Second-Coordination-Sphere Effects Reveal Electronic Structure Differences between the Mitochondrial Amidoxime Reducing Component and Sulfite Oxidase.

A combination of X-ray absorption and low-temperature electronic absorption spectroscopies has been used to probe the geometric and electronic structures of the human mitochondrial amidoxime reducing component enzyme (hmARC1) in the oxidized Mo(VI) and reduced Mo(IV) forms. Extended X-ray absorption fine structure analysis revealed that oxidized enzyme possesses a 5-coordinate [MoO2(SCys)(PDT)]- (PDT = pyranopterin dithiolene) active site with a cysteine coordinated to Mo. A 5-coordinate geometry is retained in the reduced state, with the equatorial oxo being protonated. Low-temperature electronic absorption spectroscopy of hmARC1 reveals a spectrum for the oxidized enzyme that is significantly different from what has been reported for sulfite oxidase family enzymes. Time-dependent density functional theory computations on oxidized and reduced hmARC1, and a small molecule analogue for hmARC1ox, have been used to assist us in making detailed band assignments and developing a greater understanding of enzyme electronic structure contributions to reactivity. Our understanding of the hmARCred HOMO and the LUMO of the benzamidoxime substrate reveal a potential π-bonding interaction between these redox orbitals, with two-electron occupation of the substrate LUMO along the reaction coordinate activating the O-N bond for cleavage and promoting oxygen atom transfer to the Mo site.

Active Site Structures of the Escherichia coli N-Hydroxylaminopurine Resistance Molybdoenzyme YcbX.

X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) data have been used to characterize the coordination environment for the catalytic Mo site of Escherichia coli YcbX in two different oxidation states. In the oxidized state, the Mo(VI) ion is coordinated by two terminal oxo ligands, a thiolate S atom from cysteine, and two S donors from the bidentate pyranopterin ene-1,2-dithiolate (pyranopterin dithiolene). Upon reduction, it is the more basic equatorial oxo ligand that is protonated, with a Mo-Oeq bond distance that is best described as either a short Mo4+-OH2 bond or a long Mo4+-OH bond. Mechanistic implications for substrate reduction are discussed in light of these structural details.

Combined PARP and Dual Topoisomerase Inhibition Potentiates Genome Instability and Cell Death in Ovarian Cancer.

Although ovarian cancer is a rare disease, it constitutes the fifth leading cause of cancer death among women. It is of major importance to develop new therapeutic strategies to improve survival. Combining P8-D6, a novel dual topoisomerase inhibitor with exceptional anti-tumoral properties in ovarian cancer and compounds in preclinical research, and olaparib, a PARP inhibitor targeting DNA damage repair, is a promising approach. P8-D6 induces DNA damage that can be repaired by base excision repair or homologous recombination in which PARP plays a major role. This study analyzed benefits of combining P8-D6 and olaparib treatment in 2D and 3D cultures with ovarian cancer cells. Measurement of viability, cytotoxicity and caspase activity were used to assess therapy efficacy and to calculate the combination index (CI). Further DNA damage was quantified using the biomarkers RAD51 and γH2A.X. The combinational treatment led to an increased caspase activity and reduced viability. CI values partially show synergisms in combinations at 100 nM and 500 nM P8-D6. More DNA damage accumulated, and spheroids lost their membrane integrity due to the combinational treatment. While maintaining the same therapy efficacy as single-drug therapy, doses of P8-D6 and olaparib can be reduced in combinational treatments. Synergisms can be seen in some tested combinations. In summary, the combination therapy indicates benefits and acts synergistic at 100 nM and 500 nM P8-D6.

The Novel Dual Topoisomerase Inhibitor P8-D6 Shows Anti-myeloma Activity In Vitro and In Vivo.

P8-D6 is a novel dual inhibitor of human topoisomerase I (TOP1) and II (TOP2) with broad pro-apoptotic antitumor activity. NCI-60 screening revealed markedly improved cytotoxicity of P8-D6 against solid and leukemia cell lines compared with other single and dual topoisomerase inhibitors, for example, irinotecan, doxorubicin, or pyrazoloacridine. In this study, we investigated the capacity of P8-D6 to inhibit myeloma cell growth in vitro and in vivo Growth inhibition assays demonstrated significant anti-myeloma effects against different myeloma cell lines with IC50 values in the low nanomolar range. Freshly isolated plasma cells of patients with multiple myeloma were killed by P8-D6 with similar doses. P8-D6 activated caspase 3/7 and induced significant apoptosis of myeloma cells. Supportive effects of bone marrow stromal cells on IL6-dependent INA-6 myeloma cells were abrogated by P8-D6 and apoptosis occurred in a time- and dose-dependent manner. Of note, healthy donor peripheral blood mononuclear cells and human umbilical vein endothelial cells were not affected at concentrations toxic for malignant plasma cells. Treatment of myeloma xenografts in immunodeficient SCID/beige mice by intravenous and, notably, also oral application of P8-D6 markedly inhibited tumor growths, and significantly prolonged survival of tumor-bearing mice.

Newly developed dual topoisomerase inhibitor P8-D6 is highly active in ovarian cancer.

BACKGROUND: Ovarian cancer (OvCa) constitutes a rare and highly aggressive malignancy and is one of the most lethal of all gynaecologic neoplasms. Due to chemotherapy resistance and treatment limitations because of side effects, OvCa is still not sufficiently treatable. Hence, new drugs for OvCa therapy such as P8-D6 with promising antitumour properties have a high clinical need. The benzo[c]phenanthridine P8-D6 is an effective inductor of apoptosis by acting as a dual topoisomerase I/II inhibitor. METHODS: In the present study, the effectiveness of P8-D6 on OvCa was investigated in vitro. In various OvCa cell lines and ex vivo primary cells, the apoptosis induction compared with standard therapeutic agents was determined in two-dimensional monolayers. Expanded by three-dimensional and co-culture, the P8-D6 treated cells were examined for changes in cytotoxicity, apoptosis rate and membrane integrity via scanning electron microscopy (SEM). Likewise, the effects of P8-D6 on non-cancer human ovarian surface epithelial cells and primary human hepatocytes were determined. RESULTS: This study shows a significant P8-D6-induced increase in apoptosis and cytotoxicity in OvCa cells which surpasses the efficacy of well-established drugs like cisplatin or the topoisomerase inhibitors etoposide and topotecan. Non-cancer cells were affected only slightly by P8-D6. Moreover, no hepatotoxic effect in in vitro studies was detected. CONCLUSION: P8-D6 is a strong and rapid inductor of apoptosis and might be a novel treatment option for OvCa therapy.

High Antitumor Activity of the Dual Topoisomerase Inhibitor P8-D6 in Breast Cancer.

Breast cancer constitutes the leading cause of cancer deaths among females. However, numerous shortcomings, including low bioavailability, resistance and significant side effects, are responsible for insufficient treatment. The ultimate goal, therefore, is to improve the success rates and, thus, the range available treatment options for breast cancer. Consequently, the identification, development and evaluation of potential novel drugs such as P8-D6 with seminal antitumor capacities have a high clinical need. P8-D6 effectively induces apoptosis by acting as a dual topoisomerase I/II inhibitor. This study provides an overview of the effectiveness of P8-D6 in breast cancer with both 2D monolayers and 3D spheroids compared to standard therapeutic agents. For this drug effectiveness review, cell lines and ex vivo primary cells were used and cytotoxicity, apoptosis rates and membrane integrity were examined. This study provides evidence for a significant P8-D6-induced increase in apoptosis and cytotoxicity in breast cancer cells compared to the efficacy of standard therapeutic drugs. To sum up, P8-D6 is a fast and powerful inductor of apoptosis and might become a new and suitable therapeutic option for breast cancer in the future.

The Aza-Analogous Benzo[c]phenanthridine P8-D6 Acts as a Dual Topoisomerase I and II Poison, thus Exhibiting Potent Genotoxic Properties.

The benzo[c]phenanthridine P8-D6 was recently found to suppress the catalytic activity of both human topoisomerase (Topo) I and II. Concomitantly, potent cytotoxic activity was observed in different human tumor cell lines, raising questions about the underlying mechanisms in vitro. In the present study, we addressed the question of whether P8-D6 acts as a so-called Topo poison, stabilizing the covalent Topo-DNA intermediate, thus inducing fatal DNA strand breaks in proliferating cells. In HT-29 colon carcinoma cells, fluorescence imaging revealed P8-D6 to be taken up by the cells and to accumulate in the perinuclear region. Confocal microscopy demonstrated that the compound is partially located inside the nuclei, thus reaching the potential target. In the "in vivo complex of enzyme" (ICE) bioassay, treatment of HT-29 cells with P8-D6 for 1 h significantly enhanced the proportion of Topo I and II covalently linked to the DNA in concentrations ≥1 µM, indicating effective dual Topo poisoning. Potentially resulting DNA damage was analyzed by single-cell gel electrophoresis ("comet assay"). Already at 1 h of incubation, significant genotoxic effects were observed in the comet assay in concentrations as low as 1 nM. Taken together, the present study demonstrates the high Topo-poisoning and genotoxic potential of P8-D6 in human tumor cells.

Detoxification of Trimethylamine N-Oxide by the Mitochondrial Amidoxime Reducing Component mARC.

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 Km = (30.4 ± 9.8) mM and Vmax = (100.5 ± 12.2) nmol/(mg protein·min). Nevertheless, the ubiquitous tissue expression of hmARC1 allows a continuous reduction of TMAO whereas the counter-reaction, the production of TMAO through FMO3, can take place only in the liver where FMO3 is expressed. TMAO reduction in porcine liver subfractions showed the characteristic enrichment of N-reductive activity in the outer mitochondrial membrane. TMAO reduction was also found in human cell cultures. These findings indicate the role of hmARC1 in the metabolomic pathway of TMAO, which might contribute to the prevention of CVD. This also hints at a physiological function of the molybdenum enzyme, which remains mainly unknown to date.

A Dual Topoisomerase Inhibitor of Intense Pro-Apoptotic and Antileukemic Nature for Cancer Treatment.

Classic cytotoxic drugs remain indispensable instruments in antitumor therapy due to their effectiveness and a more prevalent insensitivity toward tumor resistance mechanisms. Herein we describe the favorable properties of 6-(N,N-dimethyl-2-aminoethoxy)-11-(3,4,5-trimethoxyphenyl)pyrido[3,4-c][1,9]phenanthroline (P8-D6), a powerful inducer of apoptosis caused by an equipotent inhibition of human topoisomerase I and II activities. A broad-spectrum effect against human tumor cell lines at nanomolar concentrations, as well as strong antileukemic effects, were shown to be superior to those of marketed topoisomerase-targeting drugs and dual topoisomerase inhibitors in clinical trials. The facile four-step synthesis, advantageous drugability properties, and initial in vivo data encourage the application of P8-D6 in appropriate animal tumor models and further drug development.

11-Substituted Benzo[c]phenanthridines: New Structures and Insight into Their Mode of Antiproliferative Action.

The synthesis of various new structures of a library of 11-substituted 6-amino-11,12-dihydrobenzo[c]phenanthridines (BP) and 11-substituted 6-aminobenzo[c]phenanthridines (BP-D) is presented. These structures, further synthetic modifications, and the preparation of follow-up products which delivered about 40 new derivatives are described. Their potential as antiproliferative drug candidates was investigated by comparison of NCI 60 developmental therapeutics program (DTP) human tumor cell line screening data based on the results of in vitro tumor cell growth inhibition, including about 40 hitherto unpublished compound test results with up to 60 cancer cell lines. NCI-COMPARE studies helped to suggest the modes of action of the highly active antiproliferative drugs. These findings are supported by in vitro biological investigations showing either inhibition of tubulin polymerization and depolymerization or topoisomerase inhibition. Together with physicochemical parameters of the drug candidates, structure-activity relationships are critically discussed. Tubulin interaction or inhibition of topoisomerase I and IIα/ß activity are two rationales that can explain the antiproliferative activity observed in the NCI 60 DTP human tumor cell line screen. However, it can also be reasonably assumed that these compounds address several targets, thus prohibiting the identification of simple structure-activity relationships. The new structures described herein are thought to act as so-called multitarget drugs, thus being of special interest in the area of multidrug resistance.

Design, Synthesis, and Bioactivation of O-Glycosylated Prodrugs of the Natural Nitric Oxide Precursor N(ω)-Hydroxy-l-arginine.

Naturally occurring N(ω)-hydroxy-l-arginine (NOHA, 1) is the best substrate of NO synthases (NOS). The development of stable and bioavailable prodrugs would provide a pharmacologically valuable strategy for the treatment of cardiovascular diseases that are associated with endothelial dysfunction. To improve NOHAs druglike properties, we demonstrate that O-substitution by (glycosylic) acetal formation greatly increased the chemical stability of the hydroxyguanidine moiety and provided a nontoxic group that could be easily bioactivated by glycosidases. A straightforward synthetic concept was devised and afforded a series of diversely substituted prodrugs by O-conjugation of the hydroxyguanidine moiety with different monosaccharides. Systematic exploration of their bioactivation profile revealed that glucose-based prodrugs were more efficiently bioactivated than their galactose counterparts. NOS-dependent cytosolic NO release was quantified by automated fluorescence microscopy in a cell-based assay with murine macrophages. Glucose-based prodrugs performed particularly well and delivered cellular NO levels comparable to 1, demonstrating proof-of-concept.

One-Step Synthetic Access to Isosteric and Potent Anticancer Nitrogen Heterocycles with the Benzo[c]phenanthridine Scaffold.

A versatile one-step two-component cyclization to build new tetracyclic nitrogen heterocycles is described. Ortho-methylhetarenecarbonitrile components were condensed with aldehydes to access a large library of differently substituted ring systems. The heterocyclic core can be easily modified by variation of the position of the endocyclic nitrogen atom in the o-methylhetarenecarbonitrile substrate. The manner of the nucleophilic attack that leads to the condensation can be triggered by different electron-density distribution in the molecule induced by the position of the nitrogen atom. Taking this into account, there is an electronic preference that leads to either pyridophenanthrolines or the corresponding pyridoazacarbazoles as the main products. We demonstrate the high antitumor potential of some of our synthesized heterocycles, which is strongly dependent on the substitution pattern introduced through the aldehyde component. The position and number of endocyclic nitrogen atoms play an important role regarding cytotoxicity of the studied compounds.

Triggering the directional selectivity of a ring-closure reaction leads to pyridoazacarbazoles with anticancer properties.

We herein describe a facile and versatile synthetic route to the tetracyclic system of 6-substituted 5,6-dihydro-11H-pyrido[3,2-i]-1-azacarbazoles with promising anticancer properties. These derivatives are built up by an elegant one-step base-catalyzed synthetic procedure from commercially available building blocks. One additional step provides the corresponding skeleton hitherto unknown in the literature. The possibility to synthesize a large library of compounds with various substitution patterns utilizing this method underlines the importance of this synthetic procedure.

The pivotal role of the mitochondrial amidoxime reducing component 2 in protecting human cells against apoptotic effects of the base analog N6-hydroxylaminopurine.

N-Hydroxylated nucleobases and nucleosides as N-hydroxylaminopurine (HAP) or N-hydroxyadenosine (HAPR) may be generated endogenously in the course of cell metabolism by cytochrome P450, by oxidative stress or by a deviating nucleotide biosynthesis. These compounds have shown to be toxic and mutagenic for procaryotic and eucaryotic cells. For DNA replication fidelity it is therefore of great importance that organisms exhibit effective mechanisms to remove such non-canonical base analogs from DNA precursor pools. In vitro, the molybdoenzymes mitochondrial amidoxime reducing component 1 and 2 (mARC1 and mARC2) have shown to be capable of reducing N-hydroxylated base analogs and nucleoside analogs to the corresponding canonical nucleobases and nucleosides upon reconstitution with the electron transport proteins cytochrome b5 and NADH-cytochrome b5 reductase. By RNAi-mediated down-regulation of mARC in human cell lines the mARC-dependent N-reductive detoxication of HAP in cell metabolism could be demonstrated. For HAPR, on the other hand, the reduction to adenosine seems to be of less significance in the detoxication pathway of human cells as HAPR is primarily metabolized to inosine by direct dehydroxylamination catalyzed by adenosine deaminase. Furthermore, the effect of mARC knockdown on sensitivity of human cells to HAP was examined by flow cytometric quantification of apoptotic cell death and detection of poly (ADP-ribose) polymerase (PARP) cleavage. mARC2 was shown to protect HeLa cells against the apoptotic effects of the base analog, whereas the involvement of mARC1 in reductive detoxication of HAP does not seem to be pivotal.

In vivo SPECT imaging of [¹²³I]-labeled pentamidine pro-drugs for the treatment of human African trypanosomiasis, pharmacokinetics, and bioavailability studies in rats.

UNLABELLED: Pentamidine is an effective antiparasitic agent and approved drug for the treatment of African trypanosomiasis (sleeping sickness). However, pentamidine suffers from poor orally bioavailability and lacks central nervous system (CNS) delivery. Therefore its applicability is limited to intravenous or intramuscular treatment of the first stage of the African trypanosomiasis. For this reason, several new pentamidine pro-drugs have been developed with the aim of providing improved orally availability and CNS penetration. AIM: this work aims to measure and to compare the distribution, bioavailability, and ability to cross the blood-brain barrier of [(123)I]-labeled pentamidine and its pro-drugs, N,N'-dihydroxypentamidine and N,N'­bis(succinyloxy) pentamidine, using SPECT (single photon emission computed tomography) after intravenously and per orally administration in rats. METHODS: a total of 60 male Sprague Dawley rats were examined. Each [(123)I]-labeled substance (n=3) was applied to 12 rats (n=6 i.v. and n=6 orally). In two additional test series both [(123)I]iodopentamidine (n=6) and N,N'-bis(succinyloxy)-[(123)I]iodopentamidine (n=6) were administered orally together with the non-radioactive homologues. To evaluate the in vivo stability of the labeled compounds, [(123)I]NaI solution was administered intravenously (n=6) and orally (n=6). In vivo SPECT images were acquired after 30 min, 4h, and 24h and blood samples were taken over 24h. The SPECT images were fusioned with previously acquired magnetic resonance images. After the last SPECT the rats were perfused, sacrificed and the organ γ-radiation levels were determined with a γ-counter. Analysis and quantification of the reconstructed SPECT images was performed using the region of interest technique. RESULTS AND CONCLUSION: the data showed a highly improved oral bioavailability of the [(123)I]-labeled pro-drugs compared to [(123)I]-labeled pentamidine. While [(123)I]iodopentamidine was mainly renally eliminated the pro-drugs were primarily metabolized in the liver and underwent biliary elimination. Considering pentamidine's nephrotoxicity this feature has to be seen as an advantage of the pro-drug principle. Moreover, a significantly higher concentration in the brain was detected after intravenous injection of N,N'-dihydroxy[(123)I]iodopentamidine compared to [(123)I]iodopentamidine. The feasibility of an effective treatment of second stage African trypanosomiasis, in which the parasites already infected the brain, with the herein investigated pro-drugs remains to be clarified with infected animals in additional in vivo studies.

The N-reductive system composed of mitochondrial amidoxime reducing component (mARC), cytochrome b5 (CYB5B) and cytochrome b5 reductase (CYB5R) is regulated by fasting and high fat diet in mice.

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.

The involvement of mitochondrial amidoxime reducing components 1 and 2 and mitochondrial cytochrome b5 in N-reductive metabolism in human cells.

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.

Detection of fumarate-glutathione adducts in the portal vein blood of rats: evidence for rapid dimethylfumarate metabolism.

Dimethylfumarate (DMF), the essential ingredient of the drug product Fumaderm®, is used to treat psoriasis with a recognized favorable long-term safety profile. Interestingly, the mode of action and the pharmacokinetics of DMF in psoriasis or multiple sclerosis are not fully explored. It is known that DMF as an α,ß-unsaturated carboxylic acid ester forms an adduct with the antioxidant glutathione in vitro via a Michael-type addition within a very short period of time. In addition, it was shown that this reaction also takes place in vivo since the mercapturic acid of DMF was detected in urine of psoriasis patients after oral intake of Fumaderm®. To verify the hypothesis that DMF reacts with GSH already in or even before entering the portal vein blood an in vivo study in rats was initiated and portal vein blood was analyzed for the presence of DMF, MMF, GS-DMS and break down products, after DMF was given directly into the small intestine. The results show that no free DMF could be detected in the rat portal vein blood at any time point. MMF was the dominant metabolite and GS-DMS was also detectable in portal vein blood. In the rat mucosa the glutathione adducts of DMF and MMF were present. The data obtained provide evidence that the modulation of immune-mediated inflammatory pathways responsible for development of psoriasis and MS are targeted by DMF regulating redox-sensitive pathways for which the reaction with glutathione by DMF plays a crucial role.

Activation of the anti-cancer agent upamostat by the mARC enzyme system.

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. 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. 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.

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.