Human Aldehyde Oxidase Research Articles

Identification and characterization of erlotinib as a potent mechanism-based inactivator of human aldehyde oxidase

Identification and characterization of erlotinib as a potent mechanism-based inactivator of human aldehyde oxidase

Abstract 7127: An ATM inhibitor: ZN-B-2262 in combination with radiation/ADCs containing topoisomerase inhibitors for the treatment of solid tumors

Abstract Background: ZN-B-2262 is a best-in-class, IND-approved, small molecule serine/threonine kinase receptor inhibitor targeting Ataxia Telangiectasia Mutated (ATM), which plays a key role in DNA damage response (DDR) associated with double-strand breaks (DSB). Radiotherapy (RT) and topoisomerase inhibition can directly cause DSB. ZN-B-2262 is expected to enhance the therapeutic effect of RT, or antibody drug conjugates (ADCs) containing topoisomerase inhibitors by suppressing DSB DNA repair and blocking checkpoint controls. Methods: The inhibition of isolated recombinant ATM, ATR, mTOR, DNA-PK, PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ enzymes was evaluated using In-Cell-Western assay to determine the effect of ZN-B-2262 on ATM enzyme activity by detecting phosp-KAP1 (Ser824) expression. The inhibition of phospho-ATM (Ser1981) was evaluated by ATM assay on HT29 cells using a High-Content Imaging System (HCS). Therapeutic efficacy, as measured by tumor growth inhibition (TGI), was evaluated using ZN-B-2262 in combination with DS-1062 (Trop2 ADC), irinotecan or RT in MDA-MB-468 triple negative breast cancer (TNBC), SW620 colorectal cancer, and Fadu head and neck squamous cell carcinoma (HNSCC) subcutaneous xenograft murine models, respectively. In addition, ZN-B-2262 was tested for Human Aldehyde Oxidase (AO) activity. Safety margins were determined based on the ratio of unbound AUC obtained from 28-day GLP animal toxicology studies and predicted human PK at efficacious dose. Results: ZN-B-2262 exhibited strong inhibition against ATM with the IC50 at 4.4 nM. No inhibition effect was observed on other IKK family enzymes with IC50 value >10 uM. ZN-B-2262 showed high inhibition activity on phosp-KAP1 with IC50 of 13.25 nM and on phospho-ATM with IC50 of 21.5 nM. In the MDA-MB-468 xenograft efficacy study, ZN-B-2262 combined with a clinically relevant dose of DS-1062 showed significantly superior tumor inhibition effects compared to DS-1062 alone. In the SW620 xenograft efficacy study, in combination with irinotecan, ZN-B-2262 showed synergistic tumor inhibition effects. In the Fadu xenograft efficacy study, ZN-B-2262 demonstrated synergistic anti-tumor activity when combined with RT. Sustained and enhanced anti-tumor efficacy was observed post withdrawal of ZN-B-2262 combined with RT, which was not observed after stopping treatment of RT alone. Based on 28-day GLP tox in rat and dog, the predicted human safety margin is 25- to 50-fold. Conclusion: ZN-B-2262, in combination with ADCs containing topoisomerase inhibitors, (e.g., Dato-Dxd), or RT, is expected to be a best-in-class (possibly first-in-class) compound, for the treatment of solid tumors. In particular, ZN-B-2262 should demonstrate improved PK and minimal inter-patient PK variability compared to current ATM clinical candidates since ZN-B-2262 is not an AO substrate. Citation Format: Ding Zhou, Zheng Wang, Yan (Pita) Liu, Tingting Fu, Ziqiang (Zack) Cheng. An ATM inhibitor: ZN-B-2262 in combination with radiation/ADCs containing topoisomerase inhibitors for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7127.

Dissecting Parameters Contributing to the Underprediction of Aldehyde Oxidase-Mediated Metabolic Clearance of Drugs.

We investigated the effect of variability and instability in aldehyde oxidase (AO) content and activity on the scaling of in vitro metabolism data. AO content and activity in human liver cytosol (HLC) and five recombinant human AO preparations (rAO) were determined using targeted proteomics and carbazeran oxidation assay, respectively. AO content was highly variable as indicated by the relative expression factor (REF; i.e., HLC to rAO content) ranging from 0.001 to 1.7 across different in vitro systems. The activity of AO in HLC degrades at a 10-fold higher rate in the presence of the substrate as compared with the activity performed after preincubation without substrate. To scale the metabolic activity from rAO to HLC, a protein-normalized activity factor (pnAF) was proposed wherein the activity was corrected by AO content, which revealed up to sixfold higher AO activity in HLC versus rAO systems. A similar value of pnAF was observed for another substrate, ripasudil. Physiologically based pharmacokinetic (PBPK) modeling revealed a significant additional clearance (CL; 66%), which allowed for the successful prediction of in vivo CL of four other substrates, i.e., O-benzyl guanine, BIBX1382, zaleplon, and zoniporide. For carbazeran, the metabolite identification study showed that the direct glucuronidation may be contributing to around 12% elimination. Taken together, this study identified differential protein content, instability of in vitro activity, role of additional AO clearance, and unaccounted metabolic pathways as plausible reasons for the underprediction of AO-mediated drug metabolism. Consideration of these factors and integration of REF and pnAF in PBPK models will allow better prediction of AO metabolism. SIGNIFICANCE STATEMENT: This study elucidated the plausible reasons for the underprediction of aldehyde oxidase (AO)-mediated drug metabolism and provided recommendations to address them. It demonstrated that integrating protein content and activity differences and accounting for the loss of AO activity, as well as consideration of extrahepatic clearance and additional pathways, would improve the in vitro to in vivo extrapolation of AO-mediated drug metabolism using physiologically based pharmacokinetic modeling.

Mechanistic insights into the ROS-mediated inactivation of human aldehyde oxidase.

Human aldehyde oxidase (hAOX1) is a molybdoenzyme that oxidizes aldehydes and N-heterocyclic compounds, thereby generating hydrogen peroxide (H2 O2 ) and superoxide during turnover. hAOX1 has been shown previously to be inactivated under turnover conditions by H2 O2 . Here, we investigated the effect of exogenously added H2 O2 on the activity of hAOX1. We show that exogenously added H2 O2 did not affect the enzyme activity under aerobic conditions, but completely inactivated the enzyme under anaerobic conditions. We propose that this effect is based on the reducing power of H2 O2 and the susceptibility of the reduced molybdenum cofactor (Moco) to lose the sulfido ligand. When oxygen is present, the enzyme is rapidly reoxidized. We believe that our study is significant in understanding the detailed effect of reactive oxygen species on the inactivation of hAOX1 and other molybdoenzymes.

In vitro metabolism of 2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) by aldehyde oxidase and predicting its percent contribution relative to CYP-mediated metabolism.

2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) is an anticancer drug that was discontinued owing to hepatotoxicity found in clinical studies. Metabolite analysis of CP-724,714 was conducted using human hepatocytes, where twelve oxidative metabolites and one hydrolyzed metabolite were formed. Among the three mono-oxidative metabolites, the formation of two was inhibited by adding 1-aminobenzotriazole, a pan-CYP inhibitor. In contrast, the remaining one was not affected by this inhibitor but partially inhibited by hydralazine, indicating that aldehyde oxidase (AO) was involved in metabolizing CP-724,714, which contains a quinazoline substructure, a heterocyclic aromatic quinazoline ring, known to be preferably metabolized by AO. One of the oxidative metabolites of CP-724,714 observed in human hepatocytes was also generated in recombinant human AO. Although CP-724,714 is metabolized by both CYPs and AO in human hepatocytes, the contribution level of AO could not be evaluated using its specific inhibitors because of low AO activity in in vitro human materials. Here, we present a metabolic pathway for CP-724,714 in human hepatocytes and the involvement of AO in CP-724,714 metabolism. We showed here a plausible workflow for predicting AO contribution to the metabolism of CP-724,714 based on DMPK screening data. Significance Statement CP-724,714 was identified as a substrate of aldehyde oxidase (AO) rather than xanthine oxidase. Since CP-724,714 is also metabolized by CYPs, the contribution levels of AO and CYPs in the metabolism of CP-724,714 were estimated simultaneously based on in vitro drug metabolism screening data.

Inactivation of Human Aldehyde Oxidase by Small Sulfhydryl-containing Reducing Agents.

Human aldehyde oxidase (hAOX1) is a molybdoflavoenzyme that belongs to the xanthine oxidase (XO) family. hAOX1 is involved in phase I drug metabolism, but its physiological role is not fully understood to date and preclinical studies consistently underestimated hAOX1 clearance. In the present work, we report an unexpected effect of the common sulfhydryl-containing reducing agents, e.g. dithiothreitol (DTT), on the activity of hAOX1 and mouse aldehyde oxidases. We demonstrate that this effect is due to the reactivity of the sulfido ligand bound at the molybdenum cofactor (Moco) with the sulfhydryl groups. The sulfido ligand coordinated to the Mo atom in the XO family of enzymes plays a crucial role in the catalytic cycle and its removal results in the total inactivation of these enzymes. Since liver cytosols, S9 fractions and hepatocytes are commonly used to screen the drug candidates for hAOX1, our study suggests that DTT treatment of these samples should be avoided, as otherwise false negative results by an inactivated hAOX1 might be obtained. Significance Statement This work characterizes the inactivation of human aldehyde oxidase (hAOX1) by sulfhydryl-containing agents and identifies the site of inactivation. The role of DTT in the inhibition of hAOX1 should be considered for the preparation of hAOX1-containing fractions for pharmacological studies on drug metabolism and drug clearance.

In Silico Prediction of Metabolic Reaction Catalyzed by Human Aldehyde Oxidase.

Aldehyde oxidase (AOX) plays an important role in drug metabolism. Human AOX (hAOX) is widely distributed in the body, and there are some differences between species. Currently, animal models cannot accurately predict the metabolism of hAOX. Therefore, more and more in silico models have been constructed for the prediction of the hAOX metabolism. These models are based on molecular docking and quantum chemistry theory, which are time-consuming and difficult to automate. Therefore, in this study, we compared traditional machine learning methods, graph convolutional neural network methods, and sequence-based methods with limited data, and proposed a ligand-based model for the metabolism prediction catalyzed by hAOX. Compared with the published models, our model achieved better performance (ACC = 0.91, F1 = 0.77). What's more, we built a web server to predict the sites of metabolism (SOMs) for hAOX. In summary, this study provides a convenient and automatable model and builds a web server named Meta-hAOX for accelerating the drug design and optimization stage.

Mechanistic Investigation of the Time-Dependent Aldehyde Oxidase Inhibitor Hydralazine.

The anti-hypertensive agent hydralazine is a time-dependent inhibitor of the cytosolic drug-metabolizing enzyme aldehyde oxidase (AO). Glutathione (GSH) was found to suppress the inhibition of AO by hydralazine in multiple enzyme sources (human liver and kidney cytosol, human liver S9, rat liver S9, and recombinant human AO) and with different AO substrates (zoniporide, O6 -benzylguanine, and dantrolene). Hydralazine-induced AO inactivation was unaffected when GSH was added to the incubation mixture after pre-incubation of hydralazine with AO (rather than during the pre-incubation), suggesting that GSH traps a hydralazine reactive intermediate prior to enzyme inactivation. Consistent with previous reports of 1-phthalazylmercapturic acid formation when hydralazine was incubated with N-acetylcysteine, we detected a metabolite producing an MS/MS spectrum consistent with a 1-phthalazyl-GSH conjugate. O6 -Benzylguanine, an AO substrate, did not protect against hydralazine-induced AO inactivation, implying that hydralazine does not compete with O6 -benzylguanine for binding to the AO active site. Catalase also failed to protect AO from hydralazine-induced inactivation, suggesting that hydrogen peroxide is not involved. However, an allosteric AO inhibitor (thioridazine) offered some protection, indicating a catalytic role for AO in the bioactivation of hydralazine. AO inhibition by phthalazine (a substrate and inhibitor of AO and a metabolite of hydralazine) was unaffected by the presence of GSH. GSH also prevented hydralazine from inhibiting the nitro-reduction of dantrolene by AO. Furthermore, the GSH-hydralazine combination stimulated dantrolene reduction. Phthalazine inhibited only oxidation reactions, not reduction of dantrolene. Together, these results support the hypothesis that hydralazine is converted to a reactive intermediate that inactivates AO. SIGNIFICANCE STATEMENT: These studies suggest that a reactive intermediate of hydralazine plays a primary role in the mechanism of aldehyde oxidase (AO) inactivation. Inactivation was attenuated by glutathione and unaffected by catalase. Phthalazine (hydralazine metabolite) inhibited AO regardless of the presence of glutathione; however, phthalazine inhibited only oxidation reactions, while hydralazine inhibited both oxidation and reduction reactions. This report advances our mechanistic understanding of hydralazine as an AO inhibitor and provides information to facilitate appropriate use of hydralazine when probing AO metabolism.

Weak Inhibitory Effects of Anthocyanins on Human Aldehyde Oxidase Activity: An In Vitro Study.

Aldehyde oxidase (AO) plays an important role in the metabolism of antitumor and antiviral drugs, including methotrexate, favipiravir, and acyclovir. The consumption of blueberry fruits or their extracts, which contain large amounts of anthocyanins, has recently increased. The intake of large amounts of anthocyanins occurs through the frequent consumption of blueberries or their functional foods, which may result in unwanted interactions between anthocyanins and medicinal drugs. Therefore, the present study examined the inhibition of AO by anthocyanins, anthocyanidins, and blueberry extracts in human liver cytosol using a HPLC assay. A comparison of the 50% inhibitory concentration (IC50) values of the test compounds showed that anthocyanidins slightly suppressed AO activity, whereas the inhibitory effects of anthocyanins and blueberry extracts were negligible. The inhibitory activities of the anthocyanins tested were approximately 60- to 130-fold weaker than that of the positive control menadione and were almost negligible. Furthermore, they were approximately 2,000-fold less potent than that of raloxifene, a typical AO inhibitor, and, thus, unlikely to interfere with drug metabolism by AO. In addition, since the plasma concentrations of anthocyanins after their administration were generally lower than the IC50 level, the inhibition of AO substrate metabolism by anthocyanins does not appear to be severe.

Role of Human Aldehyde Oxidase in the Generation of Reactive Oxygen Species during the Metabolism of Nicotine

Nicotine is the major addictive constituent of tobacco and alternative tobacco products including electronic cigarettes (e‐cigs). While e‐cig vaping has been proposed as safer than traditional tobacco smoking, recent studies has demonstrated that these products can also trigger oxidant stress and inflammation with resultant pulmonary and cardiovascular disease. E‐cig liquids contain high levels of nicotine typically of 12 to 24 mg/ml or more and with the use of high power delivery devices high, poorly controlled levels of nicotine can be delivered. When inhaling tobacco cigarette smoke or e‐cig vape, nicotine is rapidly absorbed through the lungs. The major pathway of nicotine metabolism in humans consists of the C‐oxidation of nicotine to produce cotinine involving a two‐step process: the first step is CYP2A6‐catalyzed 5′‐oxidation to nicotine‐Δ1′(5′)‐iminium ion (NI), which is in equilibrium with 5′‐hydroxynicotine; and the second step is aldehyde oxidase (AO) catalyzed oxidation of NI to cotinine.Using both cytochrome‐c reduction and EPR spin trapping techniques, we show for the first time that the oxidation of NI by human AO produces superoxide radical anion (O2• −) with high flux rates. Purified recombinant human AO (hAO) was prepared and characterized by SDS gel electrophoresis and optical absorption spectroscopy. hAO, 40 nM was incubated in the presence of NI concentrations from 1‒50 µM. The kinetic parameters, Km and Vmax values, for superoxide generation from NI were determined to be 2.7 ± 0.4 μM and 694 ± 24 nmol min−1 mg−1 of protein, respectively. From quantitation of the measured superoxide and the amount of NI consumed, we estimate that ~60% of the oxygen consumed in this process is converted to superoxide with ~40% hydrogen peroxide. Superoxide generation was almost completely quenched by either 0.5 μM raloxifene, a potent inhibitor of AO, or 0.1 μM Copper‐Zinc‐superoxide dismutase (SOD1) with no superoxide generation detected either in the absence of either AO or nicotine‐Δ1′(5′)‐iminium ion.Therefore, human AO efficiently catalyzes the oxidation of NI to cotinine with concomitant production of reactive oxygen species. Due to the high expression levels of AO in lung tissue as well as its rapid rate of NI metabolism with low Km, AO rapidly and efficiently converts NI to cotinine with the formation of superoxide and hydrogen peroxide. Thus, nicotine metabolism will be associated with the formation of superoxide, hydrogen peroxide and secondary reactive oxygen species that can in turn cause lung oxidative stress and inflammation.

Interrogating the Inhibition Mechanisms of Human Aldehyde Oxidase by X-ray Crystallography and NMR Spectroscopy: The Raloxifene Case.

Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

The Inactivation of Human Aldehyde Oxidase 1 by Hydrogen Peroxide and Superoxide.

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 H2O2 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-O2 .--producing natural variant L438V for their time-dependent inactivation with H2O2/O2 .- 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.

Enzyme Kinetics, Pharmacokinetics, and Inhibition of Aldehyde Oxidase.

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.

Aldehyde Oxidase Contributes to All-Trans-Retinoic Acid Biosynthesis in Human Liver.

All-trans-retinoic acid (atRA) is a critical endogenous signaling molecule. atRA is predominantly synthesized from retinaldehyde by aldehyde dehydrogenase 1A1 (ALDH1A1), but aldehyde oxidase (AOX) may also contribute to atRA biosynthesis. The goal of this study was to test the hypothesis that AOX contributes significantly to atRA formation in human liver. Human recombinant AOX formed atRA from retinaldehyde (Km ∼1.5 ± 0.4 µM; kcat ∼3.6 ± 2.0 minute-1). In human liver S9 fractions (HLS9), atRA formation was observed in the absence of NAD+, suggesting AOX contribution to atRA formation. In the presence of NAD+, Eadie-Hofstee plots of atRA formation in HLS9 indicated that two enzymes contributed to atRA formation. The two enzymes were identified as AOX and ALDH1A1 based on inhibition of atRA formation by AOX inhibitor hydralazine (20%-50% inhibition) and ALDH1A1 inhibitor WIN18,446 (50%-80%inhibition). The expression of AOX in HLS9 was 9.4-24 pmol mg-1 S9 protein, whereas ALDH1A1 expression was 156-285 pmol mg-1 S9 protein measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of signature peptides. The formation velocity of atRA in the presence of NAD+ correlated significantly with the expression of ALDH1A1 and AOX protein. Taken together, the data show that both AOX and ALDH1A1 contribute to atRA biosynthesis in the human liver, with ALDH1A1 being the high-affinity, low-capacity enzyme and AOX being the low-affinity, high-capacity enzyme. The results suggest that in the case of ALDH1A dysfunction or excess vitamin A, AOX may play an important role in regulating hepatic vitamin A homeostasis and that inhibition of AOX may alter atRA biosynthesis and signaling. SIGNIFICANCE STATEMENT: This study provides direct evidence to show that human AOX converts retinaldehyde to atRA and contributes to hepatic atRA biosynthesis. The finding that AOX may be responsible for 20%-50% of overall hepatic atRA formation suggests that alterations in AOX activity via drug-drug interactions, genetic polymorphisms, or disease states may impact hepatic atRA concentrations and signaling and alter vitamin A homeostasis.

Understanding the Metabolism of Proteolysis Targeting Chimeras (PROTACs): The Next Step toward Pharmaceutical Applications.

Hetero-bifunctional PROteolysis TArgeting Chimeras (PROTACs) represent a new emerging class of small molecules designed to induce polyubiquitylation and proteasomal-dependent degradation of a target protein. Despite the increasing number of publications about the synthesis, biological evaluation, and mechanism of action of PROTACs, the characterization of the pharmacokinetic properties of this class of compounds is still minimal. Here, we report a study on the metabolism of a series of 40 PROTACs in cryopreserved human hepatocytes at multiple time points. Our results indicated that the metabolism of PROTACs could not be predicted from that of their constituent ligands. Their linkers’ chemical nature and length resulted in playing a major role in the PROTACs’ liability. A subset of compounds was also tested for metabolism by human cytochrome P450 3A4 (CYP3A4) and human aldehyde oxidase (hAOX) for more in-depth data interpretation, and both enzymes resulted in active PROTAC metabolism.

Site-Directed Mutagenesis at the Molybdenum Pterin Cofactor Site of the Human Aldehyde Oxidase: Interrogating the Kinetic Differences Between Human and Cynomolgus Monkey.

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.

Production of active recombinant human aldehyde oxidase (AOX) in the baculovirus expression vector system (BEVS) and deployment in a pre-clinical fraction-of-control AOX compound exposure assay

Human aldehyde oxidase (AOX) has emerged as a key enzyme activity for consideration in modern drug discovery. The enzyme catalyzes the oxidation of a wide variety of compounds, most notably azaheterocyclics that often form the building blocks of small molecule therapeutics. Failure to consider and assess AOX drug exposure early in the drug development cycle can have catastrophic consequences for novel compounds entering the clinic. AOX is a complex molybdopterin-containing iron-sulfur flavoprotein comprised of two identical 150 kDa subunits that has proven difficult to produce in recombinant form, and a commercial source of the purified human enzyme is currently unavailable. Thus, the potential exposure of novel drug development candidates to human AOX metabolism is usually assessed by using extracts of pooled human liver cytosol as a source of the enzyme. This can complicate the assignment of AOX-specific compound exposure due to its low activity and the presence of contaminating enzymes that may have overlapping substrate specificities. Herein is described a two-step process for the isolation of recombinant human AOX dimers to near homogeneity following production in the baculovirus expression vector system (BEVS). The deployment of this BEVS-produced recombinant human AOX as a substitute for human liver extracts in a fraction-of-control AOX compound-exposure screening assay is described. The ability to generate this key enzyme activity readily in a purified recombinant form provides for a more accurate and convenient approach to the assessment of new compound exposure to bona fide AOX drug metabolism.

Catalytic Mechanism of Human Aldehyde Oxidase

The mechanism of oxidation of N-heterocycle phthalazine to phthalazin-1(2H)-one and its associated free energy profile, catalyzed by human aldehyde oxidase (hAOX1), was studied in atomistic detail ...

Human Aldehyde Oxidase 1-Mediated Carbazeran Oxidation in Chimeric TK-NOG Mice Transplanted with Human Hepatocytes.

Carbazeran is a potent phosphodiesterase inhibitor with species-dependent metabolic profiles in rats, dogs, and humans. In this study, we investigated the aldehyde oxidase (AOX)-mediated oxidation of carbazeran to 4-oxo derivatives in chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene with humanized livers (humanized-liver mice). Liver cytosolic fractions from humanized-liver mouse effectively catalyzed carbazeran 4-oxidation with high affinity for the substrate, similar to those of the human liver cytosolic fractions and recombinant human AOX1 protein. Furthermore, hepatocytes prepared from humanized-liver mice and humans also exhibited substantial metabolism via carbazeran 4-oxidation. After a single oral administration of carbazeran (10 mg/kg), plasma levels of 4-oxo-carbazeran, N-desethyl-4-oxo-carbazeran, and 6,7-dimethoxy-1-[4-(hydroxy)-piperidino]-4-phthalazinone (three human metabolites formed via 4-oxidation) were higher in humanized-liver mice than in the control mice. In contrast, plasma levels of O-desmethylcarbazeran (a major metabolite in dogs) in control mice were higher than those in the humanized-liver mice. Relative excreted amounts of the three 4-oxidation-derived human-specific metabolites in the urine and feces were greater for humanized-liver mice than control mice, whereas the relative excreted amounts of O-desmethylcarbazeran were predominant in the urine and feces of control mice. Thus, the production of carbazeran 4-oxo derivatives was elevated in humanized-liver mice compared with control mice, in agreement with our in vitro enzyme-mediated oxidation data. These results suggest that hepatic human AOX1 functions in humanized-liver mice at the in vivo level and that humanized-liver mice may be useful for predicting drug metabolism in humans, at least with regard to human AOX1-dependent metabolism. SIGNIFICANCE STATEMENT: We found that the production of carbazeran 4-oxo derivatives was higher in humanized-liver mice than in control mice. These results were supported by the fact that carbazeran was rapidly metabolized to 4-oxo-carbazeran in humanized-liver mouse hepatocytes expressing human aldehyde oxidase 1. These results suggest that human aldehyde oxidase 1 is functional in humanized-liver mice in vivo and that chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene transplanted with human hepatocytes may be a suitable model animal for predicting aldehyde oxidase-dependent biotransformation of drugs in humans.

Retinoid X Receptor Modulators Transcriptionally Regulate Human Aldehyde Oxidase Expression

Aldehyde oxidase‐1 (AOX1) is an oxidative drug‐metabolizing enzyme. Retinoid X receptor (RXR) is a nuclear receptor that elicits various physiological functions and is an obligate partner in specific cell signaling pathways. It regulates gene expression by forming a homodimer or heterodimer with another nuclear receptor, such as retinoic acid receptor (RAR) or peroxisome proliferator‐activated receptor (PPAR). Given the central role of RXR in controlling gene expression, we hypothesized that RXR modulators regulate AOX1 expression in cultured MCF‐7 human breast adenocarcinoma cells. 9‐Cis‐retinoic acid (10 μM), a pan‐RXR and RAR agonist, increased AOX1 mRNA expression at 24, 48, 72, 96, and 120 h post‐treatment, and a plateau was achieved at 96 h. To determine the effect of RXR activation on AOX1 expression, we compared the effect of several RXR‐selective modulators and 9‐cis‐retinoic acid. Non‐cytotoxic concentrations (as assessed by lactate dehydrogenase assay) of LG100268, CD3254, LG100754, SR11237, bexarotene, and 9‐cis‐retinoic acid increased AOX1 mRNA level in a concentration‐dependent manner with EC50 values of 4, 6, 12, 39, 42, and 334 nM, and Emax values of 5, 4, 9, 5, 15, and 9‐fold, respectively. The EC50 values of bexarotene and 9‐cis‐retinoic acid were less than the plasma concentrations of bexarotene (3 μM) and 9‐cis‐retinoic acid (0.5 μM) achieved in cancer patients administered either of these drugs. In contrast to other RXR activators, LG101506, which has no effect on RXR‐RXR homodimer or RXR‐RAR heterodimer and is a selective RXR‐PPAR agonist, decreased AOX1 expression with an EC50 of 1 μM and Emax of 94% decrease. Pretreatment of cells with actinomycin D (a RNA synthesis inhibitor) abolished AOX1 induction by 9‐cis‐retinoic acid, bexarotene, CD3254, SR11237, LG100268, and LG100754, indicating that the induction occurred by a transcriptional mechanism. RXRα mRNA was 6‐fold greater than RXRβ mRNA in MCF‐7 cells. To evaluate the role of RXR isoforms in AOX1 induction, genetic knock‐down approach was used. Transfection of cells with RXRα or RXRβ siRNA decreased the specific RXR mRNA level by 70–80%. RXRα siRNA attenuated AOX1 induction by 9‐cis‐retinoic acid, bexarotene, CD3254, SR11237, LG100268, and LG100754 by 55–70%, whereas RXRβ siRNA attenuated AOX1 induction by only 15–30%. To determine whether RXR transactivates AOX1 promoter, a reporter gene assay was conducted. 9‐Cis‐retinoic acid and selective RXR activators increased luciferase activity in cells transfected with an AOX1 (−1467 to +10) promoter plasmid, indicating that RXR transactivated this region of the promoter. In conclusion, RXR is the first nuclear receptor that transcriptionally regulates AOX1 expression. RXR activators differentially regulate AOX1 expression and this occurs primarily by activating the RXRα isoform. Given that LG100268 is an agonist of RXR homodimer and has no effect on RXR‐RAR heterodimer, whereas LG100754 is a RXR homodimer antagonist and a RXR‐RAR and RXR‐PPAR heterodimer agonist, the results suggest a role of both RXR homodimer and heterodimers in AOX1 induction. Our novel findings suggest interaction between RXR modulators and AOX1‐mediated drug metabolism and toxicity.Support or Funding InformationSingapore Ministry of Education Academic Research Fund Tier 1 and Ministry of Health National Medical Research Council Grants