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Christine Beedham - One of the best experts on this subject based on the ideXlab platform.

  • Contribution of Aldehyde Oxidase, Xanthine Oxidase, and Aldehyde Dehydrogenase on the Oxidation of Aromatic Aldehydes
    Chemical research in toxicology, 2004
    Co-Authors: Georgios I. Panoutsopoulos, Demetrios Kouretas, Christine Beedham
    Abstract:

    Aliphatic Aldehydes have a high affinity toward Aldehyde dehydrogenase activity but are relatively poor substrates of Aldehyde Oxidase and xanthine Oxidase. In addition, the oxidation of xenobiotic-derived aromatic Aldehydes by the latter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of Aldehyde dehydrogenase, Aldehyde Oxidase, and xanthine Oxidase activities in the oxidation of substituted benzAldehydes in separate preparations. The incubation of vanillin, isovanillin, and protocatechuic Aldehyde with either guinea pig liver Aldehyde Oxidase, bovine milk xanthine Oxidase, or guinea pig liver Aldehyde dehydrogenase demonstrated that the three Aldehyde oxidizing enzymes had a complementary substrate specificity. Incubations were also performed with specific inhibitors of each enzyme (isovanillin for Aldehyde Oxidase, allopurinol for xanthine Oxidase, and disulfiram for Aldehyde dehydrogenase) to determine the relative contribution of each enzyme in the oxidation of these Aldehydes. Under these conditions, vanillin was rapidly oxidized by Aldehyde Oxidase, isovanillin was predominantly metabolized by Aldehyde dehydrogenase activity, and protocatechuic Aldehyde was slowly oxidized, possibly by all three enzymes. Thus, Aldehyde Oxidase activity may be a significant factor in the oxidation of aromatic Aldehydes generated from amines and alkyl benzenes during drug metabolism. In addition, this enzyme may also have a role in the catabolism of biogenic amines such as dopamine and noradrenaline where 3-methoxyphenylacetic acids are major metabolites.

  • ziprasidone metabolism Aldehyde Oxidase and clinical implications
    Journal of Clinical Psychopharmacology, 2003
    Co-Authors: Christine Beedham, J J Miceli, Scott R Obach
    Abstract:

    Ziprasidone (Geodon, Zeldox), a recently approved atypical antipsychotic agent for the treatment of schizophrenia, undergoes extensive metabolism in humans with very little (<5%) of the dose excreted as unchanged drug. Two enzyme systems have been implicated in ziprasidone metabolism: the cytosolic enzyme, Aldehyde Oxidase, catalyzes the predominant reductive pathway, and cytochrome P4503A4 (CYP3A4) is responsible for two alternative oxidation pathways. The involvement of two competing pathways in ziprasidone metabolism greatly reduces the potential for pharmacokinetic interactions between ziprasidone and other drugs. Because CYP3A4 only mediates one third of ziprasidone metabolism, the likelihood of interactions between ziprasidone and CYP3A4 inhibitors/ substrates is low. Furthermore, Aldehyde Oxidase activity does not appear to be altered when drugs or xenobiotics are coadministered. Aldehyde Oxidase, a molybdenum-containing enzyme, catalyzes the oxidation of N-heterocyclic drugs such as famciclovir and zaleplon, in addition to reducing some agents such as zonisamide. Both reactions can occur simultaneously. Although in vitro inhibitors of Aldehyde Oxidase have been identified, there are no reported clinical interactions with Aldehyde Oxidase inhibitors or inducers. There is no evidence of genetic polymorphism in Aldehyde Oxidase, and thus it not surprising that ziprasidone exposure demonstrates unimodality in humans. Aldehyde Oxidase is unrelated to the similarly named enzyme Aldehyde dehydrogenase, which is predominantly responsible for the oxidation of acetAldehyde during ethanol metabolism. Consequently, it is unlikely that there would be any pharmacokinetic interaction between ethanol and ziprasidone.

  • in vitro oxidation of famciclovir and 6 deoxypenciclovir by Aldehyde Oxidase from human guinea pig rabbit and rat liver
    Drug Metabolism and Disposition, 1997
    Co-Authors: Mohammad R. Rashidi, Stephen E Clarke, J A Smith, Christine Beedham
    Abstract:

    Famciclovir, a 9-substituted guanine derivative, is a new antiviral agent which undergoes rapid hydrolysis and oxidation in man to yield the active antiherpes agent, penciclovir. Studies with human liver cytosol have indicated that the oxidation of the penultimate metabolite, 6-deoxypenciclovir, to penciclovir is catalyzed by the molybdenum hydroxylase, Aldehyde Oxidase. In the present study the oxidation of famciclovir and 6-deoxypenciclovir with partially purified molybdenum hydroxylases from human, guinea pig, rabbit, and rat livers and bovine milk xanthine Oxidase has been investigated. Famciclovir and 6-deoxypenciclovir were oxidized predominantly to 6-oxo-famciclovir and penciclovir, respectively, by human, guinea pig, and rat liver Aldehyde Oxidase. Small amounts of 8-oxo and 6,8-dioxo-metabolites were also formed from each substrate. Famciclovir and 6-deoxypenciclovir were good substrates for rabbit liver Aldehyde Oxidase but, in each case, two major metabolites were formed. 6-Deoxypenciclovir was converted to penciclovir and 8-oxo-6-deoxypenciclovir in approximately equal quantities; famciclovir was oxidized to 6-oxo-famciclovir and a second metabolite which, on the basis of chromatographic and UV spectral data, was thought to be 8-oxo-famciclovir. Two groups of Sprague Dawley rats were identified; those containing hepatic Aldehyde Oxidase and xanthine Oxidase and those with only xanthine Oxidase. These have been designated AO-active and AO-inactive rats, respectively. Famciclovir was not oxidized by enzyme from AO-inactive rats or bovine milk xanthine Oxidase although 6-deoxypenciclovir was slowly converted to penciclovir by rat liver or milk xanthine Oxidase. Inhibitor studies showed in human, guinea pig, and rabbit liver that xanthine Oxidase did not contribute to the oxidation of famciclovir and 6-deoxypenciclovir; thus it is proposed that drug activation in vivo would be catalyzed solely by Aldehyde Oxidase.

  • substrate specificity of human liver Aldehyde Oxidase toward substituted quinazolines and phthalazines a comparison with hepatic enzyme from guinea pig rabbit and baboon
    Archives of Biochemistry and Biophysics, 1995
    Co-Authors: Christine Beedham, D J P Critchley, David J Rance
    Abstract:

    Abstract Partially purified Aldehyde Oxidase (EC 1.2.3.1) has been prepared from human, rabbit, guinea pig, and baboon liver by heat treatment and precipitation with ammonium sulfate. The interaction of 35 substituted quinazolines and phthalazines with human liver enzyme has been studied using a spectrophotometric assay. Fifteen quinazoline and 14 phthalazine derivatives were found to be substrates for human liver Aldehyde Oxidase with Km values ranging from 5 to 500 μM. The substrate specificity of the quinazolines toward rabbit, guinea pig, and baboon liver Aldehyde Oxidase has also been investigated; the reaction of substituted phthalazines with mammalian liver enzyme has been reported previously (Beedham et al., 1990, Biochem. Pharmacol. 39, 1213-1221). Oxidation products of 2-substituted (4-substituted) quinazolines with rabbit Liver Aldehyde Oxidase were identified by MS as 4-oxo (2-oxo)-quinazolines, respectively. In all cases, unsubstituted compounds gave the highest oxidation rates and the presence of lipophilic substituents presumably facilitated hydrophobic binding to the enzymes. However, there were marked differences in substrate specificity between human liver Aldehyde Oxidase and hepatic enzyme from rabbit, guinea pig, and baboon with the size of substrate being the differentiating factor, The molecular sizes of the substrates, estimated using calculated molar refractivities, ranked the size of the binding site of Aldehyde Oxidase in the order rabbit

Silke Leimkuhler - One of the best experts on this subject based on the ideXlab platform.

  • galactose Oxidase variants for the oxidation of amino alcohols in enzyme cascade synthesis
    Chemcatchem, 2015
    Co-Authors: Susanne Herter, Silke Leimkuhler, Shane Mckenna, Andrew J Carnell, Andrew R Frazer, Nicholas J Turner
    Abstract:

    The use of selected engineered galactose Oxidase (GOase) variants for the oxidation of amino alcohols to Aldehydes under mild conditions in aqueous systems is reported. GOase variant F-2 catalyses the regioselective oxidation of N-carbobenzyloxy (Cbz)-protected 3-amino-1,2-propanediol to the corresponding -hydroxyAldehyde which was then used in an aldolase reaction. Another variant, M3-5, was found to exhibit activity towards free and N-Cbz-protected aliphatic and aromatic amino alcohols allowing the synthesis of lactams such as 3,4-dihydronaphthalen-1(2H)-one, 2-pyrrolidone and valerolactam in one-pot tandem reactions with xanthine dehydrogenase (XDH) or Aldehyde Oxidase (PaoABC).

  • enzyme cascade reactions synthesis of furandicarboxylic acid fdca and carboxylic acids using Oxidases in tandem
    Green Chemistry, 2015
    Co-Authors: Shane Mckenna, Silke Leimkuhler, Susanne Herter, Nicholas J Turner, Andrew J Carnell
    Abstract:

    A one-pot tandem enzyme reaction using galactose Oxidase M3–5 and Aldehyde Oxidase PaoABC was used to convert hydroxymethylfurfural (HMF) to the pure bioplastics precursor FDCA in 74% isolated yield. A range of alcohols was also converted to carboxylic acids in high yield under mild conditions.

  • identification of crucial amino acids in mouse Aldehyde Oxidase 3 that determine substrate specificity
    PLOS ONE, 2013
    Co-Authors: Martin Mahro, Natercia F Bras, Nuno M F S A Cerqueira, Christian Teutloff, Catarina Coelho, Maria Joao Romao, Silke Leimkuhler
    Abstract:

    In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse Aldehyde Oxidase 3 (mAOX3). The sequence alignment of different Aldehyde Oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.

  • site directed mutagenesis of amino acid residues at the active site of mouse Aldehyde Oxidase aox1
    PLOS ONE, 2009
    Co-Authors: Silvia Schumann, Mineko Terao, Enrico Garattini, Miguel Saggu, Friedhelm Lendzian, Peter Hildebrandt, Silke Leimkuhler
    Abstract:

    Mouse Aldehyde Oxidase (mAOX1) forms a homodimer and belongs to the xanthine Oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Eschericia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of Aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the Aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.

Enrico Garattini - One of the best experts on this subject based on the ideXlab platform.

  • site directed mutagenesis of amino acid residues at the active site of mouse Aldehyde Oxidase aox1
    PLOS ONE, 2009
    Co-Authors: Silvia Schumann, Mineko Terao, Enrico Garattini, Miguel Saggu, Friedhelm Lendzian, Peter Hildebrandt, Silke Leimkuhler
    Abstract:

    Mouse Aldehyde Oxidase (mAOX1) forms a homodimer and belongs to the xanthine Oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Eschericia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of Aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the Aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.

  • the Aldehyde Oxidase gene cluster in mice and rats Aldehyde Oxidase homologue 3 a novel member of the molybdo flavoenzyme family with selective expression in the olfactory mucosa
    Journal of Biological Chemistry, 2004
    Co-Authors: Mami Kurosaki, Maria Monica Barzago, Antonio Bastone, Mario Salmona, Mineko Terao, Davide Bernardinello, Enrico Garattini
    Abstract:

    Mammalian molybdo-flavoenzymes are Oxidases requiring FAD and molybdopterin (molybdenum cofactor) for their catalytic activity. This family of proteins was thought to consist of four members, xanthine oxidoreductase, Aldehyde Oxidase 1 (AOX1), and the Aldehyde Oxidase homologues 1 and 2 (AOH1 and AOH2, respectively). Whereas the first two enzymes are present in humans and various other mammalian species, the last two proteins have been described only in mice. Here, we report on the identification, in both mice and rats, of a novel molybdo-flavoenzyme, AOH3. In addition, we have cloned the cDNAs coding for rat AOH1 and AOH2, demonstrating that this animal species has the same complement of molybdo-flavoproteins as the mouse. The AOH3 cDNA is characterized by remarkable similarity to AOX1, AOH1, AOH2, and xanthine oxidoreductase cDNAs. Mouse AOH3 is selectively expressed in Bowman's glands of the olfactory mucosa, although small amounts of the corresponding mRNA are present also in the skin. In the former location, two alternatively spliced forms of the AOH3 transcript with different 3′-untranslated regions were identified. The general properties of AOH3 were determined by purification of mouse AOH3 from the olfactory mucosa. The enzyme possesses Aldehyde Oxidase activity and oxidizes, albeit with low efficiency, exogenous substrates that are recognized by AOH1 and AOX1. The Aoh3 gene maps to mouse chromosome 1 band c1 and rat chromosome 7 in close proximity to the Aox1, Aoh1, and Aoh2 loci and has an exon/intron structure almost identical to that of the other molybdo-flavoenzyme genes in the two species.

  • regulation and biochemistry of mouse molybdo flavoenzymes the dba 2 mouse is selectively deficient in the expression of Aldehyde Oxidase homologues 1 and 2 and represents a unique source for the purification and characterization of Aldehyde Oxidase
    Journal of Biological Chemistry, 2004
    Co-Authors: Ruth Vila, Mami Kurosaki, Maria Monica Barzago, Metodej Kolek, Antonio Bastone, Laura Colombo, Mario Salmona, Mineko Terao, Enrico Garattini
    Abstract:

    Mouse molybdo-flavoenzymes consist of xanthine oxidoreductase, Aldehyde Oxidase (AOX1), and two recently identified proteins, AOH1 and AOH2 (Aldehyde Oxidase homologues 1 and 2). Here we demonstrate that CD-1, C57BL/6, 129/Sv, and other mouse strains synthesize high levels of AOH1 in the liver and AOH2 in the skin. By contrast, the DBA/2 and CBA strains are unique, having a selective deficit in the expression of the AOH1 and AOH2 genes. DBA/2 animals synthesize trace amounts of a catalytically active AOH1 protein. However, relative to CD-1 animals, an over 2 log reduction in the steady-state levels of liver AOH1 mRNA, protein, and enzymatic activity is observed in basal conditions and following administration of testosterone. The DBA/2 mouse represents a unique opportunity to purify AOX1 and compare its enzymatic characteristics to those of the AOH1 protein. The spectroscopy and biochemistry of AOX1 are very similar to those of AOH1 except for a differential sensitivity to the non-competitive inhibitory effect of norharmane. AOX1 and AOH1 oxidize an overlapping set of Aldehydes and heterocycles. For most compounds, the substrate efficiency (Vmax/Km) of AOX1 is superior to that of AOH1. Alkylic alcohols and acetAldehyde, the toxic metabolite of ethanol, are poor substrates of both enzymes. Consistent with this, the levels of acetAldehyde in the livers of ethanol administered CD-1 and DBA/2 mice are similar, indicating that neither enzyme is involved in the in vivo biotransformation of acetAldehyde.

  • cloning of the cdnas coding for two novel molybdo flavoproteins showing high similarity with Aldehyde Oxidase and xanthine oxidoreductase
    Journal of Biological Chemistry, 2000
    Co-Authors: Mineko Terao, Mami Kurosaki, Mario Salmona, Giuliana Saltini, Silvia Demontis, Massimiliano Marini, Enrico Garattini
    Abstract:

    Abstract The cDNAs coding for two novel mouse molybdo-flavoproteins, AOH1 and AOH2 (AldehydeOxidase homolog 1 and 2), were isolated. The AOH1 and AOH2 cDNAs code for polypeptides of 1336 amino acids. The two proteins have similar primary structure and show striking amino acid identity with Aldehyde Oxidase and xanthine oxidoreductase, two other molybdo-flavoenzymes. AOH1 and AOH2 contain consensus sequences for a molybdopterin-binding site and two distinct 2Fe-2S redox centers. In its native conformation, AOH1 has a molecular weight consistent with a homotetrameric structure. Transfection of the AOH1 and AOH2 cDNAs results in the production of proteins with phenanthridine but not hypoxanthine oxidizing activity. Furthermore, the AOH1 protein has benzAldehyde oxidizing activity with electrophoretic characteristics identical to those of a previously identified Aldehyde Oxidase isoenzyme (Holmes, R. S. (1979) Biochem. Genet. 17, 517–528). The AOH1 transcript is expressed in the hepatocytes of the adult and fetal liver and in spermatogonia. In liver, the AOH1 protein is synthesized in a gender-specific fashion. The expression of AOH2 is limited to keratinized epithelia and the basal layer of the epidermis and hair folliculi. The selective cell and tissue distribution of AOH1 and AOH2 mRNAs is consistent with the localization of the respective protein products.

Jeffrey P. Jones - One of the best experts on this subject based on the ideXlab platform.

  • site directed mutagenesis at the molybdenum pterin cofactor site of the human Aldehyde Oxidase interrogating the kinetic differences between human and cynomolgus monkey
    Drug Metabolism and Disposition, 2020
    Co-Authors: Armina Abbasi, Carolyn A Joswigjones, Jeffrey P. Jones
    Abstract:

    The estimation of the drug clearance by Aldehyde Oxidase (AO) has been complicated because of this enzyme’s atypical kinetics and species and substrate specificity. Since human AO (hAO) and cynomolgus monkey AO (mAO) have a 95.1% sequence identity, cynomolgus monkeys may be the best species for estimating AO clearance in humans. Here, O6-benzylguanine (O6BG) and dantrolene were used under anaerobic conditions, as oxidative and reductive substrates of AO, respectively, to compare and contrast the kinetics of these two species through numerical modeling. Whereas dantrolene reduction followed the same linear kinetics in both species, the oxidation rate of O6BG was also linear in mAO and did not follow the already established biphasic kinetics of hAO. In an attempt to determine why hAO and mAO are kinetically distinct, we have altered the hAO V811 and F885 amino acids at the oxidation site adjacent to the molybdenum pterin cofactor to the corresponding alanine and leucine in mAO, respectively. Although some shift to a more monkey-like kinetics was observed for the V811A mutant, five more mutations around the AO cofactors still need to be investigated for this purpose. In comparing the oxidative and reductive rates of metabolism under anaerobic conditions, we have come to the conclusion that despite having similar rates of reduction (4-fold difference), the oxidation rate in mAO is more than 50-fold slower than hAO. This finding implies that the presence of nonlinearity in AO kinetics is dependent upon the degree of imbalance between the rates of oxidation and reduction in this enzyme. SIGNIFICANCE STATEMENT Although they have as much as 95.1% sequence identity, human and cynomolgus monkey Aldehyde Oxidase are kinetically distinct. Therefore, monkeys may not be good estimators of drug clearance in humans.

  • A Novel Reaction Mediated by Human Aldehyde Oxidase: Amide Hydrolysis of GDC-0834
    Drug Metabolism and Disposition, 2015
    Co-Authors: Jasleen K. Sodhi, Susan Wong, Donald S. Kirkpatrick, Lichuan Liu, S. Cyrus Khojasteh, Cornelis E. C. A. Hop, John T. Barr, Jeffrey P. Jones, Jason S. Halladay
    Abstract:

    GDC-0834, a Bruton’s tyrosine kinase inhibitor investigated as a potential treatment of rheumatoid arthritis, was previously reported to be extensively metabolized by amide hydrolysis such that no measurable levels of this compound were detected in human circulation after oral administration. In vitro studies in human liver cytosol determined that GDC-0834 (R)-N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo- 4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b] thiophene-2-carboxamide) was rapidly hydrolyzed with a CLint of 0.511 ml/min per milligram of protein. Aldehyde Oxidase (AO) and carboxylesterase (CES) were putatively identified as the enzymes responsible after cytosolic fractionation and mass spectrometry-proteomics analysis of the enzymatically active fractions. Results were confirmed by a series of kinetic experiments with inhibitors of AO, CES, and xanthine Oxidase (XO), which implicated AO and CES, but not XO, as mediating GDC-0834 amide hydrolysis. Further supporting the interaction between GDC-0834 and AO, GDC-0834 was shown to be a potent reversible inhibitor of six known AO substrates with IC50 values ranging from 0.86 to 1.87 μM. Additionally, in silico modeling studies suggest that GDC-0834 is capable of binding in the active site of AO with the amide bond of GDC-0834 near the molybdenum cofactor (MoCo), orientated in such a way to enable potential nucleophilic attack on the carbonyl of the amide bond by the hydroxyl of MoCo. Together, the in vitro and in silico results suggest the involvement of AO in the amide hydrolysis of GDC-0834.

  • in vitro oxidative metabolism of 6 mercaptopurine in human liver insights into the role of the molybdoflavoenzymes Aldehyde Oxidase xanthine Oxidase and xanthine dehydrogenase
    Drug Metabolism and Disposition, 2014
    Co-Authors: Kanika V Choughule, Carlo Barnaba, Carolyn A Joswigjones, Jeffrey P. Jones
    Abstract:

    Anticancer agent 6-mercaptopurine (6MP) has been in use since 1953 for the treatment of childhood acute lymphoblastic leukemia (ALL) and inflammatory bowel disease. Despite being available for 60 years, several aspects of 6MP drug metabolism and pharmacokinetics in humans are unknown. Molybdoflavoenzymes such as Aldehyde Oxidase (AO) and xanthine Oxidase (XO) have previously been implicated in the metabolism of this drug. In this study, we investigated the in vitro metabolism of 6MP to 6-thiouric acid (6TUA) in pooled human liver cytosol. We discovered that 6MP is metabolized to 6TUA through sequential metabolism via the 6-thioxanthine (6TX) intermediate. The role of human AO and XO in the metabolism of 6MP was established using the specific inhibitors raloxifene and febuxostat. Both AO and XO were involved in the metabolism of the 6TX intermediate, whereas only XO was responsible for the conversion of 6TX to 6TUA. These findings were further confirmed using purified human AO and Escherichia coli lysate containing expressed recombinant human XO. Xanthine dehydrogenase (XDH), which belongs to the family of xanthine oxidoreductases and preferentially reduces nicotinamide adenine dinucleotide (NAD+), was shown to contribute to the overall production of the 6TX intermediate as well as the final product 6TUA in the presence of NAD+ in human liver cytosol. In conclusion, we present evidence that three enzymes, AO, XO, and XDH, contribute to the production of 6TX intermediate, whereas only XO and XDH are involved in the conversion of 6TX to 6TUA in pooled HLC.

  • inhibition of xanthine Oxidase by the Aldehyde Oxidase inhibitor raloxifene implications for identifying molybdopterin nitrite reductases
    Nitric Oxide, 2014
    Co-Authors: Eric R Weidert, Jeffrey P. Jones, Kanika V Choughule, Scott O Schoenborn, Nadiezhda Cantumedellin, Eric E Kelley
    Abstract:

    Abstract Sources of nitric oxide alternative to nitric oxide synthases are gaining significant traction as crucial mediators of vessel function under hypoxic inflammatory conditions. For example, capacity to catalyze the one electron reduction of nitrite ( NO 2 - ) to ·NO has been reported for hemoglobin, myoglobin and molybdopterin-containing enzymes including xanthine oxidoreductase (XOR) and Aldehyde Oxidase (AO). For XOR and AO, use of selective inhibition strategies is therefore crucial when attempting to assign relative contributions to nitrite-mediated ·NO formation in cells and tissue. To this end, XOR inhibition has been accomplished with application of classic pyrazolopyrimidine-based inhibitors allo/oxypurinol or the newly FDA-approved XOR-specific inhibitor, Uloric® (febuxostat). Likewise, raloxifene, an estrogen receptor antagonist, has been identified as a potent (Ki = 1.0 nM) inhibitor of AO. Herein, we characterize the inhibition kinetics of raloxifene for XOR and describe the resultant effects on inhibiting XO-catalyzed ·NO formation. Exposure of purified XO to raloxifene (PBS, pH 7.4) resulted in a dose-dependent (12.5–100 μM) inhibition of xanthine oxidation to uric acid. Dixon plot analysis revealed a competitive inhibition process with a Ki = 13 μM. This inhibitory process was more effective under acidic pH; similar to values encountered under hypoxic/inflammatory conditions. In addition, raloxifene also inhibited anoxic XO-catalyzed reduction of NO 2 - to NO (EC50 = 64 μM). In contrast to having no effect on XO-catalyzed uric acid production, the AO inhibitor menadione demonstrated potent inhibition of XO-catalyzed NO 2 - reduction (EC50 = 60 nM); somewhat similar to the XO-specific inhibitor, febuxostat (EC50 = 4 nM). Importantly, febuxostat was found to be a very poor inhibitor of human AO (EC50 = 613 μM) suggesting its usefulness for validating XO-dependent contributions to NO 2 - reduction in biological systems. Combined, these data indicate care should be taken when choosing inhibition strategies as well as inhibitor concentrations when assigning relative NO 2 - reductase activity of AO and XOR.

Shigeyuki Kitamura - One of the best experts on this subject based on the ideXlab platform.

  • XANTHINE Oxidase-CATALYZED METABOLISM OF 2-NITROFLUORENE, A CARCINOGENIC AIR POLLUTANT, IN RAT SKIN
    2020
    Co-Authors: Osamu Ueda, Shigeyuki Kitamura, Koji Ohashi, And Kazumi Sugihara, Shigeru Ohta
    Abstract:

    ABSTRACT: The reductive metabolism of 2-nitrofluorene, a carcinogenic air pollutant, in rat skin microsomes and cytosol was investigated. 2-Nitrofluorene was reduced to the corresponding amine by the microsomes with NADPH and by the cytosol with 2-hydroxypyrimidine or 4-hydroxypyrimidine under anaerobic conditions. The cytosolic activity was much higher than that of skin microsomes. The 2-or 4-hydroxypyrimidine-linked nitroreductase activity was inhibited by oxypurinol and (؉/؊)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazolo[1,5-a]-1,3,5-triazine-4(1H)-one (BOF-4272), inhibitors of xanthine Oxidase, but not by menadione, chlorpromazine and isovanillin, inhibitors of Aldehyde Oxidase. When skin cytosol was applied to a DEAE-cellulose column, the fractions containing xanthine Oxidase exhibited a marked 2-hydroxypyrimidine-linked nitroreductase activity. In contrast, the Aldehyde Oxidase fraction showed little activity. Nitroreductase fractions obtained by ion exchange chromatography showed a band in Western blotting analysis using anti-rat xanthine Oxidase. Moreover, the xanthine Oxidase fraction exhibited a significant nitroreductase activity in the presence of 2-hydroxypyrimidine, 4-hydroxypyrimidine or hypoxanthine, and these activities were inhibited by inhibitors of xanthine Oxidase. These results indicated that reduction of 2-nitrofluorene in the skin was mainly catalyzed by xanthine Oxidase

  • variability of zaleplon 5 Oxidase activity in mice and humans and inhibition by raloxifene
    Drug Metabolism Letters, 2017
    Co-Authors: Chiaki Tanoue, Kazumi Sugihara, Naoto Uramaru, Yoko Watanabe, Yoshitaka Tayama, Shigeru Ohta, Shigeyuki Kitamura
    Abstract:

    Background: Zaleplon (ZAL) is a sedative-hypnotic agent, which is mainly metabolized to inactive 5-oxidized zaleplon (5-oxo-ZAL) and N-des-ethylated ZAL (des-ethyl-ZAL) in mice and humans. The former reaction is considered to be catalyzed by Aldehyde Oxidase present in liver cytosol. Methods: Here, we examined sex and strain differences of ZAL metabolism to 5-oxo-ZAL among four strains of mice, as well as the inter-individual variation in humans, in order to evaluate the variability of 5-oxo-ZAL-forming activity and its relationship with Aldehyde Oxidase activity. In mice, the activity in C57BL/6J strain was the highest, followed by C3H/He and BALB/c. The activity in DBA/2J was the lowest, being 2.3-fold lower than that of C57BL/6J mice. The activity of male mice was higher than that of female mice. Large inter-individual variations were observed among humans, with a range of 10- fold. Raloxifene, an inhibitor of Aldehyde Oxidase, markedly decreased the formation of 5-oxo-ZAL by liver cytosol of mice and humans. Further, the plasma level of 5-oxo-ZAL in mice was decreased when raloxifene was co-administered with ZAL. Results: Our results indicate that the formation of 5-oxo-ZAL from ZAL is mainly catalyzed by Aldehyde Oxidase in mice and humans, and the variability of 5-oxo-ZAL formation is due primarily to differences of Aldehyde Oxidase activity. Conclusion: High inter-individual variability of ZAL 5-Oxidase activity and potential for interaction of ZAL with other medicines that are inhibitors of Aldehyde Oxidase should be taken into consideration in clinical usage of ZAL.

  • prediction of human metabolism of the sedative hypnotic zaleplon using chimeric mice transplanted with human hepatocytes
    Xenobiotica, 2013
    Co-Authors: Chiaki Tanoue, Kazumi Sugihara, Naoto Uramaru, Yoko Watanabe, Yoshitaka Tayama, Shigeru Ohta, Toru Horie, Shigeyuki Kitamura
    Abstract:

    1. Human chimeric mice (h-PXB mice) having humanized liver, constructed by transplantation of human hepatocytes, were evaluated as an experimental model for predicting human drug metabolism. Metabolism of zaleplon in h-PXB mice was compared with that in rat chimeric mice (r-PXB mice) constructed by transplantation of rat hepatocytes. 2. Zaleplon is metabolized to 5-oxo-zaleplon by Aldehyde Oxidase and to desethyl-zaleplon by cytochrome P450 (CYP3A4) in rat and human liver preparations. 3. Liver S9 fraction of h-PXB mice metabolized zaleplon to 5-oxo-zaleplon and desethyl-zaleplon in similar amounts. However, liver S9 fractions of r-PXB and control (urokinase-type plasminogen activator-transgenic severe combined immunodeficient) mice predominantly metabolized zaleplon to desethyl-zaleplon. 5-Oxo-zaleplon was detected as a minor metabolite. 4. Oxidase activity of h-PXB mouse liver cytosol toward zaleplon was about 10-fold higher than that of r-PXB or control mice. In contrast, activities for desethyl-zaleplon formation were similar in liver microsomes from these mice, as well as rat and human liver microsomes. 5. In vivo, the level of 5-oxo-zaleplon in plasma of h-PXB mice was about 7-fold higher than that in r-PXB or control mice, in agreement with the in vitro data. Thus, Aldehyde Oxidase in h-PXB mice functions as human Aldehyde Oxidase, both in vivo and in vitro. 6. In contrast, the plasma level of desethyl-zaleplon in r-PXB and control mice was higher than that in h-PXB mice. 7. These results suggest h-PXB mice with humanized liver could be a useful experimental model to predict Aldehyde Oxidase- and CYP3A4-mediated drug metabolism in humans.

  • Strain Difference of Oxidative Metabolism of the Sedative-hypnotic Zaleplon by Aldehyde Oxidase and Cytochrome P450 In Vivo and In Vitro in Rats
    Drug metabolism and pharmacokinetics, 2013
    Co-Authors: Chiaki Tanoue, Kazumi Sugihara, Naoto Uramaru, Yoko Watanabe, Yoshitaka Tayama, Shigeru Ohta, Shigeyuki Kitamura
    Abstract:

    The in vivo and in vitro metabolism of the sedative-hypnotic agent zaleplon (ZAL) to 5-hydroxylated ZAL (5-oxo-ZAL) and N-desethylated ZAL (desethyl-ZAL) was studied in four strains of rats. Incubation of ZAL with liver microsomes afforded desethyl-ZAL via cytochrome P450-catalyzed reaction, with little strain difference. In contrast, incubation of ZAL with liver cytosol afforded 5-oxo-ZAL with marked strain differences. ZAL hydroxylase activity was well correlated with Aldehyde Oxidase activity in these strains. The highest level of 5-oxo-ZAL and the highest activity of Aldehyde Oxidase were observed in cytosol from Sea:SD rats, followed by Jcl:SD rats, while Crj:SD and WKA/Sea rats showed low levels. When ZAL was administered to Sea:SD and WKA/Sea rats, both 5-oxo-ZAL and desethyl-ZAL were detected in blood as the major in vivo metabolites. However, the concentration of 5-oxo-ZAL was far higher in Sea:SD rats than in WKA/Sea rats, while that of desethyl-ZAL was far lower in Sea:SD rats. The levels of 5-oxo-ZAL in blood were closely correlated with the strain differences of cytosolic ZAL hydroxylase activity and benzAldehyde Oxidase activity. Our results indicate that variability in the formation of 5-oxo-ZAL from ZAL in vivo in various strains of rats is primarily due to strain differences of hepatic Aldehyde Oxidase activity.

  • developmental changes of Aldehyde Oxidase activity and protein expression in human liver cytosol
    Drug Metabolism and Pharmacokinetics, 2012
    Co-Authors: Yoshitaka Tayama, Kazumi Sugihara, Shigeyuki Kitamura, Seigo Sanoh, Katsushi Miyake, Shigeru Ohta
    Abstract:

    Summary: Aldehyde Oxidase (AO) plays a role in metabolizing many drugs, such as methotrexate and 6-mercaptopurine. We previously showed that AO activity in rat liver rapidly increases from birth, reaching a plateau within 4 weeks, and is regulated at the protein expression level. However, developmental changes of AO activity and protein expression in human liver have not been reported. Here, we investigated the developmental changes and variability of AO in 16 human livers (13 children ranging from 13 days to 12 years old and 3 adults, 17, 34 and 45 years old). Young children (13 days to 4 months after birth) showed little liver AO activity, evaluated in terms of the activities for oxidation of N -1-methylnicotinamide to N -1-methyl-2-pyridone-5-carboxamide and N -1-methyl-4-pyridone-3-carboxamide in liver cytosol. However, these Oxidase activities were markedly increased after 4 months, reaching the adult level by about 2 years of age. The AO band density in immunoblotting analysis was well correlated with the AO activity among all subjects (p 2 = 0.771). Therefore, AO activity in the liver of young children is regulated at the AO protein expression level. Thus, as in rats, the AO activity in humans rapidly increases soon after birth, and is regulated at the protein expression level.