Drug residue formation from ronidazole, a 5-nitroimidazole. VI. Lack of mutagenic activity of reduced metabolites and derivatives of ronidazole

The potential toxicity of ronidazole residues present in the tissues of food-producing animals was assessed using the Ames mutagenicity test. Since ronidazole is activated by reduction, reduced derivatives of ronidazole and metabolites formed by enzymatic reduction of ronidazole were tested for mutagenicity. When tested at levels several orders of magnitude higher than that at which ronidazole was mutagenic, 5-amino-4-S-cysteinyl-1,2- dimethylimidazole , a product of the dithionite reduction of ronidazole in the presence of cysteine, the 5-N-acetylamino derivative of ronidazole and 5-amino-1,2- dimethylimidazole all lacked mutagenic activity in Ames strain TA100. The metabolites of ronidazole formed by the incubation of ronidazole with microsomes under anaerobic conditions were also not mutagenic. These data demonstrate that although ronidazole is a potent mutagen, residues from it which may be present in the tissues of food-producing animals lack any mutagenic activity.     

Drug residue formation from ronidazole, a 5-nitroimidazole. III. Studies on the mechanism of protein alkylation in vitro

Ronidazole (1-methyl-5-nitroimidazole-2-methanol carbamate) is reductively metabolized by liver microsomal and purified NADPH-cytochrome P-450 reductase preparations to reactive metabolites that covalently bind to tissue proteins. Kinetic experiments and studies employing immobilized cysteine or blocked cysteine thiols have shown that the principal targets of protein alkylation ara cysteine thiols. Furthermore, ronidazole specifically radiolabelled with 14C in the 4,5-ring, N-methyl or 2-methylene positions give rise to equivalent apparent covalent binding suggesting that the imidazole nucleus is retained in the bound residue. In contrast, the carbonyl-14C-labeled ronidazole gives approx. 6--15-fold less apparent covalent binding indicating that the carbamoyl group is lost during the reaction leading to the covalently bound metabolite. The conversion of ronidazole to reactive metabolite(s) is quantitative and reflects the amazing efficiency by which this compound is activated by microsomal enzymes. However, only about 5% of this metabolite can be accounted for as protein-bound products under the conditions employed in these studies. Consequently, approx. 95% of the reactive ronidazole metabolite(s) can react with other constituents in the reaction media such as other thiols or water. Based on these results, a mechanism is proposed for the metabolic activation of ronidazole.     

Nitro analogs of substrates for argininosuccinate synthetase and argininosuccinate lyase

The nitro analogs of aspartate and argininosuccinate were synthesized and tested as substrates and inhibitors of argininosuccinate synthetase and argininosuccinate lyase, respectively. The Vmax for 3-nitro-2-aminopropionic acid was found to be 60% of the maximal rate of aspartate utilization in the reaction catalyzed by argininosuccinate synthetase. Only the nitronate form of this substrate, in which the C-3 hydrogen is ionized, was substrate active, indicating a requirement for a negatively charged group at the beta carbon. The V/K of the nitro analog of aspartate was 85% of the value of aspartate after correcting for the percentage of the active nitronate species. The nitro analog of argininosuccinate, N3-(L-1-carboxy-2-nitroethyl)-L-arginine, was a strong competitive inhibitor of argininosuccinate lyase but was not a substrate. The pH dependence of the observed pKi was consistent with the ionized carbon acid (pK = 8.2) in the nitronate configuration as the inhibitory material. The pH-independent pKi of 2.7 microM is 20 times smaller than the Km of argininosuccinate at pH 7.5. These results suggest that the tighter binding of the nitro analog relative to the substrate is due to the similarity in structure to a carbanionic intermediate in the reaction pathway.     

Drug residue formation from ronidazole, a 5-nitroimidazole. I. Characterization of in vitro protein alkylation

The metabolic activation of [14C]ronidazole by rat liver enzymes to metabolite(s) bound to macromolecules was investigated. The alkylation of protein by [14C]ronidazole metabolite(s) was catalyzed most efficiently by rat liver microsomes, in the absence of oxygen utilizing NADPH as a source of reducing equivalents. Based on a comparison of total ronidazole metabolized versus the amount bound to microsomal protein, approximately one molecule alkylates microsomal protein for every 20 molecules of ronidazole metabolized. Protein alkylation was strongly inhibited by sulfhydryl-containing compounds such as cysteine and glutathione whereas methionine had no effect. Based on HPLC analysis of ronidazole, cysteine was found not to inhibit microsomal metabolism of ronidazole ruling out a decrease in the rate of production of the reactive metabolite(s) as the mechanism of cysteine inhibition.     

Fatty acid acylation of the fusion glycoprotein of human respiratory syncytial virus

We describe the covalent attachment of palmitate to the fusion glycoprotein of respiratory syncytial virus and the identification of the attachment site. Labeling of respiratory syncytial virus-infected Vero cells with [3H]palmitate, followed by the purification and subsequent analysis of the fusion glycoprotein in conjunction with polyacrylamide gel electrophoresis, demonstrated that the fatty acid is covalently attached to the F1 subunit of the fusion glycoprotein. The bound palmitate was sensitive to 1 M hydroxylamine at neutral pH. In addition, the release of palmitate label by reduction with sodium borohydride showed that the palmitate is linked to the protein through a thioester bond. Isolation of a radiolabeled peptide from a tryptic digest of the protein and subsequent amino-terminal sequence analysis revealed that the cysteine residue (amino acid residue 550) within the anchor sequence, located at the carboxyl terminus of the F1 subunit, is the covalent attachment site for palmitate.     

Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme

The antibiotics nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant approximately 10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-nitrofurazone complex, nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.     

Structural arrangement in the alpha 2-macroglobulin--thrombin complex

The cysteine sulfhydryl groups of alpha 2-macroglobulin (alpha 2M) generated upon thrombin complex formation are in contact with the proteinase surface as evidenced by singlet--singlet energy transfer measurements from N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid-labeled thiol functions of alpha 2M to fluorescein isothiocyanate-labeled thrombin. The thrombin-alpha 2M binding is normally covalent, but the presence of hydroxylamine during the reaction leads to the formation of a non-covalent complex. The transfer energy determinations show that the alpha 2M binding sites of thrombin are quite similar, whatever covalent or non-covalent binding occurs.     

Effects of species, MFO inducers and conjugation agents on the in vitro covalent binding of 14C-3-methylindole metabolite in liver and lung tissues

The degree of tissue covalent binding of 14C-3-methylindole metabolite in goat and rat pretreated with phenobarbital or 3-methylcholanthrene was compared. The effect of conjugating agents, i.e. glutathione (GSH), cysteine and sulfate, in reducing the degree of tissue covalent binding was measured. The degree of tissue covalent binding was significantly higher in the lung than the liver of goats. In rats, covalent binding was higher in the liver than the lung. Glutathione and cysteine were effective in decreasing the degree of in vitro covalent binding in both liver and lung tissues of goat and rat.     

A structure-function study of ligand recognition by CD22beta

B-cell-specific CD22 is a member of a group of cell adhesion molecules within the immunoglobulin superfamily that display binding to glycans with terminal sialic acid residues. Binding of endogenous ligands to CD22 triggers B-cell activation and proliferation. It is therefore conceivable that high affinity ligands for CD22 may be of value as inhibitors of B-cell activation in allergy and chronic inflammation. In this study, we aimed to delineate the structural requirements for ligand binding to CD22. A library of 20 mono-, di-, and trisaccharide analogs of the basic binding motif Neu5Ac(alpha2,6)Lac was synthesized and screened for affinity for CD22beta. In general, CD22 ligand recognition appeared to be rather tolerant with respect to structural modifications of the anomeric sugar on a mono-, di-, and trisaccharide level, although affinity was increased by the presence of a nitro aromatic group at C-2. The most potent monovalent ligand, Neu5Ac-4-nitrobenzoyl-Glc, was selected to generate multivalent ligands based on either a glutamate or Tris cluster core. All multivalent ligands displayed at least a 10-fold increased affinity for CD22 compared with the corresponding monovalent glycoside. Interestingly, a maximal gain in affinity was already obtained for bivalent ligands, regardless of the terminal glycoside. A trivalent Tris-based cluster of Neu5Ac-4-nitrobenzoyl-Glc displayed a 300-fold higher affinity compared with the basic binding motif, which makes it, to our knowledge, the most potent antagonist for CD22 yet synthesized. As our in vitro fluorescence-activated cell sorting studies demonstrated efficient cellular uptake of a CD22 substrate, the most potent ligand in this study may hold promise as a homing device for immunomodulatory compounds and cytostatics.     

Sequence-specific and mechanism-based crosslinking of Dcm DNA cytosine-C5 methyltransferase of E. coli K-12 to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine.

The product of the dcm gene is the only DNA cytosine-C5 methyltransferase of Escherichia coli K-12; it catalyses transfer of a methyl group from S-adenosyl methionine (SAM) to the C-5 position of the inner cytosine residue of the cognate sequence CCA/TGG. Sequence-specific, covalent crosslinking of the enzyme to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine is demonstrated. This reaction is abolished if serine replaces the cysteine at residue #177 of the enzyme. These results lend strong support to a catalytic mechanism in which an enzyme sulfhydryl group undergoes Michael addition to the C5-C6 double bond, thus activating position C-5 of the substrate DNA cytosine residue for electrophilic attack by the methyl donor SAM. The enzyme is capable of self-methylation in a DNA-independent reaction requiring SAM and the presence of cysteine at position #177.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest: I. The Influence of Solvent

The electrochemical properties of three nitroimidazoles, a nitropyrazole, a nitrofuran and three nitroben-zenoid compounds have been extensively investigated in a range of solvents. The reduction pathway for the nitro group is independent of the cyclic function to which it is attached, but is strongly influenced by the nature of the solvent. In aqueous media, generally, a single, irreversible 4-electron reduction occurs to give the hydroxylamine. In aprotic media (dimethylformamide, methylene chloride or dimethylsulphoxide), a reversible one-electron reduction takes place to form a stable nitro radical anion. At more negative values, a further 3-electron reduction occurs, irreversibly to give the hydroxylamine. In mixed aqueous-organic systems, intermediate behaviour is found, with the reversibility of the RNO₂/RNO₂^- couple increasing with addition of organic medium. The control of the reduction pathway, by changing the electrolytic medium is discussed in relation to the biological activities of the drugs and identification of the short-lived reduction intermediate responsible for DNA damage.     

Synthesis and antiviral evaluation of acyclic azanucleosides developed from sulfanilamide as a lead structure

The acyclic azanucleosides with 2-, 3-, or 4-aminobenzenesulfonyl function at the nitrogen atom of the sugar mimic were prepared by coupling of 2-, 3-, or 4-nitro-N-(2-pivaloyloxyethyl)-N-(pivaloyloxymethyl)benzenesulfonamide with the silylated pyrimidine nucleobases followed by the reduction of the nitro group with sodium dithionite in aqueous solution or the palladium-catalysed transfer hydrogenation. The azanucleosides were evaluated for, but found to be devoid of, activity against several RNA- and DNA-viruses in vitro.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest: I. The Influence of Solvent

The electrochemical properties of three nitroimidazoles, a nitropyrazole, a nitrofuran and three nitroben-zenoid compounds have been extensively investigated in a range of solvents. The reduction pathway for the nitro group is independent of the cyclic function to which it is attached, but is strongly influenced by the nature of the solvent. In aqueous media, generally, a single, irreversible 4-electron reduction occurs to give the hydroxylamine. In aprotic media (dimethylformamide, methylene chloride or dimethylsulphoxide), a reversible one-electron reduction takes place to form a stable nitro radical anion. At more negative values, a further 3-electron reduction occurs, irreversibly to give the hydroxylamine. In mixed aqueous-organic systems, intermediate behaviour is found, with the reversibility of the RNO₂/RNO₂^? couple increasing with addition of organic medium. The control of the reduction pathway, by changing the electrolytic medium is discussed in relation to the biological activities of the drugs and identification of the short-lived reduction intermediate responsible for DNA damage.     

Reaction of 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase with hydrogen peroxide. Formation of a covalent flavin-protein linkage

Hydrogen peroxide reacts with 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase to yield a long wavelength intermediate (lambda max = 360, 620 nm) which can be isolated in stable form on removal of excess H2O2. The blue flavin derivative slowly decays in a second peroxide-dependent reaction to yield a new flavin product lacking long wavelength absorbance (lambda max = 408, 472 nm). This final peroxide-modified enzyme binds p-hydroxybenzoate with a 10-fold lower affinity than does the native enzyme; furthermore, substrate binding leads to the inhibition of enzyme reduction by NADPH. Trichloroacetic acid treatment of the final peroxide-modified enzyme results in the quantitative conversion of the bound flavin to free FAD. However, gel filtration of the modified enzyme in guanidine hydrochloride at neutral pH leads to the co-elution of protein and modified flavin. The nondenatured peroxide product reacts rapidly with hydroxylamine to yield 2-NHOH-substituted FAD. These observations indicate that the secondary reaction of peroxide with the blue intermediate from 2-thio-FAD p-hydroxybenzoate hydroxylase results in the formation of an acid-labile covalent flavin-protein linkage within the enzyme active site, involving the flavin C-2 position.     

Analysis of transmembrane segment 7 of the dipeptide transporter hPepT1 by cysteine-scanning mutagenesis

To investigate the involvement of transmembrane segment 7 (TMS7) of hPepT1 in forming the putative central aqueous channel through which the substrate traverses, we individually mutated each of the 21 amino acids in TMS7 to a cysteine and analyzed the mutated transporters using the scanning cysteine accessibility method. Y287C- and M292C-hPepT1 did not express at the plasma membrane. Out of the remaining 19 transporters, three (F293C-, L296C-, and F297C-hPepT1) showed negligible glycyl-sarcosine (gly-sar) uptake activity and may play an important role in defining the overall hPepT1 structure. K278C-hPepT1 showed approximately 40% activity and the 15 other transporters exhibited more than 50% gly-sar uptake when compared with wild type (WT)-hPepT1. Gly-sar uptake for the 16 active transporters containing cysteine mutations was then measured in the presence of 2.5 mM 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 1 mM [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET). Gly-sar uptake was significantly inhibited for each of the 16 single cysteine mutants in the presence of 2.5 mM MTSEA. In contrast, significant inhibition of uptake was only observed for K278C-, M279C-, V280C-, T281C-, M284C-, L286C-, P291C-, and D298C-hPepT1 in the presence of 1 mM MTSET. MTSET modification of R282C-hPepT1 resulted in a significant increase in gly-sar uptake. To investigate this further, we mutated WT-hPepT1 to R282A-, R282E-, and R282K-hPepT1. R282E-hPepT1 showed a 43% reduction in uptake activity, whereas R282A- and R282K-hPepT1 had activities comparable with WT-hPepT1, suggesting a role for the Arg-282 positive charge in substrate translocation. Most of the amino acids that were MTSET-sensitive upon cysteine mutation, including R282C, are located toward the intracellular end of TMS7. Hence, our results suggest that TMS7 of hPepT1 is relatively solvent-accessible along most of its length but that the intracellular end of the transmembrane domain is particularly so. From a structure-function perspective, we speculate that the extracellular end of TMS7 may shift following substrate binding, providing the basis for channel opening and substrate translocation.     

The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.

The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions.     

Peptide ketobenzoxazole inhibitors bound to cathepsin K

Potent inhibitors of human cysteine proteases of the papain family have been made and assayed versus a number of relevant family members. We describe the synthesis of peptide alpha-ketoheterocyclic inhibitors that occupy binding subsites S1'-S3 of the cysteine protease substrate recognition cleft and that form a reversible covalent bond with the Cys 25 nucleophile. X-ray crystal structures of cathepsin K both unbound and complexed with inhibitors provide detailed information on protease/inhibitor interactions and suggestions for the design of tight-binding, selective molecules.     

Mutagenicity in Salmonella typhimurium TA98 and TA100 of nitroso and respective hydroxylamine compounds

Five aromatic nitroso compounds were prepared and their mutagenicity in Salmonella typhimurium strains TA98 and TA100 compared with that of the corresponding hydroxylamines and the previously studied nitroarenes. A remarkable correspondence of the dose-response curves was observed between the nitroso and the respective hydroxylamine compounds. This effect could be observed in TA98 and TA100. It was only marginally dependent on the metabolical activation by rat liver S9-mix. Even the presence of a bulky alkyl substituent either near to the functional group, or far away from it, previously shown to considerably influence the mutagenic properties of nitroarenes, does not remarkably affect the properties of the nitroso and hydroxylamine species. The similarity between the latter two is likely to be due to a fast reduction of the nitrosoarenes to the hydroxylamine species under the test conditions. It seems that enzymes are not responsible for that reduction step, because sterical crowding near the functional group does not influence that behaviour.The test results of the aromatic hydroxylamines bearing a bulky substituent show that there are at least two ways to influence the mutagenicity of an aromatic nitro compound by such a group. A substituent near the functional group (ortho-position) disturbs the enzymatic reduction of the nitro group, because 3-tert-butyl-4-hydroxylaminobiphenyl and its corresponding nitroso compound are highly mutagenic, whereas 3-tert-butyl-4-nitrobiphenyl was previously shown to be inactive even after addition of S9-mix. In contrast, 4'-tert-butyl-4-hydroxylaminobiphenyl with the tert-butyl group "far away" from the hydroxylamino functionality clearly shows decreased mutagenic activity suggesting a different influence of a substituent in that position. In addition, the substance shows only little cell toxicity even at higher concentrations. Both effects could be due to a reduced effective dose of the hydroxylamine in the cells compared to the non-alkylated compound, caused by a faster degradation of the hydroxylamine or a hindered interaction between that substance and the cells.     

Metabolites of procainamide and practolol inhibit complement components C3 and C4.

Drug-induced systemic lupus erythematosus arises from toxic side-effects of administration of hydralazine, isoniazid, procainamide and practolol. Hydralazine and isoniazid are nucleophilic drugs and inhibit the covalent binding reaction of complement components, C3 and C4, an effect likely to lead to deposition of immune complexes (a feature of systemic lupus erythematosus). Procainamide and practolol do not themselves inhibit C3 and C4. A range of metabolites and putative metabolites of procainamide and practolol were synthesized, and tested for their ability to inhibit the covalent binding reactions of C3 and C4. The highly nucleophilic hydroxylamine metabolite of procainamide was strongly inhibitory in both tests, as was a putative hydroxylamine metabolite of practolol. These studies indicate a potential role for the hydroxylamine metabolites in mediating the toxic side-effects of procainamide and practolol, and emphasize the need for adequate measurements of hydroxylamine metabolites in human tissue.     

Reactions of para-substituted nitrosobenzenes with human hemoglobin

Bio-monitoring the covalent binding of nitrosoarenes to the SH groups of human hemoglobin has been proposed as a reliable approach to get an integral parameter for exposure control and possibly risk assessment of persons exposed to aromatic amines and nitro compounds. Availability of nitrosoarenes to bind to the cysteine residues is greatly influenced by the competition of hemoglobin iron with nitrosoarenes. In contrast to earlier reports, we found that nitrosobenzene has a 14 fold higher affinity for "stripped" human hemoglobin than oxygen. The binding mode is similar to gaseous ligands and exhibits the same free energy of cooperation and sensitivity to heterotropic effectors like inositol hexaphosphate. To elucidate the electronic influence of para substituents, 4-chloronitrosobenzene, 4-nitrosotoluene and 4-nitrosophenetole were tested. A linear free energy relationship was found for all equilibrium parameters with a reaction constant rho = 3, when using Hammett sigma p constants. Similarly, the apparent second order rate constants for binding of para-substituted nitrosobenzenes to the cysteine residues (Cys beta 93) in hemoglobin followed the Hammett relationship with lg k-lg k0 = 1.7 X sigma p (r2 = 0.99). In case of 4-chloronitrosobenzene covalent binding proceeded biphasically and a "semimercaptal"-like intermediate was observed. The affinities for hemoglobin iron and for the SH groups were highest with 4-chloronitrosobenzene and lowest with 4-nitrosophenetole. All nitrosobenzenes were capable to produce ferrihemoglobin. In the absence of oxygen, 4-chloronitrosobenzene hemoglobin decayed with formation of ferrihemoglobin. Presumably the nitroxide radical anion is formed as an intermediate which comproportionates into the azoxy derivative. It is assumed that the efficiency of the microscopic compartmentation of nitrosoarenes by binding to hemoglobin iron has important impacts on the toxicokinetics of these compounds.