Bernd Clement

Identification of the Missing Component in the Mitochondrial Benzamidoxime Prodrug-converting System as a Novel Molybdenum Enzyme

Amidoximes can be used as prodrugs for amidines and related functional groups to enhance their intestinal absorption. These prodrugs are reduced to their active amidines. Other N-hydroxylated structures are mutagenic or responsible for toxic effects of drugs and are detoxified by reduction. In this study, a N-reductive enzyme system of pig liver mitochondria using benzamidoxime as a model substrate was identified. A protein fraction free from cytochrome b5 and cytochrome b5 reductase was purified, enhancing 250-fold the minor benzamidoxime-reductase activity catalyzed by the membrane-bound cytochrome b5/NADH cytochrome b5 reductase system. This fraction contained a 35-kDa protein with homologies to the C-terminal domain of the human molybdenum cofactor sulfurase. Here it was demonstrated that this 35-kDa protein contains molybdenum cofactor and forms the hitherto ill defined third component of the N-reductive complex in the outer mitochondrial membrane. Thus, the 35-kDa protein represents a novel group of molybdenum proteins in eukaryotes as it forms the catalytic part of a three-component enzyme complex consisting of separate proteins. Supporting these findings, recombinant C-terminal domain of the human molybdenum cofactor sulfurase exhibited N-reductive activity in vitro, which was strictly dependent on molybdenum cofactor.

Reduction of N-hydroxylated compounds: Amidoximes (N-hydroxyamidines) as pro-drugs of amidines

In order to examine the importance of metabolic cycles and in particular of reductions of N-hydroxylated compounds, the reversible metabolism at the amidine, guanidine, and amidinohydrazone nitrogen atoms of various drugs and model compounds was investigated. Many of these N-oxygenated metabolites are very easily reduced back into the starting materials. A comparison of the kinetic data for the N-hydroxylation and reduction suggests that the reduction should predominate in vivo. This could be verified by in vivo studies. Thus, N-hydroxylated amidines (amidoximes) can be used as pro-drugs of amidines. Because of their strong basicity, amidines, guanidines, and amidinohydrazones are protonated under physiological conditions, are very hydrophilic, and are usually not absorbed from the gastrointestinal tract. The N-hydroxylated derivatives of amidines (amidoximes), guanidines (N-hydroxyamidines), and amidinohydrazones (N-hydroxyamidinohydrazones) are less basic because of the introduction of the oxygen atom. They are absorbed from the gastrointestinal tract and then reduced to the active amidines, guanidines, and amidinohydrazones. The pro-drug principle was originally developed in our laboratory for pentamidine and then applied to other amidines such as sibrafiban and melagatran (ximelagatran). The enzymatic basis of N-oxidative processes is very well understood, whereas reductions have been less extensively investigated. We purified an enzyme system from pig and human liver consisting of cytochrome b5, its reductase, and a P450 enzyme, which is involved in the reduction of the N-hydroxylated compounds. Similar activities were found in all species studied so far. Furthermore, comparable reductive reactions could also be demonstrated with microsomal fractions from organs other than liver. In addition, mitochondria are highly capable of performing the reductions of these N-hydroxylated compounds. Thus, several organs and cell organelles are involved in the reduction explaining the extensive reduction of the pro-drugs in vivo underlying the suitability of the concept for drug development.

The Fourth Molybdenum Containing Enzyme mARC: Cloning and Involvement in the Activation of N-Hydroxylated Prodrugs

The recently discovered mammalian molybdoprotein mARC1 is capable of reducing N-hydroxylated compounds. Upon reconstitution with cytochrome b(5) and b(5) reductase, benzamidoxime, pentamidine, and diminazene amidoximes, N-hydroxymelagatran, guanoxabenz, and N-hydroxydebrisoquine are efficiently reduced. These substances are amidoxime/N-hydroxyguanidine prodrugs, leading to improved bioavailability compared to the active amidines/guanidines. Thus, the recombinant enzyme allows prediction about in vivo reduction of N-hydroxylated prodrugs. Furthermore, the prodrug principle is not dependent on cytochrome P450 enzymes.

In Vivo Spin Trapping of Glyceryl Trinitrate–Derived Nitric Oxide in Rabbit Blood Vessels and Organs

BACKGROUND The objectives of this study were (1) to assess glyceryl trinitrate (GTN)-derived nitric oxide (NO) formation in vascular tissues and organs of anesthetized rabbits in vivo, (2) to establish a correlation between tissue NO levels and a biological response, and (3) to verify biotransformation of GTN to NO by cytochrome P-450. METHODS AND RESULTS NO was trapped in tissues in vivo as a stable paramagnetic mononitrosyl-iron-diethyldithiocarbamate complex [NOFe(DETC)2]. After removal of the tissues, NO was determined by cryogenic electron spin resonance spectroscopy. NO formation in vitro was assessed by spin trapping and by activation of soluble guanylyl cyclase. The GTN-elicited decrease in coronary perfusion pressure was monitored in isolated, constant-flow perfused rabbit hearts. NO was not detected in control tissues. In GTN-treated rabbits, NO formation was higher in organs than in vascular tissues and higher in venous than in arterial vessels. In isolated hearts, ventricular NO levels and decreases in coronary perfusion pressure achieved by GTN were closely correlated. Purified cytochrome P-450 catalyzed NO formation from GTN in a P-450-NADPH reductase- and NADPH-dependent fashion. CONCLUSIONS Since GTN-derived NO formation in myocardial tissue correlates to the GTN-elicited vasodilator response, we conclude that GTN-derived NO detected in vivo correlates with the systemic effects of GTN. Therefore, the higher rate of NO formation detected in veins compared with arteries explains the preferential venodilator activity of GTN. High NO formation in cytochrome P-450-rich organs in vivo and efficient NO formation from GTN by cytochrome P-450 in vitro highlights the importance of this pathway for NO formation from GTN in the intact organism.

Detection of Metabolites of Fumaric Acid Esters in Human Urine: Implications for Their Mode of Action

Medvecz, Rachel Sajó, Réka Lepesi-Benk + o, Zsolt Tulassay, Mária Katona, Zsófia Hatvani, Antal Blazsek and Sarolta Kárpáti Semmelweis University, Department of Dermatology, Venerologie and Dermatooncology, Budapest, Hungary; Hungarian Academy of Sciences Semmelweis University Molecular Medicine Research Group, Budapest, Hungary and Szentágothai Regional Knowledge Centre, Semmelweis University, Budapest, Hungary E-mail: bona@bor.sote.hu

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b5, and NADH/FAD-dependent cytochrome b5 reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo- or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.

Cytochrome P450 dependent N-hydroxylation of a guanidine (debrisoquine), microsomal catalysed reduction and further oxidation of the N-hydroxy-guanidine metabolite to the urea derivative: Similarity with the oxidation of arginine to citrulline and nitric oxide

The microsomal N-hydroxylation of the strongly basic guanidinium group (debrisoquine) to N-hydroxyguanidine (N-hydroxydebrisoquine) and the retroreduction of the N-hydroxyguanidine are demonstrated for the first time. The reduction of the N-hydroxyguanidine by liver homogenates and hepatocytes is catalysed by a microsomal NADH-dependent system that is strongly inhibited by hydroxylamine or N-methylhydroxylamine. In the presence of these alternate substrates for the reductase the microsomal catalysed N-hydroxylation of debrisoquine is readily characterized. The oxidation was inhibited by antibodies against NADPH cytochrome P450 reductase and the role of the P450 monooxygenase was further verified by studies with partially purified and purified P450 2C3 reconstituted systems. The transformation of N-hydroxydebrisoquine to the corresponding urea derivative was also detected in in vitro experiments with microsomal fractions and enriched P450 fractions as well as with flavin-containing monooxygenase (FMO). Experiments with catalase, superoxide dismutase and H2O2 have shown that the H2O2 or O2-, respectively, formed from the respective enzyme and the substrate, apparently participated in the reaction. Whereas the N-hydroxylation of the guanidine involves the usual monooxygenase activity of cytochrome P450 the resultant N-hydroxyguanidine decouples monooxygenases (cytochrome P450, FMO) and the H2O2 and, above all, O2- thus formed transform the N-hydroxyguanidine further to the corresponding urea derivative. The possibility for the N-hydroxylation of non-physiological guanidines to N-hydroxyguanidines and subsequent oxidative conversion to the respective urea is comparable to the physiological transformation of arginine to citrulline via N-hydroxyarginine with the liberation of nitric oxide (endothelial derived relaxing factor) and could, therefore, contribute to the efficacy of drugs containing guanidine and similar functional groups.

ISOLATION AND CHARACTERIZATION OF THE PROTEIN COMPONENTS OF THE LIVER MICROSOMAL O2-INSENSITIVE NADH-BENZAMIDOXIME REDUCTASE

Drugs containing strong basic nitrogen functional groups can be N-oxygenated to genotoxic products. While the reduction of such products is of considerable toxicological significance, most in vitro studies have focused on oxygen-sensitive reductase systems. However, an oxygen-insensitive microsomal hydroxylamine reductase consisting of NADH, cytochromeb 5, its reductase, and a third unidentified protein component has been known for some time (Kadlubar, F. F., and Ziegler, D. M. (1974) Arch. Biochem. Biophys. 162, 83–92). This report describes the isolation and identification of all of the components required for the reconstitution of an oxygen-insensitive liver microsomal system capable of catalyzing the efficient reduction of primary N-hydroxylated structures such as amidines, guanidines, amidinohydrazones, and similar functional groups. In addition to cytochrome b 5 and its reductase, the reconstituted system requires phosphatidylcholine and a P450 isoenzyme that has been purified to homogeneity from pig liver. The participation of cytochrome b 5 and NADH cytochrome b 5 reductase in cytochrome P450-dependent biotransformations has previously only been described for oxidative processes. The data presented suggest that this system may be an important catalyst in the reduction of genotoxicN-hydroxylated nitrogen components in liver. Their facile reduction by cellular NADH may be the reason whyN-hydroxylated products can be missed by studies in vivo. Furthermore, the enzyme system is involved in the reduction of amidoximes and similar functional groups, which can be used as prodrug functionalities for amidines and related groups.

Reduction of N ω -hydroxy-L-arginine by the mitochondrial amidoxime reducing component (mARC)

NOSs (nitric oxide synthases) catalyse the oxidation of L-arginine to L-citrulline and nitric oxide via the intermediate NOHA (N(ω)-hydroxy-L-arginine). This intermediate is rapidly converted further, but to a small extent can also be liberated from the active site of NOSs and act as a transportable precursor of nitric oxide or potent physiological inhibitor of arginases. Thus its formation is of enormous importance for the nitric-oxide-generating system. It has also been shown that NOHA is reduced by microsomes and mitochondria to L-arginine. In the present study, we show for the first time that both human isoforms of the newly identified mARC (mitochondrial amidoxime reducing component) enhance the rate of reduction of NOHA, in the presence of NADH cytochrome b₅ reductase and cytochrome b₅, by more than 500-fold. Consequently, these results provide the first hints that mARC might be involved in mitochondrial NOHA reduction and could be of physiological significance in affecting endogenous nitric oxide levels. Possibly, this reduction represents another regulative mechanism in the complex regulation of nitric oxide biosynthesis, considering a mitochondrial NOS has been identified. Moreover, this reduction is not restricted to NOHA since the analogous arginase inhibitor NHAM (N(ω)-hydroxy-N(δ)-methyl-L-arginine) is also reduced by this system.

On the mechanism of nitric oxide formation upon oxidative cleavage of CN(OH) bonds by NO-synthases and cytochromes P450

Microsomal liver cytochromes P450 catalyze the oxidative cleavage of the C = NOH bond of many ketoximes, amidoximes and guanidoximes, and NO synthases catalyze the oxidation of N omega-hydroxy-L-arginine to citrulline and NO. All these oxidations appear to be performed either by the FE(II) O2 complex of these hemoproteins or by O2.- which is formed by its decomposition. This leads to a unifying view of the mechanisms of P450- and NOS-dependent oxidative cleavage of C = NOH bonds, the relative contribution of Fe(II) O2.- being very different in NO-synthase and cytochromes P450.