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N-(4-Aminobutyl)-N′-(2-methoxyethyl)guanidine (8a) is a potent inhibitor targeting the hDDAH-1 active site (Ki = 18 μM) and derived from a series of guanidine- and amidine-based inhibitors. Its nonamino acid nature leads to high selectivities toward other enzymes of the nitric oxide-modulating system. Crystallographic data of 8a-bound hDDAH-1 illuminated a unique binding mode. Together with its developed N-hydroxyguanidine prodrug 11, 8a will serve as a most widely applicable, pharmacological tool to target DDAH-1-associated diseases.

The mitochondrial amidoxime-reducing component (MARC) is a mammalian molybdenum-containing enzyme. All annotated mammalian genomes harbor two MARC genes, MARC1 and MARC2, which share a high degree of sequence similarity. Both molybdoenzymes reduce a variety of N-hydroxylated compounds. Besides their role in N-reductive drug metabolism, only little is known about their physiological functions. In this study, we characterized an existing KO mouse model lacking the functional MARC2 gene and fed a high-fat diet and also performed in vivo and in vitro experiments to characterize reductase activity toward known MARC substrates. MARC2 KO significantly decreased reductase activity toward several N-oxygenated substrates, and for typical MARC substrates, only small residual reductive activity was still detectable in MARC2 KO mice. The residual detected reductase activity in MARC2 KO mice could be explained by MARC1 expression that was hardly unaffected by KO, and we found no evidence of significant activity of other reductase enzymes. These results clearly indicate that MARC2 is mainly responsible for N-reductive biotransformation in mice. Striking phenotypical features of MARC2 KO mice were lower body weight, increased body temperature, decreased levels of total cholesterol, and increased glucose levels, supporting previous findings that MARC2 affects energy pathways. Of note, the MARC2 KO mice were resistant to high-fat diet–induced obesity. We propose that the MARC2 KO mouse model could be a powerful tool for predicting MARC-mediated drug metabolism and further investigating MARC's roles in energy homeostasis.

The mitochondrial amidoxime reducing component is a recently discovered molybdenum enzyme in mammals which, in concert with the electron transport proteins cytochrome b5 and NADH cytochrome b5 reductase, catalyzes the reduction of N-oxygenated structures. This three component enzyme system plays a major role in N-reductive drug metabolism. Belonging to the group of N-hydroxylated structures, hydroxamic acids are also potential substrates of the mARC-system. Hydroxamic acids show a variety of pharmacological activities and are therefore often found in drug candidates. They can also exhibit toxic properties as is the case for many aryl hydroxamic acids formed during the metabolism of arylamides. Biotransformation assays using recombinant human proteins, subcellular porcine tissue fractions as well as human cell culture were performed. Here the mARC-dependent reduction of the model compound benzhydroxamic acid is reported in addition to the reduction of three drugs. In comparison with other known substrates of the molybdenum depending enzyme system (e.g., amidoxime prodrugs) the conversion rates measured here are slower, thereby reflecting the mediocre metabolic stability and oral bioavailability of distinct hydroxamic acids. Moreover, the toxic N-hydroxylated metabolite of the analgesic phenacetin, N-hydroxyphenacetin, is not reduced by the mARC-system under the chosen conditions. This confirms the high toxicity of this component, as it needs to be detoxified by other pathways. This work highlights the need to monitor the N-reductive metabolism of new drug candidates by the mARC-system when evaluating the metabolic stability of hydroxamic acid-containing structures or the potential risks of toxic metabolites.

Although known for years, the toxic effects of trimethylamine N-oxide (TMAO), a physiological metabolite, were just recently discovered and are currently under investigation. It is known that elevated TMAO plasma levels correlate with an elevated risk for cardiovascular disease (CVD). Even though there is a general consensus about the existence of a causal relationship between TMAO and CVD, the underlying mechanisms are not fully understood. TMAO is an oxidation product of the hepatic flavin-containing monooxygenases (FMO), mainly of isoform 3, and it is conceivable that humans also have an enzyme reversing this toxification by reducing TMAO to its precursor trimethylamine (TMA). All prokaryotic enzymes that use TMAO as a substrate have molybdenum-containing cofactors in common. Such molybdenum-containing enzymes also exist in mammals, with the so-called mitochondrial amidoxime reducing component (mARC) representing the most recently discovered mammalian molybdenum enzyme. The enzyme has been found to exist in two isoforms, mARC1 and mARC2, both being capable of reducing a variety of N-oxygenated compounds, including nonphysiological N-oxides. To investigate whether the two isoforms of this enzyme are able to reduce and detoxify TMAO, we developed a suitable analytical method and tested TMAO reduction with a recombinant enzyme system. We found that one of the two recombinant human mARC proteins, namely, hmARC1, reduces TMAO to TMA. The N-reductive activity is relatively low and identified via the kinetic parameters with K

As recently as 2006 the mitochondrial amidoxime reducing component (mARC) was identified as the fourth and last Mo enzyme present in humans. Its physiological role remains unknown. mARC is capable of reducing a variety of N-hydroxylated compounds such as amidoximes to their corresponding amidine and there is considerable interest in this enzyme from a pharmaceutical perspective. mARC is a target for N-hydroxylated pro-drugs that may be reductively activated intracellularly to release potent drugs such as cationic amidinium ions, which exhibit a broad spectrum of activity as antithrombotics and against various bacteria and parasites. In the quest for a rapid screen of new mARC substrates and inhibitors we present an electrochemical method which utilizes the natural electron partner of mARC, cytochrome b5, coupled to an electrochemical electrode. Mediated electron transfer from the electrode via cytochrome b5 to mARC results in a catalytic current in the presence of substrate. Keywords: mARC, Molybdenum, Enzyme, Voltammetry, Cytochrome

Acta crystallographica / F 74(6), 337 - 344 (2018). doi:10.1107/S2053230X18006921, The human mitochondrial amidoxime reducing component (hmARC) is a molybdenum cofactor-dependent enzyme that is involved in the reduction of a diverse range of N-hydroxylated compounds of either physiological or xenobiotic origin. In this study, the use of a fusion-protein approach with T4 lysozyme (T4L) to determine the structure of this hitherto noncrystallizable enzyme by X-ray crystallography is described. A set of four different hmARC-T4L fusion proteins were designed. Two of them contained either an N-terminal or a C-terminal T4L moiety fused to hmARC, while the other two contained T4L as an internal fusion partner tethered to the hmARC enzyme between two predicted secondary-structure elements. One of these internal fusion constructs could be expressed and crystallized successfully. The hmARC-T4L crystals diffracted to 1.7 Å resolution using synchrotron radiation and belonged to space group P2$_1$2$_1$2$_1$ with one molecule in the asymmetric unit. Initial attempts to solve the structure by molecular replacement using T4L did not result in electron-density distributions that were sufficient for model building and interpretation of the hmARC moiety. However, this study emphasizes the utility of the T4L fusion-protein approach, which can be used for the crystallization and structure determination of membrane-bound proteins as well as soluble proteins., Published by Blackwell, Oxford [u.a.]

The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.

The mitochondrial amidoxime-reducing component (mARC) is a recently discovered molybdenum-containing enzyme in mammalians. Upon reconstitution with the electron transport proteins, cytochrome b(5) and its reductase, this molybdenum enzyme is capable of reducing N-hydroxylated compounds. It was named mARC because the N-reduction of amidoxime structures was initially studied using this isolated mitochondrial enzyme. All hitherto analyzed mammalian genomes harbor two mARC genes: molybdenum cofactor (Moco) sulferase C-terminal domain MOSC1 and MOSC2. Proteins encoded by these genes represent the simplest eukaryotic molybdenum enzymes, in that they bind only the Moco. It is also suggested that they are members of a new family of molybdenum enzymes. mARC and its N-reductive enzyme system plays a major role in drug metabolism, especially in the activation of so-called "amidoxime-prodrugs" and in the detoxification of N-hydroxylated xenobiotics, though its physiological relevance is largely unknown.

Purification of the mitochondrial enzyme responsible for reduction of N-hydroxylated amidine prodrugs led to the identification of two newly discovered mammalian molybdenum-containing proteins, the mitochondrial amidoxime reducing components mARC-1 and mARC-2 (Gruenewald et al., 2008). These 35-kDa proteins represent a novel group of molybdenum proteins in eukaryotes as they form a molybdenum cofactor-dependent enzyme system consisting of three separate proteins (Havemeyer et al., 2006). Each mARC protein reduces N-hydroxylated compounds after reconstitution with the electron transport proteins cytochrome b(5) and b(5) reductase. In continuation of our drug metabolism investigations (Havemeyer et al., 2006; Gruenewald et al., 2008), we present data from reconstituted enzyme systems with recombinant human and native porcine enzymes showing the reduction of N-hydroxy-sulfonamides (sulfohydroxamic acids) to sulfonamides: the N-hydroxy-sulfonamide N-hydroxy-valdecoxib (N-hydroxy-4-[5-methyl-3-phenyl-4-isoxazolyl]-benzenesulfonamide) represents a novel cyclooxygenase (COX)-2 inhibitor and is therefore a drug candidate in the treatment of diseases associated with rheumatic inflammation, pain, and fever. It was synthesized as an analog of the known COX-2 inhibitor valdecoxib (4-[5-methyl-3-phenyl-4-isoxazolyl]-benzenesulfonamide) (Talley et al., 2000). N-Hydroxy-valdecoxib had low in vitro COX-2 activity but showed significant analgesic activity in vivo and a prolonged therapeutic effect compared with valdecoxib (Erdélyi et al., 2008). In this report, we demonstrate that N-hydroxy-valdecoxib is enzymatically reduced to its pharmacologically active metabolite valdecoxib. Thus, N-hydroxy-valdecoxib acts as prodrug that is activated by the molybdenum-containing enzyme mARC.

In previous studies, it was shown that liver microsomes from rabbit, rat, pig, and human are involved in the reduction of N-hydroxylated amidines, guanidines, and amidinohydrazones of various drugs and model compounds (Drug Metab Rev 34: 565-579). One responsible enzyme system, the microsomal benzamidoxime reductase, consisting of cytochrome b5, its reductase, and a cytochrome P450 isoenzyme, was isolated from pig liver microsomes (J Biol Chem 272:19615-19620). Further investigations followed to establish whether such enzyme systems are also present in microsomes of other organs such as brain, lung, and intestine. In addition, the mitochondrial reduction in human and porcine liver and kidney preparations was studied. The reductase activities were measured by following the reduction of benzamidoxime to benzamidine, guanoxabenz to guanabenz, and Ro 48-3656 ([[1-[(2S)-2-[[4-[(hydroxyamino)iminomethyl]benzoyl]amino]-1-oxopropyl]-4-piperidinyl]oxy]-acetic acid) to Ro 44-3888 ([[1-[(2S)-2-[[4-(aminoiminomethyl)benzoyl]amino]-1-oxopropyl]-4-piperidinyl]oxy]-acetic acid). Interestingly, preparations of all tested organs were capable of reducing the three compounds. The highest specific rates were found in kidney followed by liver, brain, lung, and intestine, and usually the mitochondrial reduction rates were superior. From the determined characteristics, similarities between the enzyme systems in the different organs and organelles were detected. Furthermore, properties of the benzamidoxime reductase located in the outer membrane of pig liver mitochondria were studied. In summary, these results demonstrate that in addition to the microsomal reduction, mitochondria are involved to a great extent in the activation of amidoxime prodrugs. The importance of extrahepatic metabolism in the reduction of N-hydroxylated prodrugs is demonstrated.

The “mitochondrial amidoxime reducing component” (mARC) is the most recently discovered molybdenum-containing enzyme in mammals. All mammalian genomes studied to date contain two mARC genes: MARC1 and MARC2. The proteins encoded by these genes are mARC-1 and mARC-2 and represent the simplest form of eukaryotic molybdenum enzymes, only binding the molybdenum cofactor. In the presence of NADH, mARC proteins exert N-reductive activity together with the two electron transport proteins cytochrome b5 type B and NADH cytochrome b5 reductase. This enzyme system is capable of reducing a great variety of N-hydroxylated substrates. It plays a decisive role in the activation of prodrugs containing an amidoxime structure, and in detoxification pathways, e.g., of N-hydroxylated purine and pyrimidine bases. It belongs to a group of drug metabolism enzymes, in particular as a counterpart of P450 formed N-oxygenated metabolites. Its physiological relevance, on the other hand, is largely unknown. The aim of this article is to summarize our current knowledge of these proteins with a special focus on the mammalian enzymes and their N-reductive activity.

The mitochondrial amidoxime reducing component (mARC) is a molybdenum-containing enzyme and capable of reducing N-hydroxylated structures such as amidoxime prodrugs. In this study, we tested the involvement of mARC in the reduction of N-oxides (amitriptyline-N-oxide, nicotinamide-N-oxide), oximes ((E)-/(Z)-2,4,6-trimethylacetophenonoxime) and a N-hydroxyamidinohydrazone (guanoxabenz). All groups are reduced by mARC proteins, and the enzymes are therefore involved in the interconversion of N-oxygenated metabolites originating from cytochrome P450s and flavin-containing monooxygenases. In addition, these structures open up further options for serving as prodrugs. Thus, with respect to these reactions, testing of candidates with N-oxygenated structures should not solely be carried out in microsomal enzyme sources but as well in mitochondria. However, differences in the reduction of oximes and N-oxides between the two isoforms, namely mARC1 and mARC2, were detectable; N-oxides are exclusively reduced by mARC1. We therefore assume differences between the so far unknown 3D structures of the two proteins.

The mitochondrial amidoxime reducing component mARC is the fourth mammalian molybdenum enzyme. The protein is capable of reducing N-oxygenated structures, but requires cytochrome b5 and cytochrome b5 reductase for electron transfer to catalyze such reactions. It is well accepted that the enzyme is involved in N-reductive drug metabolism such as the activation of amidoxime prodrugs. However, the endogenous function of the protein is not fully understood. Among other functions, an involvement in lipogenesis is discussed. To study the potential involvement of the protein in energy metabolism, we tested whether the mARC protein and its partners are regulated due to fasting and high fat diet in mice. We used qRT-PCR for expression studies, Western Blot analysis to study protein levels and an N-reductive biotransformation assay to gain activity data. Indeed all proteins of the N-reductive system are regulated by fasting and its activity decreases. To study the potential impact of these changes on prodrug activation in vivo, another mice experiment was conducted. Model compound benzamidoxime was injected to mice that underwent fasting and the resulting metabolite of the N-reductive reaction, benzamidine, was determined. Albeit altered in vitro activity, no changes in the metabolite concentration in vivo were detectable and we can dispel concerns that fasting alters prodrug activation in animal models. With respect to high fat diet, changes in the mARC proteins occur that result in increased N-reductive activity. With this study we provide further evidence that the endogenous function of the mARC protein is linked with lipid metabolism.

1. Upamostat (Mesupron®) is a new small molecule serine protease inhibitor. The drug candidate was developed to inhibit the urokinase-type plasminogen activator (uPA) system, which plays a major role in tumor invasion and metastasis. Upamostat is currently in clinical development as an anti-metastatic and non-cytotoxic agent against pancreatic and breast cancer.2. Upamostat is the orally available amidoxime- (i.e. hydroxyamidine-) prodrug of the pharmacologically active form, WX-UK1. In this study, the reductive enzymatic activation of upamostat to its corresponding amidine WX-UK1 was analyzed.3. The recently discovered molybdenum enzyme “mitochondrial Amidoxime Reducing Component” (mARC) catalyses together with its electron transport proteins cytochrome b5 and NADH cytochrome b5 reductase the reduction of N-hydroxylated prodrugs. In vitro biotransformation assays with porcine subcellular fractions and the reconstituted human enzymes demonstrate an mARC-dependent N-reduction of upamostat.

The "mitochondrial Amidoxime Reducing Component" (mARC) is the newly discovered fourth molybdenum enzyme in mammals. All hitherto analyzed mammals express two mARC proteins, referred to as mARC1 and mARC2. Together with their electron transport proteins cytochrome b(5) and NADH cytochrome b(5) reductase, they form a three-component enzyme system and catalyze the reduction of N-hydroxylated prodrugs. Here, we demonstrate the reductive detoxification of toxic and mutagenic N-hydroxylated nucleobases and their corresponding nucleosides by the mammalian mARC-containing enzyme system. The N-reductive activity was found in all tested tissues with the highest detectable conversion rates in liver, kidney, thyroid, and pancreas. According to the presumed localization, the N-reductive activity is most pronounced in enriched mitochondrial fractions. In vitro assays with the respective recombinant three-component enzyme system show that both mARC isoforms are able to reduce N-hydroxylated base analogues, with mARC1 representing the more efficient isoform. On the basis of the high specific activities with N-hydroxylated base analogues relative to other N-hydroxylated substrates, our data suggest that mARC proteins might be involved in protecting cellular DNA from misincorporation of toxic N-hydroxylated base analogues during replication by converting them to the correct purine or pyrimidine bases, respectively.

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 b(5), and NADH/FAD-dependent cytochrome b(5) 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.