Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.5.2 (NQO1)
 6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitroaniline mustards have potential as hypoxia-selective cytotoxic agents, with reductive metabolism activating the nitrogen mustard by converting the electron-withdrawing nitro group to an electron-donating hydroxylamine or amine. However, the parent compounds have poor aqueous solubility, and their potencies are limited by low reduction potentials (E1/2 ca. -600 mV versus the normal hydrogen electrode) and corresponding slow rates of nitro reduction. To address these limitations, a series of 4-nitroaniline mustards bearing hydrophilic side chains attached via an electron-withdrawing carboxamide group was prepared and evaluated for hypoxia-selective cytotoxicity against Chinese hamster cell lines. The N-[(N,N-dimethylamino)ethyl]carboxamide derivatives proved to have excellent aqueous solubility and improved cytotoxic potency, but their reduction potentials, while higher than the non-carboxamide compounds, were still low and little selectivity for hypoxic cells were observed. A series of carboxamides of 2,4-dinitroaniline mustard was also prepared. These compounds had reduction potentials in the desired range (E1/2 ca. -450 mV by cyclic voltammetry) and were more toxic to hypoxic than aerobic UV4 cells. The most selective compounds were 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (20, SN 23862) and its water-soluble N-[(N,N-dimethylamino)ethyl]carboxamide analogue. These showed selectivities of 60- to 70-fold for hypoxic UV4 cells. The selectivity of 20 was much superior to that of its aziridine analogue (23, CB 1954), which was only 3.6-fold more toxic to hypoxic than oxic cells in the same system. Compound 20 is a much less efficient substrate than CB 1954 for the major aerobic nitroreductase from rat Walker tumor cells, NAD(P)H:quinone oxidoreductase (DT diaphorase). Lack of aerobic bioactivation of 20 by DT diaphorases may be responsible for its higher hypoxic selectivity than that of 23.
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PMID:Hypoxia-selective antitumor agents. 5. Synthesis of water-soluble nitroaniline mustards with selective cytotoxicity for hypoxic mammalian cells. 150 7

DT-diaphorase is a unique two electron (2e) donating reductase catalyzing either bioactivation or bioprotection reactions. Using human and rodent DT-diaphorase preparations (cell extracts and purified enzyme) we have characterized the reductive metabolism of the hypoxic cell cytotoxins EO9, mitomycin C (MMC), CB 1954, and SR 4233 in vitro. Drug metabolism was assayed spectrophotometrically or by HPLC, with dicoumarol as a selective inhibitor. DNA damage was measured using an agarose gel mobility technique with plasmid pBR322 DNA. The developmental indoloquinone, EO9, was metabolized by both rat Walker and human HT29 tumor DT-diaphorases. Reduction proceeded 5-fold more efficiently with the rat than the human tumor enzyme and resulted in single-strand breaks in plasmid DNA. The structurally related MMC was metabolized much more slowly than EO9 by the rat Walker tumor enzyme and there was no detectable reaction with the human HT29 tumor DT-diaphorase. No DNA damage was seen with MMC for either enzyme. The dinitrophenylaziridine CB 1954 was reduced by both human and rat enzymes forming, preferentially, the highly toxic 4-hydroxylamine as a 4e reduction product. Rates were 3-fold lower than for the human tumor enzyme. SR 4233 was also reduced by the rat tumor enzyme predominantly via 4e reduction to the benzotriazine SR 4330, in a novel reaction mechanism. This appears to be a bioprotection pathway that bypasses the toxic 1e radical formed by other reductases. Such information may be valuable in the selection of hypoxic cell cytoxins to treat human tumors high or low in DT-diaphorase and should facilitate 'enzyme-directed' analogue development.
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PMID:The role of human and rodent DT-diaphorase in the reductive metabolism of hypoxic cell cytotoxins. 154 31

5-(Aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is the active form of CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). This hydroxylamine is formed by the bioreduction of CB 1954 by the enzyme DT diaphorase and accounts for the highly selective cytotoxicity of this compound. The reason why the hydroxylamine derivative is so cytotoxic is that, in contrast to CB 1954, it can react difunctionally as characterized by the formation of DNA-DNA interstrand crosslinks in cells treated by this agent. However, although the 4-hydroxylamine compound can produce these crosslinks in cells it cannot crosslink naked DNA (Knox et al., Biochem Pharmacol 37: 4661-4669, 1988). We show here that 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide can become a species capable of binding to DNA and producing interstrand crosslinks, by a direct, non-enzymatic reaction with either acetyl coenzyme A, butyl and propyl coenzyme A or S-acetylthiocholine. Coenzyme A itself cannot produce these effects. The major product of the reaction between the 4-hydroxylamine and thioesters was identified as 4-amino-5-(aziridin-1-yl)-2-nitrobenzamide. However, this compound is not capable of producing the above effects and the major DNA reactive species was a minor product of the reaction. It is proposed that the ultimate, DNA reactive, derivative of CB 1954 is 4-(N-acetoxy)-5-(aziridin-1-yl)-2-nitrobenzamide.
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PMID:Bioactivation of CB 1954: reaction of the active 4-hydroxylamino derivative with thioesters to form the ultimate DNA-DNA interstrand crosslinking species. 193 Feb 94

The kinetic characteristics of the diaphorase activities associated with the NADH-dependent nitrite reductase (EC 1.6.6.4) from Escherichia coli have been determined. The values of the apparent maximum velocity are similar for the reduction of Fe(CN)6(3)-and mammalian cytochrome c by NADH. These reactions may therefore have the same rate-limiting step. NAD+ activates NADH-dependent reduction of cytochrome c, and the apparent maximum velocity for this substrate increases more sharply with the concentration of NAD+ than for hydroxylamine. The simplest explanation is that NAD+ activation of hydroxylamine reduction derives solely from activation of steps involved in the reduction of cytochrome c, a flavin-mediated reaction, but these steps are only partly rate-limiting for the reduction of hydroxylamine. At 0.5 mM-NAD+, the apparent maximum velocity was 2.3 times higher for 0.1 mM-cytochrome c as substrate than for 100 mM-hydroxylamine, suggesting that the rate-limiting step during hydroxylamine reduction is a step that is not involved in cytochrome c reduction. A scheme is proposed that can account for the pattern of variation with [NAD+] of the Michaelis-Menten parameters for hydroxylamine and for NADH with hydroxylamine or cytochrome c as oxidized substrate.
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PMID:The steady state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K12. The reduction of single-electron acceptors. 628 3

Diethyl pyrocarbonate inhibited diaphorase activity of ferredoxin-NADP+ oxidoreductase with a second-order rate constant of 2 mM-1 X min-1 at pH 7.0 and 20 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity could be restored by hydroxylamine, and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Derivatization of tyrosyl residues was also evident, although with no effect on the diaphorase activity. Both NADP+ and NADPH protected the enzyme against inactivation, suggesting that the modification occurred at or near the nucleotide binding domain. The reductase lost all of its diaphorase activity after about two histidine residues had been blocked by the reagent. In differential-labeling experiments with NADP+ as protective agent, it was shown that diaphorase inactivation resulted from blocking of only one histidyl residue per mole of enzyme. Modified reductase did not bind pyridine nucleotides. Modification of the flavoprotein in the presence of NADP+, i.e., with full preservation of diaphorase activity, resulted in a significant impairment of cytochrome c reductase activity, with a second-order rate constant for inactivation of about 0.5 mM-1 X min-1. Reversal by hydroxylamine and spectroscopic data indicated that this second residue was also a histidine. Ferredoxin afforded only slight protection against this inhibition. Conversely, carbethoxylation of the enzyme did not affect complex formation with the ferrosulfoprotein. Redox titration of the modified reductase with NADPH and with reduced ferredoxin suggested that the second histidine might be located in the electron pathway between FAD and ferredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Essential histidyl residues of ferredoxin-NADP+ oxidoreductase revealed by diethyl pyrocarbonate inactivation. 668 70

Liver cytosolic fractions are known to catalyze the reduction of certain C-nitroso compounds to their corresponding hydroxylamines and amines. Alcohol dehydrogenase (ADH), NAD(P)H:quinone oxidoreductase, and xanthine and aldehyde oxidases have been implicated as C-nitroso reductases. To probe the role of these cytosolic enzymes in the reduction of C-nitroso compounds we have studied the effects of classical inhibitors of these enzymes on the ability of liver cytosolic fractions from ADH+ and ADH- deermice to reduce p-nitrosophenol to p-aminophenol. Pyrazole, a potent inhibitor of ADH, inhibited NADH-p-nitrosophenol reduction by ADH+ cytosol by > 85%. Thus, ADH contributes substantially to NADH-C-nitroso reduction by cytosol from ADH+ deermice. The NAD(P)H:quinone oxidoreductase inhibitor, dicumarol, inhibited NADH-dependent p-aminophenol formation by about 25%; however, dicumarol potently inhibited the NADPH-dependent formation (90-95%). As expected, cytosol from ADH- deermice did not catalyze pyrazole-sensitive (ADH-dependent) C-nitroso reduction with NADH as the cofactor. Both NADPH- and NADH-p-nitrosophenol reduction by ADH- cytosol were inhibited > 90% by dicumarol. The xanthine oxidase/aldehyde oxidase inhibitor, allopurinol, was without effect on NAD(P)H cytosolic p-nitrosophenol reduction from ADH- and ADH+ deermice under either aerobic or anaerobic conditions. Our findings suggest that in the ADH+ animal, ADH contributes significantly to NADH-dependent C-nitroso reduction by cytosol relative to NAD(P)H:quinone oxidoreductase. NADPH-dependent p-nitrosophenol reduction by liver cytosol of ADH+ animals is mostly dicumarol-sensitive, which implicates NAD(P)H:quinone oxidoreductase as the major NADPH-dependent activity. In ADH- deermice, both NADH- and NADPH-dependent p-nitrosophenol reduction are essentially dicumarol-sensitive (NAD(P)H:quinone oxidoreductase-dependent). Because the toxic expression of C-nitroso compounds is mediated by hydroxylamine intermediates, the present data indicate the importance of considering the role of ADH in the toxic sequelae of nitro and nitroso arenes.
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PMID:p-nitrosophenol reduction by liver cytosol from ADH-positive and -negative deermice (Peromyscus maniculatus). 753 87

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives. In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine. This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA. In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases. Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E. coli enzyme and cofactor (NADH). Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme. None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells. For those compounds which were substrates for the E. coli nitroreductase, there was a positive correlation between kcat and IC50 ratio. Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1. This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system.
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PMID:Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase. 766 63

5-(Aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, the active form of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), can react spontaneously with oxygen, and in aqueous solution yields 5-(aziridin-1-yl)-2-nitro-4-nitrosobenzamide and hydrogen peroxide. Mild biological reducing agents such as NAD(P)H, reduced thiols and ascorbic acid rapidly re-reduced the nitroso compound to the hydroxylamine. Both compounds were equally efficient at inducing cytotoxicity and DNA interstrand crosslinking in cells when exposed in phosphate-buffered saline (PBS). Neither agent was capable of inducing cross-links in isolated DNA. When acetyl coenzyme A was included in the incubation, crosslink formation was seen with the hydroxylamine, but not with the nitroso compound. Thus, the nitroso compound is acting as a prodrug for the hydroxylamine, and needs to be reduced to this compound to exert its cytotoxic effects. In vivo anti-tumour tests showed that neither compound was effective in its own right. This may be due to the rapid reduction of the nitroso to the hydroxylamine, and the reaction of the hydroxylamine with serum proteins. The chemical synthesis of the 5-(aziridin-1-yl)-2-nitro-4-nitrosobenzamide, and an improved synthesis of 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is described. These results emphasize the potential efficacy of the in situ activation of prodrugs such as CB1954 either by endogenous enzymes such as DT diaphorase, or by antibody directed enzyme prodrug therapy (ADEPT).
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PMID:Identification, synthesis and properties of 5-(aziridin-1-yl)-2-nitro-4-nitrosobenzamide, a novel DNA crosslinking agent derived from CB1954. 837 33

Walker cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). The basis of the sensitivity is that CB 1954 forms DNA interstrand crosslinks in Walker cells but not in insensitive cells. Crosslink formation is due to the aerobic reduction of CB 1954 to form 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide by the enzyme DT diaphorase. The 4-hydroxylamine can not crosslink DNA directly but requires further activation by a non-enzymatic reaction with a thioester (such as acetyl coenzyme A). As predicted from their measured DT diaphorase activities, a number of rat hepatoma and hepatocyte cell lines are also sensitive to CB 1954. However, no CB 1954-sensitive tumours or cell lines of human origin have been found. This is because the rate of reduction of CB 1954 by the human form of DT diaphorase is much lower than that of the Walker enzyme (ratio of kcat = 6.4). To overcome this intrinsic resistance of human cells towards CB 1954 a number of strategies have been developed. First, analogues have been developed that are more rapidly reduced by the human form of CB 1954. Second, the cytotoxicity of CB 1954 can be potentiated by reduced pyridinium compounds. Third, a CB 1954 activating enzyme can be targeted to human tumours by conjugating it to an antibody (ADEPT). A nitroreductase enzyme has been isolated from E. coli that can bioactivate CB 1954 much more rapidly than Walker DT diaphorase and is very suitable for ADEPT. Thus CB 1954 may have a role in the therapy of human tumours.
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PMID:The bioactivation of CB 1954 and its use as a prodrug in antibody-directed enzyme prodrug therapy (ADEPT). 837 21

We have synthesized a number of nitrobenzimidazoles containing nitro groups in the benzene ring and found that they acted as relatively efficient substrates for rat liver DT-diaphorase (EC 1.6.99.2), their reactivity exceeding reactivities of nitrofurans and nitrobenzenes. Nitrobenzimidazoles were competitive with NADPH inhibitors of DT-diaphorase in menadione reductase reactions, their inhibition constant being unchanged in the presence of dicumarol and being increased in the presence of 2',5'-ADP. These data indicate that the poor reactivity of nitrobenzimidazoles and other nitroaromatics in comparison to quinones could be determined by their binding in the adenosine-phosphate binding region of the NADPH-binding site, whereas quinones bind at the nicotinamide-binding pocket at the vicinity of FAD of DT-diaphorase. The reduction of 4,5,6-trinitrobenzimidazol-2-one by DT-diaphorase most probably involves reduction of 5-nitro group to 5-nitroso or 5-hydroxylamine derivative at the initial step. A certain parallelism existed between reactivities of nitrobenzimidazoles toward DT-diaphorase and their reactivities in single-electron reduction by Anabaena ferredoxin:NADP+ reductase (EC 1.18.1.2) and Saccharomyces cerevisiae flavocytochrome b2 (EC 1.1.2.3), the latter being determined by electronic factors. However, we suppose that the relatively high reactivity of polinitrobenzimidazoles toward DT-diaphorase was due not only to electronic effects, but also to a sterical crowding of nitrogroups by each other. The toxicity of nitrobenzimidazoles to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) with a moderate amount of DT-diaphorase (260 U/mg protein) is partly prevented by dicumarol. That points out to partial determination of nitrobenzimidazole cytotoxicity by their reduction by DT-diaphorase. Another important factor of nitrobenzimidazole toxicity to this cell line was oxidative stress, catalyzed by single-electron transferring enzymes.
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PMID:Nitrobenzimidazoles as substrates for DT-diaphorase and redox cycling compounds: their enzymatic reactions and cytotoxicity. 934 69


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