion. Fourteen percent of the reducing equivalents donated from allopurinol or xanthine reacted with oxygen to form superoxide radicals. Superoxide dismutase prevented the reduction of cytochrome C by these substrates. At higher xanthine oxidase concentrations, or at lower temperatures, more of the 20 microM allopurinol was converted to oxypurinol during the initial reaction. At lower xanthine oxidase concentrations, or higher temperatures, less conversion occurred. At pH 7.8, the amount of superoxide radicals produced from allopurinol and xanthine was nearly doubled. These results indicate that allopurinol is a conventional substrate that generates superoxide radicals during its oxidation by xanthine oxidase. Oxypurinol did not produce superoxide radicals.     

Effect of anti-inflammatory drugs on xanthine oxidase and xanthine oxidase induced depolymerization of hyaluronic acid

The inhibitory effect of various anti-inflammatory drugs on the xanthine oxidase derived depolymerization of hyaluronic acid was studied. The depolymerization was assayed by repeated viscosity measurements. By using a low xanthine oxidase activity, the decrease in viscosity with time followed first order reaction kinetics and was therefore suitable for kinetic analysis. The xanthine oxidase activity was monitored by assay of O2-consumption with a Clark-electrode and by assay of urate production. We present evidence that salicylic, acetylsalicylic, gentisic and azodisalicylic acid and sulfasalazine inhibit the production of oxygen-derived free radicals by xanthine oxidase. We found that sulfapyridine, 5-aminosalicylic acid, allopurinol, mannitol, glucuronic acid and N-acetylglucosamine in addition to the earlier studied drugs, paracetamol, ibuprofen, benoxaprofen and gentisic acid exert their effect via scavenging of free radicals. These drugs had very little effect on the enzyme activity.     

丹参二萜醌对黄嘌呤氧化酶活性的抑制作用(英文)

目的黄嘌呤氧化酶(XOD)抑制剂对痛风或其他XOD诱导的疾病有潜在的治疗作用,因此探讨了从丹参(Salvia miltiorrhiza Bge.)中分离的丹参二萜醌---隐丹参酮(CT)和次甲丹参酮(MT)对XOD的抑制作用。方法在分子氧存在的条件下,XOD催化黄嘌呤产生尿酸和超氧阴离子。在黄嘌呤/XOD的反应媒介中加入CT或MT,通过测量波长290nm处吸光度的增加测定尿酸形成速率。结果CT和MT对XOD有抑制作用,酶动力学曲线Dixon图显示抑制方式是竞争型。CT和MT的Ki值分别为17.8和25.9μmol.L-1,抑制活性与浓度正相关。CT和MT的IC50值分别为70和67μmol.L-1,阳性对照别黄嘌呤醇的IC50值为60μmol.L-1。结论CT和MT对XOD活性有抑制作用,对痛风或其他XOD诱导的疾病可能有一定的治疗作用。     

Inhibition of superoxide dismutase by 2-methoxyoestradiol analogues and oestrogen derivatives: structure-activity relationships

Superoxide dismutases catalyse the dismutation of highly reactive superoxide ions to produce hydrogen peroxide and several lines of evidence suggest that these enzymes play important roles in the development and response to treatment of human cancers. For example, Mn-containing superoxide dismutase is frequently overexpressed in various cancer types and can contribute to resistance to apoptosis. 2-Methoxyoestradiol is a naturally occurring metabolic product of 17beta-oestradiol that inhibits tubulin polymerization and possesses growth inhibitory and cytotoxic activity in vitro and in vivo. More recently 2-methoxyoestradiol has also been shown to inhibit superoxide dismutase (SOD) in a tetrazolium salt based enzyme assay, suggesting that oestrogen derivatives might be useful starting points for the development of effective, non-toxic enzyme inhibitors. Here we have tested the SOD inhibiting activity of a range of oestrogen derivatives to determine structural features important for enzyme inhibition.     

Inhibitors of folate biosynthesis. 1. Inhibition of dihydroneopterin aldolase by pteridine derivatives.

2-Amino-6-carboxamido-7,8-dihydropteridin-4-one and 2-amino-6-hydroxymethyl-7,7-dimethyl-7,8-dihydropteridin-4-one have been shown to be good inhibitors of Escherichia coli dihydroneopterin aldolase, an early enzyme of de novo folate biosynthesis.     

Nitroreduction of 5-nitrofuran derivatives by rat liver xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase

Rat liver cytosol and microsomes catalyzed the nitroreduction of N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide (NFTA), a potent carcinogen for the mouse, rat, hamster and dog, and formed a metabolite capable of binding to protein. The cytosol nitroreductase was NADH or hypoxanthine dependent and strongly inhibited by a low concentration of allopurinol. Partial purification of the cytosol nitroreductase resulted in the parallel purification of nitroreductase and xanthine oxidase. Furthermore, NFTA, as well as 11 other nitrofurans, was reduced by purified milk xanthine oxidase. The metabolite formed was capable of binding to protein. These observations suggested that the cytosol nitroreductase activity was due to xanthine oxidase. The microsomal nitroreductase, which is NADPH dependent, was probably NADPH-cytochrome c reductase. Microsomal nitroreductase activity paralleled NADPH-cytochrome c reductase activity in rats pretreated with allylisopropylacetamide (AIA), phenobarbital or 3-methylcholanthrene (3-MC), but did not parallel the level of microsomal cytochrome P-450. A partial purification of the microsomal nitroreductase resulted in the parallel purification of nitroreductase and NADPH-cytochrome c reductase. The NFTA metabolite formed by the partially purified enzyme was capable of binding to protein. Other nitrofurans also were reduced by the same enzyme preparation. Hence, microsomal nitroreductase activity may be due to NADPH-cytochrome c reductase.     

Inhibition of rat liver monoamine oxidase by α-methyl- and N-propargyl-amine derivatives

The inhibition of rat liver monoamine oxidase by a number of N-propargyl and α-methyl amine derivatives has been examined. The results indicate that α-methyl-substituted primary and secondary amine derivatives tend to show selectivity as reversible inhibitors towards the A-form of the enzyme. The structural features that result in selectivity in irreversible inhibitors are less easy to define and substitution of an N-propargyl group into a compound that is a selective reversible inhibitor of monoamine oxidase will not necessarily result in retention of that selectivity. Replacement of the acetylenic group in a B-selective irreversible inhibitor by an ethylenic group resulted in a compound that was a reversible inhibitor showing slight selectivity for the A-form of the enzyme.     

Induction of alkaline phosphatase activity in HeLa cells. Inhibition by xanthine derivatives and thermostability studies.

The induction of alkaline phosphate activity in HeLa cells by 5-iodo-2′-deoxyuridine (IUdR) or hydrocortisone was inhibited in a dose-dependent manner by the addition of the xanthine derivatives caffeine, theophylline or 3-isobutyl-1-methylxanthine to the culture medium during the 72 hr of the induction. Pretreatment with theophylline from ?24 to 0 hr or treatment from 0 to 24 hr with any of the xanthine derivatives was ineffective in inhibiting alkaline phosphatase induction produced by treatment with IUdR from 0 to 72 hr. The induction of alkaline phosphatase activity produced by treatment with hydrocortisone from 0 to 72 hr was inhibited by pretreatment with theophylline from ?24 to 0 hr, although this inhibition was only about 60 per cent as great as that seen with treatment from 0 to 72 hr. As judged by heat inactivation studies, IUdR predominantly increased the heat-labile form of alkaline phosphatase activity, while hydrocortisone predominantly increased the heat-stable form. Regardless of the inducer used, the xanthine derivatives mainly decreased the heat-stable form of alkaline phosphatase activity. Treatment with imidazole over a 72-hr period produced over a 2-fold induction of alkaline phosphatase activity, which could be inhibited completely by concurrent treatment with theophylline.