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Diquat (DQ) poisoning has garnered attention in recent years, primarily due to the rising incidence of cases worldwide, coupled with the absence of a viable antidote for its treatment. Despite the fact that diquat monopyridone (DQ-M) has been identified as a significant metabolite of DQ, the enzyme responsible for its formation remains unknown. In this study, we have identified aldehyde oxidase (AOX) as a vital enzyme involved in DQ oxidative metabolism. The metabolism of DQ to DQ-M was significantly inhibited by AOX inhibitors including raloxifene and hydralazine. The source of oxygen incorporated into DQ-M was proved to be from water through a H2 18 O incubation experiment which further corroborated DQ-M formation via AOX metabolism. The product of DQ-M in vitro generated by fresh rat tissues co-incubation was consistent with its AOX expression. The result of the molecular docking analysis of DQ and AOX protein showed that DQ is capable of binding to AOX. Furthermore, the cytotoxicity of DQ was significantly higher than DQ-M at the same concentration tested in six cell types. This work is the first to uncover the involvement of aldehyde oxidase, a non-cytochrome P450 enzyme, in the oxidative metabolic pathway of diquat, thus providing a potential target for the development of detoxification treatment., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aldehyde oxidase (AOX) and other related molybdenum-containing enzymes are known to oxidize the C-H bonds of aromatic rings. This process contributes to the metabolism of pharmaceutical compounds and, therefore, is of vital importance to drug pharmacokinetics. The present work describes an automated computational workflow and its use for the prediction of intrinsic reactivity of small aromatic molecules toward a minimal model of the active site of AOX. The workflow is based on quantum chemical transition state searches for the underlying single-step oxidation reaction, where the automated protocol includes identification of unique aromatic C-H bonds, creation of three-dimensional reactant and product complex geometries via a templating approach, search for a transition state, and validation of reaction end points. Conformational search on the reactants, products, and the transition states is performed. The automated procedure has been validated on previously reported transition state barriers and was used to evaluate the intrinsic reactivity of nearly three hundred heterocycles commonly found in approved drug molecules. The intrinsic reactivity of more than 1000 individual aromatic carbon sites is reported. Stereochemical and conformational aspects of the oxidation reaction, which have not been discussed in previous studies, are shown to play important roles in accurate modeling of the oxidation reaction. Observations on structural trends that determine the reactivity are provided and rationalized.

Aldehyde oxidase (AOX) is a cytosolic drug-metabolizing enzyme which has attracted increasing attention in drug development due to its high hepatic expression, broad substrate profile and species differences. In contrast, there is limited information on the presence and activity of AOX in extrahepatic tissues including ocular tissues. Because several ocular drugs are potential substrates for AOX, we performed a comprehensive analysis of the AOX1 expression and activity profile in seven ocular tissues from humans, rabbits, and pigs. AOX activities were determined using optimized assays for the established human AOX1 probe substrates 4-dimethylamino-cinnamaldehyde (DMAC) and phthalazine. Inhibition studies were undertaken in conjunctival and retinal homogenates using well-established human AOX1 inhibitors menadione and chlorpromazine. AOX1 protein contents were quantitated with targeted proteomics and confirmed by immunoblotting. Overall, DMAC oxidation rates varied over 10-fold between species (human ˃˃ rabbit ˃ pig) and showed 2- to 6-fold differences between tissues from the same species. Menadione seemed a more potent inhibitor of DMAC oxidation across species than chlorpromazine. Human AOX1 protein levels were highest in the conjunctiva, followed by most posterior tissues, whereas anterior tissues showed low levels. The rabbit AOX1 expression was high in the conjunctiva, retinal pigment epithelial (RPE), and choroid while lower in the anterior tissues. Quantification of pig AOX1 was not successful but immunoblotting confirmed the presence of AOX1 in all species. DMAC oxidation rates and AOX1 contents correlated quite well in humans and rabbits. This study provides, for the first time, insights into the ocular expression and activity of AOX1 among multiple species., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

This is an overview of the metabolic reactions of drugs, natural products, physiological compounds, and other (general) chemicals catalyzed by flavin monooxygenase (FMO), monoamine oxidase (MAO), NAD(P)H quinone oxidoreductase (NQO), and molybdenum hydroxylase enzymes (aldehyde oxidase (AOX) and xanthine oxidoreductase (XOR)), including roles as substrates, inducers, and inhibitors of the enzymes. The metabolism and bioactivation of selected examples of each group (i.e., drugs, "general chemicals," natural products, and physiological compounds) are discussed. We identified a higher fraction of bioactivation reactions for FMO enzymes compared to other enzymes, predominately involving drugs and general chemicals. With MAO enzymes, physiological compounds predominate as substrates, and some products lead to unwanted side effects or illness. AOX and XOR enzymes are molybdenum hydroxylases that catalyze the oxidation of various heteroaromatic rings and aldehydes and the reduction of a number of different functional groups. While neither of these two enzymes contributes substantially to the metabolism of currently marketed drugs, AOX has become a frequently encountered route of metabolism among drug discovery programs in the past 10-15 years. XOR has even less of a role in the metabolism of clinical drugs and preclinical drug candidates than AOX, likely due to narrower substrate specificity., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mammalian aldehyde oxidases (AOX) are molybdo-flavoenzymes of pharmacological and pathophysiologic relevance that are involved in phase I drug metabolism and, as a product of their enzymatic activity, are also involved in the generation of reactive oxygen species. So far, the physiologic role of aldehyde oxidase 1 in the human body remains unknown. The human enzyme hAOX1 is characterized by a broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into their corresponding carboxylic acids, and hydroxylating various heteroaromatic rings. The enzyme uses oxygen as terminal electron acceptor to produce hydrogen peroxide and superoxide during turnover. Since hAOX1 and, in particular, some natural variants produce not only H 2 O 2 but also high amounts of superoxide, we investigated the effect of both ROS molecules on the enzymatic activity of hAOX1 in more detail. We compared hAOX1 to the high-O 2 .- -producing natural variant L438V for their time-dependent inactivation with H 2 O 2 /O 2 .- during substrate turnover. We show that the inactivation of the hAOX1 wild-type enzyme is mainly based on the production of hydrogen peroxide, whereas for the variant L438V, both hydrogen peroxide and superoxide contribute to the time-dependent inactivation of the enzyme during turnover. Further, the level of inactivation was revealed to be substrate-dependent: using substrates with higher turnover numbers resulted in a faster inactivation of the enzymes. Analysis of the inactivation site of the enzyme identified a loss of the terminal sulfido ligand at the molybdenum active site by the produced ROS during turnover. SIGNIFICANCE STATEMENT: This work characterizes the substrate-dependent inactivation of human aldehyde oxidase 1 under turnover by reactive oxygen species and identifies the site of inactivation. The role of ROS in the inhibition of human aldehyde oxidase 1 will have a high impact on future studies., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Aldehyde oxidase (AO) has emerged as an important drug metabolizing enzyme over the last decade. Several compounds have failed in the clinic because the clearance or toxicity was underestimated by preclinical species. Human AO is much more active than rodent AO, and dogs do not have functional AO. Metabolic products from AO-catalyzed oxidation are generally nonreactive and often they have much lower solubility. AO metabolism is not limited to oxidation as AO can also catalyze reduction of oxygen and nitrite. Reduction of oxygen leads to the reactive oxygen species (ROS) superoxide radical anion and hydrogen peroxide. Reduction of nitrite leads to the formation of nitric oxide with potential pharmacological implications. AO is also reported to catalyze the reductive metabolism of nitro-compounds, N-oxides, sulfoxides, isoxazoles, isothiazoles, nitrite, and hydroxamic acids. These reductive transformations may cause toxicity due to the formation of reactive metabolites. Moreover, the inhibition kinetics are complex, and multiple probe substrates should be used when assessing the potential for DDIs. Finally, AO appears to be amenable to computational predictions of both regioselectivity and rates of reaction, which holds promise for virtual screening., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Aldehyde oxidases (AOXs) are a subfamily of cytosolic molybdo-flavoenzymes that play critical roles in the detoxification and degradation of chemicals. Active AOXs, such as AOX1 and AOX2, have been identified and functionally analyzed in insect antennae but are rarely reported in other tissues. This is the first study to isolate and characterize the cDNA that encodes aldehyde oxidase 5 (BmAOX5) in the pheromone gland (PG) of the silkworm, Bombyx mori. The size of BmAOX5 cDNA is 3,741 nucleotides and includes an open reading frame, which encodes a protein of 1,246 amino acid residues. The theoretical molecular weight and isoelectric point of BmAOX5 are approximately 138 kDa and 5.58, respectively. BmAOX5 shares a similar primary structure with BmAOX1 and BmAOX2, containing two [2Fe-2S] redox centers, a FAD-binding domain, and a molybdenum cofactor (MoCo)-binding domain. RT-PCR revealed BmAOX5 to be particularly highly expressed in the PG (including ovipositor) of the female silkworm moth, and the expression was further confirmed by in situ hybridization, AOX activity staining, and anti-BmAOX5 western blotting. Further, BmAOX5 was shown to metabolize aromatic aldehydes, such as benzaldehyde, salicylaldehyde, and vanillic aldehyde, and fatty aldehydes, such as heptaldehyde and propionaldehyde. The maximum reaction rate (Vmax) of benzaldehyde as substrate was 21 mU and Km was 1.745 mmol/liter. These results suggested that BmAOX5 in the PG could metabolize aldehydes in the cytoplasm for detoxification or participate in the degradation of aldehyde pheromone substances and odorant compounds to identify mating partners and locate suitable spawning sites., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.)

Gefitinib and erlotinib are epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) with activity against metastatic non-small cell lung cancer. Aldehyde oxidase-1 (AOX1) is a cytosolic drug-metabolizing enzyme. We conducted an experimental and molecular docking study on the effect of gefitinib, erlotinib, and select metabolites on the in vitro catalytic activity of AOX1, as assessed by carbazeran 4-oxidation, and determined the impact of AOX1 inhibition on hepatic metabolism of zaleplon and methotrexate. Gefitinib, desmorpholinopropylgefitinib, erlotinib, desmethylerlotinib, and didesmethylerlotinib inhibited human hepatic cytosolic carbazeran 4-oxidation by a competitive mode, with inhibition constants in submicromolar or low micromolar concentrations. Desmethylgefitinib did not affect AOX1 catalytic activity. A similar pattern was obtained when investigated with human kidney cytosol or recombinant AOX1. The differential effect of gefitinib on human, rat, and mouse hepatic AOX1 catalytic activity suggests species-dependent chemical inhibition of AOX1. Erlotinib was considerably more potent than gefitinib in decreasing hepatic cytosolic zaleplon 5-oxidation and methotrexate 7-oxidation. Molecular docking analyses provided structural insights into the interaction between EGFR-TKIs and AOX1, with key residues and bonds identified, which provided favorable comparison and ranking of potential inhibitors. Based on the US Food and Drug Administration guidance to assess the risk of drug-drug interactions, the calculated R 1 values indicate that further investigations are warranted to determine whether gefitinib and erlotinib impact AOX1-mediated drug metabolism in vivo. Overall, erlotinib desmethylerlotinib, didesmethylerlotinib, gefitinib, and desmorpholinopropylgefitinib are potent inhibitors of human AOX1 catalytic function and hepatic metabolism of zaleplon and methotrexate, potentially affecting drug efficacy or toxicity. SIGNIFICANCE STATEMENT: As epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib are first-line pharmacotherapy for metastatic non-small cell lung cancer. Our experimental findings indicate that clinically relevant concentrations of gefitinib, desmorpholinopropylgefitinib, erlotinib, desmethylerlotinib, and didesmethylerlotinib, but not desmethylgefitinib, inhibit human aldehyde oxidase (AOX1) catalytic activity and hepatic cytosolic metabolism of zaleplon and methotrexate. Molecular docking analysis provide structural insights into the key AOX1 interactions with these EGFR-TKIs. Our findings may trigger improved strategies for new EGFR-TKI design and development., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Aldehyde oxidases (AOXs) are a small group of enzymes belonging to the larger family of molybdo-flavoenzymes, along with the well-characterized xanthine oxidoreductase. The two major types of reactions that are catalyzed by AOXs are the hydroxylation of heterocycles and the oxidation of aldehydes to their corresponding carboxylic acids. Different animal species have different complements of AOX genes. The two extremes are represented in humans and rodents; whereas the human genome contains a single active gene ( AOX1 ), those of rodents, such as mice, are endowed with four genes ( Aox1-4 ), clustering on the same chromosome, each encoding a functionally distinct AOX enzyme. It still remains enigmatic why some species have numerous AOX enzymes, whereas others harbor only one functional enzyme. At present, little is known about the physiological relevance of AOX enzymes in humans and their additional forms in other mammals. These enzymes are expressed in the liver and play an important role in the metabolisms of drugs and other xenobiotics. In this review, we discuss the expression, tissue-specific roles, and substrate specificities of the different mammalian AOX enzymes and highlight insights into their physiological roles., (© 2020 Terao et al.)

The in vivo monitoring of ascorbic acid (AA) following physiological and pathological events is of great importance because AA plays a critical role in brain functions. The conventional electrochemical sensors (ECSs) usually suffered from poor selectivity and sluggish electron transfer kinetics for cerebral AA oxidation. The exploitation of ECSs adapt to the electrochemical detection (ECD)-microdialysis system, here we reported a facile ratiometric electrochemical sensor (RECS) for in vivo/online repetitive measurements of cerebral AA in brain microdiaysate. The sensor were constructed by careful electrodeposition of graphene oxide (GO) onto glassy carbon (GC) electrodes. Methylene blue (MB) was electrostatically adsorbed onto the GO surface as a built-in reference to achieve ratiometric detection of AA. The subsequent proper electroreduction treatment was able to readily facilitate the oxidation of AA at a relatively negative potential (-100 mV) and the oxidation of MB at separated potential (-428 mV). The in vitro experiments demonstrated that the RECS exhibited high sensitivity (detection limit: 10 nM), selectivity, and stability toward AA determination, enabling the in vivo/online repetitive measurement of cerebral AA in brain microdiaysate with high reliability. As a result, the designed RECS was successfully applied in the ECD-microdialysis system to in vivo/online repetitive monitoring the dynamic change of cerebral AA in the progress of the global cerebral ischemia/reperfusion events. More, the microinjection of endogenous AA and AA oxidase (AAOx) verified the reliability of the proposed RECS for in vivo/online repetitive cerebral AA detection. This proposed sensor filled the gap that no rational electrochemical sensor has been developed for the ECD-microdialysis system since its creation by the Mao group in 2005, which provided a reliable and effective method for brain chemistry research.

Human AOX1 is a member of the mammalian aldehyde oxidase (AOX) family of enzymes and it is an emerging cytosolic enzyme involved in phase I drug-metabolism, bio-transforming a number of therapeutic agents and xenobiotics. The current trend in drug-development is to design molecules which are not recognized and inactivated by CYP450 monooxygenases, the main drug-metabolizing system, to generate novel therapeutic agents characterized by optimal pharmacokinetic and pharmacodynamic properties. Unfortunately, this has resulted in a substantial enrichment in molecules which are recognized and metabolized by AOXs. The observation has raised interest in the generation of tools capable of predicting AOX-dependent drug-metabolism of novel molecules during the early phases of drug development. Such tools are likely to reduce the number of failures occurring at the clinical and late phase of the drug development process. The current review describes different in silico , in vitro and in vivo methods for the prediction of AOX metabolizing ability and focuses on the existing drawbacks and challenges associated with these approaches.

Tamoxifen, raloxifene, and nafoxidine are selective estrogen receptor modulators (SERMs) reported to inhibit the catalytic activity of human aldehyde oxidase 1 (AOX1). How these drugs interact with AOX1 and whether other SERMs inhibit this drug-metabolizing enzyme are not known. Therefore, a detailed in vitro and in silico study involving parent drugs and their analogs was conducted to investigate the effect of specific SERMs, particularly acolbifene, bazedoxifene, and lasofoxifene on AOX1 catalytic activity, as assessed by carbazeran 4-oxidation, an AOX1-selective catalytic marker. The rank order in the potency (based on IC 50 values) of AOX1 inhibition by SERMs was raloxifene > bazedoxifene ∼ lasofoxifene > tamoxifen > acolbifene. Inhibition of liver cytosolic AOX1 by bazedoxifene, lasofoxifene, and tamoxifen was competitive, whereas that by raloxifene was noncompetitive. Loss of 1-azepanylethyl group increased the inhibitory potency of bazedoxifene, whereas the N -oxide group decreased it. The 7-hydroxy group and the substituted pyrrolidine ring attached to the tetrahydronaphthalene structure contributed to AOX1 inhibition by lasofoxifene. These results are supported by molecular-docking simulations in terms of predicted binding modes, encompassing binding orientation and efficiency, and analysis of key interactions, particularly hydrogen bonds. The extent of AOX1 inhibition by bazedoxifene was increased by estrone sulfate and estrone. In summary, SERMs differentially inhibited human AOX1 catalytic activity. Structural features of bazedoxifene and lasofoxifene contributed to AOX1 inhibition, whereas those of acolbifene rendered it considerably less susceptible to AOX1 inhibition. Overall, our novel biochemical findings and molecular-docking analyses provide new insights into the interaction between SERMs and AOX1. SIGNIFICANCE STATEMENT: Aldehyde oxidase (AOX1) is a molybdo-flavoprotein and has emerged as a drug-metabolizing enzyme of potential therapeutic importance because drugs have been identified as AOX1 substrates. Selective estrogen receptor modulators (SERM), which are drugs used to treat and prevent various conditions, differentially inhibit AOX1 catalytic activity. Structural features of bazedoxifene and lasofoxifene contribute to AOX1 inhibition, whereas those of acolbifene render it considerably less susceptible to AOX1 inhibition. Our novel biochemical findings, together with molecular- docking analyses, provide new insights into the differential inhibitory effect of SERMs on the catalytic activity of human AOX1, how SERMs bind to AOX1, and increase our understanding of the AOX1 pharmacophore in the inhibition of AOX1 by drugs and other chemicals., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

( R )-2-(2-methylimidazo[2,1-b]thiazol-6-yl)-1-(2-(5-(6-methylpyrimidin-4-yl)-2,3-dihydro-1 H -inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)ethan-1-one (PF-5190457) was identified as a potent and selective inverse agonist of the ghrelin receptor [growth hormone secretagogue receptor 1a (GHS-R1a)]. The present translational bed-to-bench work characterizes the biotransformation of this compound in vivo and then further explores in vitro metabolism in fractions of human liver and primary hepatocytes. Following oral administration of PF-5190457 in a phase 1b clinical study, hydroxyl metabolites of the compound were observed, including one that had not been observed in previously performed human liver microsomal incubations. PF-6870961 was biosynthesized using liver cytosol, and the site of hydroxylation was shown to be on the pyrimidine using nuclear magnetic resonance spectroscopy. The aldehyde oxidase (AO) inhibitor raloxifene and the xanthine oxidase inhibitor febuxostat inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. However, greater inhibition was observed with raloxifene, indicating AO is a dominant enzyme in the biotransformation. The intrinsic clearance of the drug in human liver cytosol was estimated to be 0.002 ml/min per milligram protein. This study provides important novel information at three levels: 1) it provides additional new information on the recently developed novel compound PF-5190457, the first GHS-R1a blocker that has moved to development in humans; 2) it provides an example of a reverse translational approach where a discovery in humans was brought back, validated, and further investigated at the bench level; and 3) it demonstrates the importance of considering the molybdenum-containing oxidases during the development of new drug entities. SIGNIFICANCE STATEMENT: PF-5190457 is a novel ghrelin receptor inverse agonist that is currently undergoing clinical development for treatment of alcohol use disorder. PF-6870961, a major hydroxyl metabolite of the compound, was observed in human plasma, but was absent in human liver microsomal incubations. PF-6870961 was biosynthesized using liver cytosol, and the site of hydroxylation on the pyrimidine ring was characterized. Inhibitors of aldehyde oxidase and xanthine oxidase inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. This information is important for patient selection in subsequent clinical studies., Competing Interests: R.S.O. and T.F.R. are active employees at Pfizer Inc., Groton, CT., (U.S. Government work not protected by U.S. copyright.)

Human aldehyde oxidase (hAOX1) is a molybdenum dependent enzyme that plays an important role in the metabolism of various compounds either endogenous or xenobiotics. Due to its promiscuity, hAOX1 plays a major role in the pharmacokinetics of many drugs and therefore has gathered a lot of attention from the scientific community and, particularly, from the pharmaceutical industry. In this work, homology modelling, molecular docking and molecular dynamics simulations were used to study the structure of the monomer and dimer of human AOX. The results with the monomer of hAOX1 allowed to shed some light on the role played by thioridazine and two malonate ions that are co-crystalized in the recent X-ray structure of hAOX1. The results show that these molecules endorse several conformational rearrangements in the binding pocket of the enzyme and these changes have an impact in the active site topology as well as in the stability of the substrate (phthalazine). The results show that the presence of both molecules open two gates located at the entrance of the binding pocket, from which results the flooding of the active site. They also endorse several modifications in the shape of the binding pocket (namely the position of Lys893) that, together with the presence of the solvent molecules, favour the release of the substrate to the solvent. Further insights were also obtained with the assembled homodimer of hAOX1. The allosteric inhibitor (THI) binds closely to the region where the dimerization of both monomers occur. These findings suggest that THI can interfere with protein dimerization.

Human aldehyde oxidase (hAOX1) is a molybdenum enzyme with high toxicological importance, but its physiological role is still unknown. hAOX1 metabolizes different classes of xenobiotics and is one of the main drug-metabolizing enzymes in the liver, along with cytochrome P450. hAOX1 oxidizes and inactivates a large number of drug molecules and has been responsible for the failure of several phase I clinical trials. The interindividual variability of drug-metabolizing enzymes caused by single nucleotide polymorphisms (SNPs) is highly relevant in pharmaceutical treatments. In this study, we present the crystal structure of the inactive variant G1269R, revealing the first structure of a molybdenum cofactor (Moco)-free form of hAOX1. These data allowed to model, for the first time, the flexible Gate 1 that controls access to the active site. Furthermore, we inspected the thermostability of wild-type hAOX1 and hAOX1 with various SNPs (L438V, R1231H, G1269R or S1271L) by CD spectroscopy and ThermoFAD, revealing that amino acid exchanges close to the Moco site can impact protein stability up to 10 °C. These results correlated with biochemical and structural data and enhance our understanding of hAOX1 and the effect of SNPs in the gene encoding this enzyme in the human population. ENZYMES: Aldehyde oxidase (EC1.2.3.1); xanthine dehydrogenase (EC1.17.1.4); xanthine oxidase (EC1.1.3.2). DATABASES: Structural data are available in the Protein Data Bank under the accession number 6Q6Q., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Background: Aldehyde oxidase (AOX) is an important molybdenum-containing enzyme with high similarity with xanthine oxidase (XO). AOX involved in the metabolism of a large array of aldehydes and N-heterocyclic compounds and its activity is highly substrate-dependent., Objectives: The aim of this work was to study the effect of five important phenothiazine drugs on AOX activity using benzaldehyde and phenanthridine as aldehyde and N-heterocyclic substrates, respectively., Methods: The effect of trifluperazine, chlorpromazine, perphenazine, thioridazine and promethazine on rat liver AOX was measured spectrophotometrically. To predict the mode of interactions between the studied compounds and AOX, a combination of homology modeling and a molecular docking study was performed., Results: All phenothiazines could inhibit AOX activity measured either by phenanthridine or benzaldehyde with almost no effect on XO activity. In the case of benzaldehyde oxidation, the lowest and highest half-maximal inhibitory concentration (IC 50 ) values were obtained for promethazine (IC 50  = 0.9 µM), and trifluoperazine (IC 50  = 3.9 µM), respectively; whereas perphenazine (IC 50  = 4.3 µM), and trifluoperazine (IC 50  = 49.6 µM) showed the strongest and weakest inhibitory activity against AOX-catalyzed phenanthridine oxidation, respectively. The in silico findings revealed that the binding site of thioridazine is near the dimer interference, and that hydrophobic interactions are of great importance in all the tested phenothiazines., Conclusion: The five studied phenothiazine drugs showed dual inhibitory effects on AOX activity towards aldehydes and N-heterocycles as two major classes of enzyme substrates. Most of the interactions between the phenothiazine-related drugs and AOX in the binding pocket showed a hydrophobic nature.

Auxin is thought to be an important factor in the induction of galls by galling insects. We have previously shown that both galling and nongalling insects synthesize indole-3-acetic acid (IAA) from tryptophan (Trp) via two intermediates, indole-3-acetaldoxime (IAOx) and indole-3-acetaldehyde (IAAld). In this study, we isolated an enzyme that catalyzes the last step "IAAld → IAA" from a silk-gland extract of Bombyx mori. The enzyme, designated "BmIAO1", contains two 2Fe-2S iron-sulfur-cluster-binding domains, an FAD-binding domain, and a molybdopterin-binding domain, which are conserved in aldehyde oxidases. BmIAO1 causes the nonenzymatic conversion of Trp to IAAld and the enzymatic conversion of IAOx to IAA, suggesting that BmIAO1 alone is responsible for IAA production in B. mori. However, a detailed comparison of pure BmIAO1 and the crude silk-gland extract suggested the presence of other enzymes involved in IAA production from Trp. Abbreviations: BA: benzoic acid; CE: collision energy; CXP: collision cell exit potential; DP: declustering potential; IAA: indole-3-acetic acid; IBI1: IAA biosynthetic inhibitor-1; IAAld: indole-3-acetaldehyde; ICA: indole-3-carboxylic acid; IAOx: indole-3-acetaldoxime; IEtOH: indole-3-ethanol; LC-MS/MS: liquid chromatography-tandem mass spectrometry; Trp: tryptophan.

Almost all drug molecules become the substrates for oxidoreductase enzymes, get metabolized into more hydrophilic products and eliminated from the body. These metabolites sometime may be more potent, active, inactive, or toxic in nature compared to parent molecule. Xanthine oxidoreductase and aldehyde oxidase belong to molybdenum containing family and are well characterized for their structures and functions, in particular to their ability to oxidize/hydroxylate the xenobiotics. Their upregulated clinical levels causing oxidative stress are associated with pathways either directly involved in the progression of diseases, gout, or indirectly with the succession of other diseases such as diabetes, cancer, etc. Herein, we have put forth a comprehensive review on the xanthine and aldehyde oxidases pertaining to their structures, functions, pathophysiological role, and a comparative analysis of structural insights of xanthine and aldehyde oxidases' binding domains with endogenous ligands or inhibitors. Though both the enzymes are molybdenum containing and are likely to share some common pathways and interact with inhibitors in a similar manner but we have focused on structural prerequisites for inhibitor specificity to both the enzymes keeping in view of the existing X-ray structures. This review also provides futuristic implications in the design of inhibitors derived from inorganic complexes or small organic molecules considering the spatial features and structural insights of both the enzymes., (© 2017 Wiley Periodicals, Inc.)

Nitric oxide (NO) is an important gasotransmitter involved in numerous intra- and intercellular signaling events. In addition to the oxidative pathway of NO generation, which includes three NO synthase (NOS) isoforms in mammals, a reductive pathway contributes to NO generation. In this pathway, nitrite is reduced to NO by various metal-containing proteins. Among these, all members of the eukaryotic molybdenum (Mo)-dependent enzyme family were found to be able to reduce nitrite to NO. This Review focuses on the current state of research in the field of Mo-dependent nitrite reduction in eukaryotes. An overview on the five eukaryotic Mo-enzymes is given, and similarities as well as differences in their nitrite reduction mechanisms are presented and discussed in the context of physiological relevance., (© 2018 Federation of European Biochemical Societies.)

Hetero-trimeric aldehyde oxidases of bacterial origin, which use O2 to catalyse the oxidation of various aldehydes but not those of aromatic N-heterocycles, belong to the xanthine oxidase family. In the present study, the crystal structure of a recombinant aldehyde oxidase from Methylobacillus sp. KY4400 (Mb-AOX) was determined at 2.5 Å resolution. The structures of its subunits resemble those of the corresponding subunits or domains of other structurally characterised enzymes belonging to the family, and include a [4Fe-4 S] cluster in the medium subunit like that found in Escherichia coli periplasmic aldehyde oxidoreductase (EP-AOR). A funnel leading to the si-face of the isoalloxazine ring of FAD, which is narrower than those in mouse liver AOX3 and human AOX1, is also present and it is even narrower than that in EP-AOR. The environment surrounding the ring in Mb-AOX and EP-AOR is subtly different, which might account for their different abilities to use O2. A remarkable characteristic of the Mo catalytic centre in Mb-AOX is a tryptophan situated near the centre instead of the alanine present in other xanthine oxidase family members. The tryptophan residue together with other residue differences might play an important role in binding to aldehydes such as n-heptylaldehyde in Mb-AOX.

Mammalian aldehyde oxidases (AOXs) are molybdo-flavoenzymes which are present in many tissues in various mammalian species, including humans and rodents. Different species contain a different number of AOX isoforms. In particular, the reasons why mammals other than humans express a multiplicity of tissue-specific AOX enzymes is unknown. In mouse, the isoforms mAOX1, mAOX3, mAOX4 and mAOX2 are present. We previously established a codon-optimized heterologous expression systems for the mAOX1-4 isoforms in Escherichia coli that gives yield to sufficient amounts of active protein for kinetic characterizations and sets the basis in this study for site-directed mutagenesis and structure-function studies. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes on a larger number of substrates has never been performed. Here, thirty different structurally related aromatic, aliphatic and N-heterocyclic compounds were used as substrates, and the kinetic parameters of all four mAOX enzymes were directly compared. The results show that especially mAOX4 displays a higher substrate selectivity, while no major differences between mAOX1, mAOX2 and mAOX3 were identified. Generally, mAOX1 was the enzyme with the highest catalytic turnover for most substrates. To understand the factors that contribute to the substrate specificity of mAOX4, site-directed mutagenesis was applied to substitute amino acids in the substrate-binding funnel by the ones present in mAOX1, mAOX3, and mAOX2. An increase in activity was obtained by the amino acid exchange M1088V in the active site identified to be specific for mAOX4, to the amino acid identified in mAOX3.

Aldehyde oxidase (AOX) is a molibdo-flavoenzyme that has raised great interest in recent years, since its contribution in xenobiotic metabolism has not always been identified before clinical trials, with consequent negative effects on the fate of new potential drugs. The fundamental role of AOX in metabolizing xenobiotics is also due to the attempt of medicinal chemists to stabilize candidates toward cytochrome P450 activity, which increases the risk for new compounds to be susceptible to AOX nucleophile attack. Therefore, novel strategies to predict the potential liability of new entities toward the AOX enzyme are urgently needed to increase effectiveness, reduce costs, and prioritize experimental studies. In the present work, we present the most up-to-date computational method to predict liability toward human AOX (hAOX), for applications in drug design and pharmacokinetic optimization. The method was developed using a large data set of homogeneous experimental data, which is also disclosed as Supporting Information .

Nitrazepam (NZP) is a hypnotic agent that rarely causes liver injuries in humans and teratogenicity in rodents. In humans, NZP is primarily metabolized to 7-aminonitrazepam (ANZP) by reduction and subsequently to 7-acetylamino nitrazepam (AANZP) by acetylation. ANZP can be regenerated from AANZP by hydrolysis in rodents, but it is still unclear whether this reaction occurs in humans. In rodents, AANZP may be associated with teratogenicity, while in humans, it is known that drug-induced liver injuries may be caused by NZP reactive metabolite(s). In this study, we attempted to identify the enzymes responsible for NZP metabolism to obtain a basic understanding of this process and the associated metabolite toxicities. We found that the NZP reductase activity in human liver cytosol (HLC) was higher than that in human liver microsomes (HLM). We purified the responsible enzyme(s) from HLC and found that the NZP reductase was aldehyde oxidase 1 (AOX1). The role of AOX1 was confirmed by an observed increase in the NZP reductase activity upon addition of N 1 -methylnicotinamide, an electron donor of AOX1, as well as inhibition of this activity in HLC in the presence of AOX1 inhibitors. ANZP was acetylated to form AANZP by N-acetyltransferase (NAT) 2. An experiment using recombinant esterases in an inhibition study using HLM revealed that AANZP is hydrolyzed by arylacetamide deacetylase (AADAC) in the human liver. N-Hydroxylamino NZP, which is suspected to be a reactive metabolite, was detected as a conjugate with N-acetyl-l-cysteine through NZP reduction and ANZP hydroxylation reactions. In the latter reaction, the conjugate was readily formed by recombinant CYP3A4 among the various P450 isoforms tested. In sum, we found that AOX1, NAT2, AADAC, and CYP3A4 are the determinants for the pharmacokinetics of NZP and that they confer interindividual variability in sensitivity to NZP side effects., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. The physiological function of mammalian AOX isoenzymes is still unclear, however, human AOX (hAOX1) is an emerging enzyme in phase-I drug metabolism. Indeed, the number of xenobiotics acting as hAOX1 substrates is increasing. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified within the hAOX1 gene. SNPs are a major source of inter-individual variability in the human population, and SNP-based amino acid exchanges in hAOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. In this report we selected ten novel SNPs resulting in amino acid exchanges in proximity to the FAD site of hAOX1 and characterized the purified enzymes after heterologous expression in Escherichia coli. The hAOX1 variants were characterized carefully by quantitative differences in their ability to produce superoxide radical. ROS represent prominent key molecules in physiological and pathological conditions in the cell. Our data reveal significant alterations in superoxide anion production among the variants. In particular the SNP-based amino acid exchange L438V in proximity to the isoalloxanzine ring of the FAD cofactor resulted in increased rate of superoxide radical production of 75%. Considering the high toxicity of the superoxide in the cell, the hAOX1-L438V SNP variant is an eventual candidate for critical or pathological roles of this natural variant within the human population.

Aldehyde oxidase (AOX) is a metabolic enzyme catalyzing the oxidation of aldehyde and aza-aromatic compounds and the hydrolysis of amides, moieties frequently shared by the majority of drugs. Despite its key role in human metabolism, to date only fragmentary information about the chemical features responsible for AOX susceptibility are reported and only "very local" structure-metabolism relationships based on a small number of similar compounds have been developed. This study reports a more comprehensive coverage of the chemical space of structures with a high risk of AOX phase I metabolism in humans. More than 270 compounds were studied to identify the site of metabolism and the metabolite(s). Both electronic [supported by density functional theory (DFT) calculations] and exposure effects were considered when rationalizing the structure-metabolism relationship., Competing Interests: The authors declare no conflict of interest.

Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. Mammals are characterized by a complement of species-specific AOX isoenzymes, that varies from one in humans (AOX1) to four in rodents (AOX1, AOX2, AOX3 and AOX4). The physiological function of mammalian AOX isoenzymes is unknown, although human AOX1 is an emerging enzyme in phase-I drug metabolism. Indeed, the number of therapeutic molecules under development which act as AOX substrates is increasing. The recent crystallization and structure determination of human AOX1 as well as mouse AOX3 has brought new insights into the mechanisms underlying substrate/inhibitor binding as well as the catalytic activity of this class of enzymes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: Given the rising trend in medicinal chemistry strategy to reduce cytochrome P450-dependent metabolism, aldehyde oxidase (AOX) has recently gained increased attention in drug discovery programs and the number of drug candidates that are metabolized by AOX is steadily growing. Areas covered: Despite the emerging importance of AOX in drug discovery, there are certain major recognized problems associated with AOX-mediated metabolism of drugs. Intra- and inter-species variations in AOX activity, the lack of reliable and predictive animal models using the common experimental animals, and failure in the predictions of in vivo metabolic activity of AOX using traditional in vitro methods are among these issues that are covered in this article. A comprehensive review of computational human AOX (hAOX) related studies are also provided. Expert opinion: Following the recent progress in the stem cell field, the authors recommend the application of organoids technology as an effective tool to solve the fundamental problems associated with the evaluation of AOX in drug discovery. The recent success in resolving the hAOX crystal structure can too be another valuable data source for the study of AOX-catalyzed metabolism of new drug candidates, using computer-aided drug discovery methods.

Over the years, significant progress has been made in reducing metabolic instability due to cytochrome P450-mediated oxidation. High-throughput metabolic stability screening has enabled the advancement of compounds with little to no oxidative metabolism. Furthermore, high lipophilicity and low aqueous solubility of presently pursued chemotypes reduces the probability of renal excretion. As such, these low microsomal turnover compounds are often substrates for non-CYP-mediated metabolism. UGTs, esterases, and aldehyde oxidase are major enzymes involved in catalyzing such metabolism. Hepatocytes provide an excellent tool to identify such pathways including elucidation of major metabolites. To predict human PK parameters for P450-mediated metabolism, in vitro-in vivo extrapolation using hepatic microsomes, hepatocytes, and intestinal microsomes has been actively investigated. However, such methods have not been sufficiently evaluated for non-P450 enzymes. In addition to the involvement of the liver, extrahepatic enzymes (intestine, kidney, lung) are also likely to contribute to these pathways. While there has been considerable progress in predicting metabolic pathways and clearance primarily mediated by the liver, progress in characterizing extrahepatic metabolism and prediction of clearance has been slow. Well-characterized in vitro systems or in vivo animal models to assess drug-drug interaction potential and intersubject variability due to polymorphism are not available. Here we focus on the utility of appropriate in vitro studies to characterize non-CYP-mediated metabolism and to understand the enzymes involved followed by pharmacokinetic studies in the appropriately characterized surrogate species. The review will highlight progress made in establishing in vitro-in vivo correlation, predicting human clearance and avoiding costly clinical failures when non-CYP-mediated metabolic pathways are predominant.

Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme's role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified in human AOX1. SNPs are a major source of interindividual variability in the human population, and SNP-based amino acid exchanges in AOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. For the reliable analysis of the effect of amino acid exchanges in human proteins, the existence of reproducible expression systems for the production of active protein in ample amounts for kinetic, spectroscopic, and crystallographic studies is required. In our study we report an optimized expression system for hAOX1 in Escherichia coli using a codon-optimized construct. The codon-optimization resulted in an up to 15-fold increase of protein production and a simplified purification procedure. The optimized expression system was used to study three SNPs that result in amino acid changes C44W, G1269R, and S1271L. In addition, the crystal structure of the S1271L SNP was solved. We demonstrate that the recombinant enzyme can be used for future studies to exploit the role of AOX in drug metabolism, and for the identification and synthesis of new drugs targeting AOX when combined with crystallographic and modeling studies., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Mammalian aldehyde oxidases (AOXs; EC1.2.3.1) are a group of conserved proteins belonging to the family of molybdo-flavoenzymes along with the structurally related xanthine dehydrogenase enzyme. AOXs are characterized by broad substrate specificity, oxidizing not only aromatic and aliphatic aldehydes into the corresponding carboxylic acids, but also hydroxylating a series of heteroaromatic rings. The number of AOX isoenzymes expressed in different vertebrate species is variable. The two extremes are represented by humans, which express a single enzyme (AOX1) in many organs and mice or rats which are characterized by tissue-specific expression of four isoforms (AOX1, AOX2, AOX3, and AOX4). In vertebrates each AOX isoenzyme is the product of a distinct gene consisting of 35 highly conserved exons. The extant species-specific complement of AOX isoenzymes is the result of a complex evolutionary process consisting of a first phase characterized by a series of asynchronous gene duplications and a second phase where the pseudogenization and gene deletion events prevail. In the last few years remarkable advances in the elucidation of the structural characteristics and the catalytic mechanisms of mammalian AOXs have been made thanks to the successful crystallization of human AOX1 and mouse AOX3. Much less is known about the physiological function and physiological substrates of human AOX1 and other mammalian AOX isoenzymes, although the importance of these proteins in xenobiotic metabolism is fairly well established and their relevance in drug development is increasing. This review article provides an overview and a discussion of the current knowledge on mammalian AOX.

Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-Å resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-Å resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

Several drug compounds have failed in clinical trials due to extensive biotransformation by aldehyde oxidase (AOX) (EC 1.2.3.1). One of the main reasons is the difficulty in scaling clearance for drugs metabolised by AOX, from preclinical species to human. Using methotrexate as a probe substrate, we evaluated AOX metabolism in liver cytosol from human and commonly used laboratory species namely guinea pig, monkey, rat and rabbit. We found that the metabolism of methotrexate in rabbit liver cytosol was several orders of magnitude higher than any of the other species tested. The results of protein quantitation revealed that the amount of AOX1 in human liver was similar to rabbit liver. To understand if the observed differences in activity were due to structural differences, we modelled rabbit AOX1 using the previously generated human AOX1 homology model. Molecular docking of methotrexate into the active site of the enzyme led to the identification of important residues that could potentially be involved in substrate binding and account for the observed differences. In order to study the impact of these residue changes on enzyme activity, we used site directed mutagenesis to construct mutant AOX1 cDNAs by substituting nucleotides of human AOX1 with relevant ones of rabbit AOX1. AOX1 mutant proteins were expressed in Escherichia coli. Differences in the kinetic properties of these mutants have been presented in this study., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

We found a new aldehyde oxidase (ALOD), which catalyzes the conversion of glycolaldehyde to glycolate, from Burkholderia sp. AIU 129. The enzyme further oxidized aliphatic aldehydes, an aromatic aldehyde, and glyoxal, but not glycolate or alcohols. The molecular mass of this enzyme was 130 kDa, and it was composed of three different subunits (αβγ structure), in which the α, β, and γ subunits were 76 kDa, 36 kDa, and 14 kDa, respectively. The N-terminal amino acid sequences of each subunit showed high similarity to those of putative subunits of xanthine dehydrogenase. Metals (copper, iron and molybdenum) and chelating reagents (α,α'-dipyridyl and 8-hydroxyquinoline) inhibited the ALOD activity. The ALOD showed highest activity at pH 6.0 and 50°C. Twenty mM glycolaldehyde was completely converted to glycolate by incubation at 30°C for 3 h, suggesting that the ALOD found in this study would be useful for enzymatic production of glycolate., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

In this work, a combination of homology modeling and molecular dynamics (MD) simulations was used to investigate the factors that modulate substrate specificity and activity of the mouse AOX isoforms: mAOX1, mAOX2 (previously mAOX3l1), mAOX3 and mAOX4. The results indicate that the AOX isoform structures are highly preserved and even more conserved than the corresponding amino acid sequences. The only differences are at the protein surface and substrate-binding site region. The substrate-binding site of all isoforms consists of two regions: the active site, which is highly conserved among all isoforms, and a isoform-specific region located above. We predict that mAOX1 accepts a broader range of substrates of different shape, size and nature relative to the other isoforms. In contrast, mAOX4 appears to accept a more restricted range of substrates. Its narrow and hydrophobic binding site indicates that it only accepts small hydrophobic substrates. Although mAOX2 and mAOX3 are very similar to each other, we propose the following pairs of overlapping substrate specificities: mAOX2/mAOX4 and mAOX3/mAXO1. Based on these considerations, we propose that the catalytic activity between all isoforms should be similar but the differences observed in the binding site might influence the substrate specificity of each enzyme. These results also suggest that the presence of several AOX isoforms in mouse allows them to oxidize more efficiently a wider range of substrates. This contrasts with the same or other organisms that only express one isoform and are less efficient or incapable of oxidizing the same type of substrates.

Aldehyde oxidase (AO), an important enzyme in the biotransformation of drugs and xenobiotics, is inhibited by flavonoids. This enzyme can metabolize both aldehydes and N-heterocycles. In this work, a set of 15 flavonoids was assessed for inhibitory activity on the AO oxidation of vanillin as an aldehyde substrate. Spectrophotometrically determined IC50 values showed that myricetin, quercetin, and epicatechin were the most potent inhibitors. The results also revealed that the inhibition of vanillin oxidation by flavonoids was stronger than that of phenanthridine oxidation (an N-heterocyclic substrate) as reported previously. In order to investigate the important structural features responsible for the inhibitory effects of the studied flavonoids, a quantitative structure-activity relationship (QSAR) analysis was performed. This study showed that the size of the flavonoids was the most important factor inversely affecting their potencies. The QSAR model can be used more broadly to predict the AO inhibitory activity of flavonoid-like structures for application in food-drug and drug-drug interaction studies., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po(lpo)) and aldehyde oxidase-1 (Aldox-1(n1)) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Po(lpo)-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Po(lpo) allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1(n1) phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays., (© 2014. Published by The Company of Biologists Ltd.)

Quinoxaline-1,4-dioxides (QdNOs) are a class of quinoxaline derivatives that are widely used in humans or animals as drugs or feed additives. However, the metabolic mechanism, especially the involved enzymes, has not been reported in detail. In this study, the N-oxide reduction enzyme, porcine aldehyde oxidase SsAOX1 was identified and characterized. The SsAOX1 gene was cloned from pig liver through reverse-transcription polymerase chain reaction using degenerate primers, which encode a 147-kDa protein with typical aldehyde oxidase motifs, two [2Fe-2S] centers, a flavin adenine dinucleotide (FAD) binding domain, and a molybdenum cofactor domain. After heterologous expression in a prokaryote, purified SsAOX1 formed a functional homodimer under native conditions. Importantly, the SsAOX1 catalyzed the N-oxide reduction at the N1 position of three representative QdNOs (quinocetone, mequindox, and cyadox), which are commonly used as animal feed additives. SsAOX1 has the highest activity toward quinocetone, followed by mequindox and cyadox, with kcat/K(m) values of 1.94 ± 0.04, 1.27 ± 0.15, and 0.43 ± 0.09 minute(-1) μM(-1), respectively. However, SsAOX1 has the lowest substrate affinity for quinocetone, followed by the cyadox and mequindox, with K(m) values of 4.36 ± 0.56, 3.16 ± 0.48, and 2.96 ± 0.51 μM, respectively. In addition, using site-directed mutagenesis, we found that substitution of glycine 1019 with threonine endows SsAOX1 with N-oxide reductive activity at the N4 position. The goal of this study was to identify and characterize the N-oxide reduction enzyme for a class of veterinary drugs, QdNOs, which will aid in the elucidation of the metabolic pathways of QdNOs and will provide a theoretical basis for their administration and new veterinary drug design.

Aldehyde oxidase 1 (AOX1) is a cytosolic enzyme highly expressed in liver and plays a key role in metabolizing drugs containing aromatic azaheterocyclic substituents. Rapid metabolism catalyzed by AOX1 can cause a drug to exhibit high clearance, low exposure, and hence decreased efficacy or even increased toxicity (if AOX1 generated metabolites are toxic). There is a need to develop the correlation between AOX1 expression levels and AOX1-substrate clearance. A fast, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify AOX1 in human liver cytosol for the first time. This LC-MS/MS method includes a straightforward ultrafiltration fractionation step and gives great selectivity and wide dynamic range (5.2 pM to 20.7 nM). The AOX1 levels in human liver cytosols of 20 donors were quantified using this method to investigate individual differences in AOX1 expression. No significant individual or gender differences in AOX1 levels were observed, although male donors exhibited a broader distribution than female donors (0.74-2.30 pmol/mg versus 0.74-1.69 pmol/mg, respectively). The AOX1 protein levels measured by LC-MS/MS were consistent with those measured by an enzyme-linked immunosorbent assay. Several donors have a normal AOX1 protein level but low enzyme activity, which might be due to cofactor deficiency, single nucleotide polymorphism, or homodimer dissociation. Cytosols from donors with chronic alcohol consumption had low AOX1-catalyzed carbazeran oxidation activities (<51 µl/min per milligram compared with a median of 455 µl/min per milligram), but preserved similar AOX1 protein expression levels (approximately 15% less than the median value).

1. Flavonoids are a group of polyphenolic plant metabolites most commonly known for their antioxidant activities. They also show inhibitory activities on molybdo-flavoenzymes family of enzymes which are involved in biotransformation of some exogenous and endogenous chemicals. Most notably, aldehyde oxidase (AO), a member of this family, is responsible for metabolism of some therapeutic agents. On the other hand, there are some therapeutics which inhibit AO. As flavonoids are ubiquitous in human diet and have potential to interact with AO, it is important to investigate their effects at the molecular details. 2. The inhibitory effects of 15 flavonoids on the activity of rat liver AO were assessed. Quantitative structure-activity relationship studies were performed using genetic algorithm coupled partial least square and stepwise multiple linear regression methods to elucidate the important structural properties responsible for the observed inhibitory effects. To further understand the mode of interaction between these flavonoids and AO, a homology model of the enzyme was built and flavonoids were docked into its active site. The most important amino acids involved in the interactions were identified. 3. Quercetin, myricetin and genistein were the most potent inhibitors establishing favorable interactions with the enzyme. However, the glycosylated flavonoids showed relatively weaker inhibition which may be attributed to their hindered binding into the active site of AO by bulky sugar groups.

Antennae-specific odorant-degrading enzymes (ODEs) are postulated to inactivate odorant molecules after they convey their signal. Different classes of insect ODEs are specific to esters, alcohols, and aldehydes--the major functional groups of female-produced, hydrophobic sex pheromones from moth species. Esterases that rapidly inactive acetate and other esters have been well-studied, but less is known about aldehyde oxidases (AOXs). Here we report cloning of an aldehyde oxidase, AtraAOX2, from the antennae of the navel orangeworm (NOW), Amyelois transitella, and the first activity characterization of a recombinant insect AOX. AtraAOX2 gene spans 3,813 bp and encodes a protein with 1,270 amino acid residues. AtraAOX2 cDNA was expressed in baculovirus-infected insect Sf21 cells as a ≈280 kDa homodimer with 140 kDa subunits. Recombinant AtraAOX2 degraded Z11Z13-16Ald and plant volatile aldehydes as substrates. However, as expected for aldehyde oxidases, recombinant AtraAOX2 did not show specificity for Z11Z13-16Ald, the main constituent of the sex pheromone, but showed high activity for plant volatile aldehydes. Our data suggest AtraAOX2 might be involved in degradation of a diversity of aldehydes including sex pheromones, plant-derived semiochemicals, and chemical cues for oviposition sites. Additionally, AtraAOX2 could protect the insect's olfactory system from xenobiotics, including pesticides that might reach the sensillar lymph surrounding the olfactory receptor neurons.

Introduction: Aldehyde oxidases (AOXs) are molybdo-flavoenzymes that oxidize aromatic aldehydes into the corresponding carboxylic acids and heterocycles into hydroxylated derivatives. AOXs have broad substrate specificity and are present in the liver of humans and many experimental animals. These enzymes play an important role in Phase I metabolism of drugs and xenobiotics of toxicological interest., Areas Covered: Preclinical studies on the AOX-dependent metabolism of new drug candidates are problematic. Furthermore, there is a general lack of reliable in silico methodologies to predict whether a new organic molecule is an AOX substrate. In vitro systems, for the design of high- or medium-throughput screening tests, to identify AOX substrates have many limitations. In vivo studies on AOX-dependent metabolism in animal models, on the other hand, are difficult because the complement of liver AOXs in humans and popular experimental animals is different. The authors discuss the possible ways to overcome all these problems., Expert Opinion: The significance of AOXs as drug-metabolizing enzymes is increasing, as the current strategies of organic synthesis designed to avoid cytochrome P450 (CYP450)-dependent metabolism tend to enrich for new chemical structures efficiently oxidized by these enzymes. There is need for reliable methods to screen for, predict, and validate AOX-dependent metabolism of new drug candidates.

Metabolism by aldehyde oxidase (AO) has been responsible for a number of drug failures in clinical trials. The main reason is the clearance values for drugs metabolized by AO are underestimated by allometric scaling from preclinical species. Furthermore, in vitro human data also underestimates clearance. We have developed the first in silico models to predict both in vitro and in vivo human intrinsic clearance for 8 drugs with just two chemical descriptors. These models explain a large amount of the variance in the data using two computational estimates of the electronic and steric features of the reaction. The in vivo computational models for human metabolism are better than in vitro preclinical animal testing at predicting human intrinsic clearance. Thus, it appears that AO is amenable to computational prediction of rates, which may be used to guide drug discovery, and predict pharmacokinetics for clinical trials.

While most enzyme-catalyzed reactions are adequately described by Michaelis-Menten kinetics, Aldehyde Oxidase (AOX) metabolism might exhibit atypical kinetics due to possible substrate inhibition. Ignoring this phenomenon may lead to erroneous estimates of kinetic parameters and over simplification of the enzyme mechanism. In this study, in vitro metabolism data for 3 AOX substrates exhibiting varying degrees of substrate inhibition were analyzed with the following kinetic models: A) Michaelis-Menten (naïve) model; B) Substrate inhibition (empirical) model; and C) Twobinding site (mechanistic) model. The application of this mechanistic model is a novel interpretation for kinetic analysis of AOX metabolism whereby substrate can presumably bind to two enzymes' active site(s). Unlike the other models, this mechanistic model quantitatively captures the degree of substrate inhibition observed. Analysis by this model showed: A) All tested substrates have simultaneous access to the metabolic and inhibitory site of the enzyme with Ks (binding affinity for inhibitory site) greater (1.3- to 28-fold) than Km (binding affinity for metabolic site); B) Dissociation constants for binding of a second substrate in either the productive and nonproductive enzyme conformations decreased with factor α ranging from 2.58 to 15.6 between compounds; and C) In addition, a drastic decrease (from 64%-98%) in the metabolism rates between compounds was exhibited by factor β (ranging from 0.02-0.36). Overall, the mechanistic two-binding site model best fitted the experimental data. Moreover, the observed differences between kinetic parameters generated by these models highlight the importance of appropriate model selection to adequately fit the substrate inhibition kinetics of AOX metabolism.

Introduction: Aldehyde oxidase (AO) is a drug-metabolizing molybdo-flavoenzyme with profound species differences in expression and activity toward various substrates. The contribution of this enzyme to the metabolism and clearance of heterocyclic-containing xenobiotics appears to have increased in recent years, but has not always been identified prior to clinical studies. As a result, drug candidates have been negatively impacted in development., Areas Covered: This review provides the most recent in vitro and in vivo strategies for the drug metabolism-pharmacokinetic (DMPK) scientist. The review details approaches for confirmation of AO as an operable metabolic pathway, estimating clearance and fraction of total metabolism, and identification of an appropriate surrogate species for human AO activity for evaluating safety of clinically relevant metabolites., Expert Opinion: As the role of AO in metabolism of new drug molecules continues to emerge, it is critical that DMPK scientists have the most updated methodologies to enable formulation of a thorough experimental plan to understand the potential implications of this metabolic pathway. Whether it is higher-than-expected clearance, contributing to an unfavorable half-life, or the formation of an AO-derived disproportionate human metabolite (DHM), such a plan would serve to minimize complications or attrition of drug candidates due to unforeseen issues in the clinic.

Background: Aldehyde oxidases have pharmacological relevance, and AOX3 is the major drug-metabolizing enzyme in rodents., Results: The crystal structure of mouse AOX3 with kinetics and molecular docking studies provides insights into its enzymatic characteristics., Conclusion: Differences in substrate and inhibitor specificities can be rationalized by comparing the AOX3 and xanthine oxidase structures., Significance: The first aldehyde oxidase structure represents a major advance for drug design and mechanistic studies. Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 Å. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.

The transition element molybdenum (Mo) needs to be complexed by a special cofactor in order to gain catalytic activity. With the exception of bacterial Mo-nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor Moco, which in different variants is the active compound at the catalytic site of all other Mo-containing enzymes. In eukaryotes, the most prominent Mo-enzymes are nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and the mitochondrial amidoxime reductase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also requires iron, ATP and copper. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco-carrier/binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms. In humans, Moco deficiency is a severe inherited inborn error in metabolism resulting in severe neurodegeneration in newborns and causing early childhood death. This article is part of a Special Issue entitled: Cell Biology of Metals., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We have cloned a gene encoding an aldehyde oxidase (ALOD) oxidized glyoxal but not glyoxylic acid from Pseudomonas sp. AIU 362. The ALOD gene contained an open reading frame consisting of 888 nucleotides corresponding to 295 amino acid residues. The deduced amino acid sequence exhibited a high similarity to those of 3-hydroxyisobutyrate dehydrogenases (3-HIBDHs). We expressed the cloned gene as an active product in Escherichia coli BL21 cells. The productivity (total units per culture broth volume) of the recombinant ALOD expressed in E. coli BL21 was 20,000-fold higher than that of ALOD in Pseudomonas sp. AIU 362. The recombinant ALOD exhibited ALOD activity and 3-HIBDH activity. The 3-HIBDH from Pseudomonas putida KT2440 also exhibited ALOD activity. Thus, the ALOD from Pseudomonas sp. AIU 362 and 3-HIBDH from P. putida KT2440 were classified into the same enzyme group., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Aldehyde oxidase (AO) is a molybdenum-containing enzyme distributed throughout the animal kingdom and capable of metabolising a wide range of aldehydes and N-heterocyclic compounds. Although metabolism by this enzyme in man is recognised to have significant clinical impact where human AO activity was not predicted by screening in preclinical species, there is very little reported literature offering real examples where drug discoverers have successfully designed away from AO oxidation. This article reports on some strategies adopted in the Pfizer TLR7 agonist programme to successfully switch off AO metabolism that was seen principally in the rat., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

N-{trans-3-[(5-Cyano-6-methylpyridin-2-yl)oxy]-2,2,4,4-tetramethylcyclobutyl}imidazo[1,2-a]pyrimidine-3-carboxamide (1) was recently identified as a full antagonist of the androgen receptor, demonstrating excellent in vivo tumor growth inhibition in castration-resistant prostate cancer (CRPC). However, the imidazo[1,2-a]pyrimidine moiety is rapidly metabolized by aldehyde oxidase (AO). The present paper describes a number of medicinal chemistry strategies taken to avoid the AO-mediated oxidation of this particular system. Guided by an AO protein structure-based model, our investigation revealed the most probable site of AO oxidation and the observation that altering the heterocycle or blocking the reactive site are two of the more effective strategies for reducing AO metabolism. These strategies may be useful for other drug discovery programs.