Human Aldehyde Oxidase Research Articles

Pyrido[3,4-d]pyrimidin-4(3H)-one metabolism mediated by aldehyde oxidase is blocked by C2-substitution

1. We have previously described C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one derivatives as cell permeable inhibitors of the KDM4 and KDM5 subfamilies of JmjC histone lysine demethylases.2. Although exemplar compound 1 exhibited moderate clearance in mouse liver microsomes, it was highly cleared in vivo due to metabolism by aldehyde oxidase (AO). Similar human and mouse AO-mediated metabolism was observed with the pyrido[3,4-d]pyrimidin-4(3H)-one scaffold and other C8-substituted derivatives.3. We identified the C2-position as the oxidation site by LC-MS and 1H-NMR and showed that C2-substituted derivatives are no longer AO substrates.4. In addition to the experimental data, these observations are supported by molecular modelling studies in the human AO protein crystal structure.

Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms.

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.

Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase.

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.

A novel reaction mediated by human aldehyde oxidase: amide hydrolysis of GDC-0834.

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.

Inhibition of human aldehyde oxidase activity by diet-derived constituents: structural influence, enzyme-ligand interactions, and clinical relevance.

The mechanistic understanding of interactions between diet-derived substances and conventional medications in humans is nascent. Most investigations have examined cytochrome P450-mediated interactions. Interactions mediated by other phase I enzymes are understudied. Aldehyde oxidase (AO) is a phase I hydroxylase that is gaining recognition in drug design and development programs. Taken together, a panel of structurally diverse phytoconstituents (n = 24) was screened for inhibitors of the AO-mediated oxidation of the probe substrate O(6)-benzylguanine. Based on the estimated IC50 (<100 μM), 17 constituents were advanced for Ki determination. Three constituents were described best by a competitive inhibition model, whereas 14 constituents were described best by a mixed-mode model. The latter model consists of two Ki terms, Kis and Kii, which ranged from 0.26-73 and 0.80-120 μM, respectively. Molecular modeling was used to glean mechanistic insight into AO inhibition. Docking studies indicated that the tested constituents bound within the AO active site and elucidated key enzyme-inhibitor interactions. Quantitative structure-activity relationship modeling identified three structural descriptors that correlated with inhibition potency (r(2) = 0.85), providing a framework for developing in silico models to predict the AO inhibitory activity of a xenobiotic based solely on chemical structure. Finally, a simple static model was used to assess potential clinically relevant AO-mediated dietary substance-drug interactions. Epicatechin gallate and epigallocatechin gallate, prominent constituents in green tea, were predicted to have moderate to high risk. Further characterization of this uncharted type of interaction is warranted, including dynamic modeling and, potentially, clinical evaluation.

Aldehyde oxidase activity in fresh human skin.

Human aldehyde oxidase (AO) is a molybdoflavoenzyme that commonly oxidizes azaheterocycles in therapeutic drugs. Although high metabolic clearance by AO resulted in several drug failures, existing in vitro-in vivo correlations are often poor and the extrahepatic role of AO practically unknown. This study investigated enzymatic activity of AO in fresh human skin, the largest organ of the body, frequently exposed to therapeutic drugs and xenobiotics. Fresh, full-thickness human skin was obtained from 13 individual donors and assayed with two specific AO substrates: carbazeran and zoniporide. Human skin explants from all donors metabolized carbazeran to 4-hydroxycarbazeran and zoniporide to 2-oxo-zoniporide. Average rates of carbazeran and zoniporide hydroxylations were 1.301 and 0.164 pmol⋅mg skin(-1)⋅h(-1), resulting in 13 and 2% substrate turnover, respectively, after 24 hours of incubation with 10 μM substrate. Hydroxylation activities for the two substrates were significantly correlated (r(2) = 0.769), with interindividual variability ranging from 3-fold (zoniporide) to 6-fold (carbazeran). Inclusion of hydralazine, an irreversible inhibitor of AO, resulted in concentration-dependent decrease of hydroxylation activities, exceeding 90% inhibition of carbazeran 4-hydroxylation at 100 μM inhibitor. Reaction rates were linear up to 4 hours and well described by Michaelis-Menten enzyme kinetics. Comparison of carbazeran and zoniporide hydroxylation with rates of triclosan glucuronidation and sulfation and p-toluidine N-acetylation showed that cutaneous AO activity is comparable to tested phase II metabolic reactions, indicating a significant role of AO in cutaneous drug metabolism. To our best knowledge, this is the first report of AO enzymatic activity in human skin.

The crystal structure of human Aldehyde Oxidase in native and inhibited forms

Aldehyde oxidases (AOX; E.C. 1.2.3.1) are molybdo-flavoenzymes with broad substrate specificity, oxidizing aldehydes and N-heterocycles. AOX belongs to the xanthine oxidase (XO) family of Mo-containing enzymes. The true physiological function of AOX is still unknown, although it is recognized to play a role in the metabolism of compounds with medicinal and toxicological relevance [1]. AOX importance has increased in recent years since it is substituting Cyt-P450 as the central drug-metabolizing system in humans. We have solved the 3D structure of mouse AOX3 to 2.9 Å resolution [2] that was the first structure of an aldehyde oxidase, providing important evidences on substrate and inhibitor specificities between AOX and XO. The complement of AOX proteins in mammals varies from one in humans (hAOX1) to four in rodents (mAOX1, mAOX3, mAOX4 and mAOX3L1) as a result of evolutionary genetic events. Due to this unusual complement of AOX genes in different animal species, conclusions regarding protein metabolism in humans cannot be taken exclusively from the mouse model. Using the human aldehyde oxidase (hAOX1) purified after heterologous expression in Escherichia coli we were able to crystallize it and solve its 3D structure to 2.7 Å resolution (submitted). In addition to the native protein we also solved the structure of an inhibited form of the enzyme to 2.6Å resolution. Analysis of the protein active site and comparison with the structure of the mouse isoform (mAOX3) allowed us to identity, for the first time, the unique features that characterize hAOX1 as an important drug-metabolizing enzyme. In spite of the similarities of both enzymes, they show marked and relevant differences at the Mo active site, substrate tunnel as well as at the FAD site. The ensemble of these structures provides important insights into the role of aldehyde oxidases, contributing to elucidate the clinical metabolism implications of hAOX1 in humans which has particular relevance for novel drug design studies.

Cynomolgus monkey as a surrogate for human aldehyde oxidase metabolism of the EGFR inhibitor BIBX1382.

BIBX1382 was an epidermal growth factor receptor inhibitor under clinical investigation for treatment of cancer. This candidate possessed an attractive preclinical absorption, distribution, metabolism, and excretion profile, yet failed in clinical studies due in part to poor oral exposure, resulting from extensive metabolism by aldehyde oxidase (AO). In vitro metabolism studies were performed in liver cytosol and cryopreserved hepatocytes from multiple species. In addition, a pharmacokinetic study was performed in cynomolgus monkey for comparison with the reported human pharmacokinetics of BIBX1382. Estimated hepatic clearance of BIBX1382 in rhesus (42 ml/min per kg) and cynomolgus monkey (43 ml/min per kg) liver cytosol was comparable to human (≥93% of liver blood flow). Metabolite identification after incubation of BIBX1382 in liver cytosol fortified with the AO inhibitor raloxifene confirmed that AO is involved in the formation of the predominant metabolite (BIBU1476, M1) in cynomolgus monkey. After intravenous and oral administration of BIBX1382 to cynomolgus monkeys, high plasma clearance (118 ml/min per kg) and low oral exposure (C(max) = 12.7 nM and 6% oral bioavailability) was observed, with the exposure of M1 exceeding BIBX1382 after oral dosing. This pharmacokinetic profile compared favorably with the human clinical data of BIBX1382 (plasma clearance 25-55 ml/min per kg and 5% oral bioavailability). Thus, it appears that cynomolgus monkey represents a suitable surrogate for the observed human AO metabolism of BIBX1382. To circumvent clinical failures due to uncharacterized metabolism by AO, in vitro studies in the appropriate subcellular fraction, followed by pharmacokinetic and toxicokinetic studies in the appropriately characterized surrogate species should be conducted for substrates of AO.

In vitro oxidative metabolism of 6-mercaptopurine in human liver: insights into the role of the molybdoflavoenzymes aldehyde oxidase, xanthine oxidase, and xanthine dehydrogenase.

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

Production of Recombinant Human Aldehyde Oxidase in Escherichia coli and Optimization of Its Application for the Preparative Synthesis of Oxidized Drug Metabolites

AbstractRecombinant human aldehyde oxidase (AO) was expressed in Escherichia coli. Different cell disruption methods and conditions of cell culture in shake flasks and bioreactors and of biotransformation on an analytical scale were tested to optimize the synthesis of oxidized AO drug metabolites. The volumetric productivity was increased 24‐fold by optimizing the cell culture conditions. The highest yield was achieved in a 25 L stirred tank bioreactor under non‐oxygen‐limited conditions and high lactose feed rate. Suspensions of highly concentrated and well‐aerated whole cells at neutral pH and relatively low temperatures led to the best conversion. The solvent for the substrate and the buffering agent for the biotransformation had an important effect. In a biotransformation with AO, 210 mg of famciclovir was converted to diacetyl penciclovir a yield of 82 %. The optimized protocol represents a viable method for the preparative synthesis of oxidized AO metabolites of drugs.

Inhibition of xanthine oxidase by the aldehyde oxidase inhibitor raloxifene: Implications for identifying molybdopterin nitrite reductases

Sources of nitric oxide alternative to nitric oxide synthases are gaining significant traction as crucial mediators of vessel function under hypoxic inflammatory conditions. For example, capacity to catalyze the one electron reduction of nitrite (NO2-) to ·NO has been reported for hemoglobin, myoglobin and molybdopterin-containing enzymes including xanthine oxidoreductase (XOR) and aldehyde oxidase (AO). For XOR and AO, use of selective inhibition strategies is therefore crucial when attempting to assign relative contributions to nitrite-mediated ·NO formation in cells and tissue. To this end, XOR inhibition has been accomplished with application of classic pyrazolopyrimidine-based inhibitors allo/oxypurinol or the newly FDA-approved XOR-specific inhibitor, Uloric® (febuxostat). Likewise, raloxifene, an estrogen receptor antagonist, has been identified as a potent (Ki=1.0nM) inhibitor of AO. Herein, we characterize the inhibition kinetics of raloxifene for XOR and describe the resultant effects on inhibiting XO-catalyzed ·NO formation. Exposure of purified XO to raloxifene (PBS, pH 7.4) resulted in a dose-dependent (12.5–100μM) inhibition of xanthine oxidation to uric acid. Dixon plot analysis revealed a competitive inhibition process with a Ki=13μM. This inhibitory process was more effective under acidic pH; similar to values encountered under hypoxic/inflammatory conditions. In addition, raloxifene also inhibited anoxic XO-catalyzed reduction of NO2- to NO (EC50=64μM). In contrast to having no effect on XO-catalyzed uric acid production, the AO inhibitor menadione demonstrated potent inhibition of XO-catalyzed NO2- reduction (EC50=60nM); somewhat similar to the XO-specific inhibitor, febuxostat (EC50=4nM). Importantly, febuxostat was found to be a very poor inhibitor of human AO (EC50=613μM) suggesting its usefulness for validating XO-dependent contributions to NO2- reduction in biological systems. Combined, these data indicate care should be taken when choosing inhibition strategies as well as inhibitor concentrations when assigning relative NO2- reductase activity of AO and XOR.

Absolute Quantification of Aldehyde Oxidase Protein in Human Liver Using Liquid Chromatography–Tandem Mass Spectrometry

The function of the enzyme human aldehyde oxidase (AOX1) is uncertain; however, recent studies have implicated significant biochemical involvement in humans. AOX1 has also rapidly become an important drug-metabolizing enzyme. Until now, quantitation of AOX1 in complex matrices such as tissue has not been achieved. Herein, we developed and employed a trypsin digest and subsequent liquid chromatography-tandem mass spectrometry analysis to determine absolute amounts of AOX1 in human liver. E. coli expressed human purified AOX1 was used to validate the linearity, sensitivity, and selectivity of the method. Overall, the method is highly efficient and sensitive for determination of AOX1 in cytosolic liver fractions. Using this method, we observed substantial batch-to-batch variation in AOX1 content (21-40 pmol AOX1/mg total protein) between various pooled human liver cytosol preparations. We also observed interbatch variation in Vmax (3.3-4.9 nmol min(-1) mg(-1)) and a modest correlation between enzyme concentration and activity. In addition, we measured a large difference in kcat/Km, between purified (kcat/Km of 1.4) and human liver cytosol (kcat/Km of 15-20) indicating cytosol to be 11-14 times more efficient in the turnover of DACA than the E. coli expressed purified enzyme. Finally, we discussed the future impact of this method for the development of drug metabolism models and understanding the biochemical role of this enzyme.

Evaluation of Rhesus Monkey and Guinea Pig Hepatic Cytosol Fractions as Models for Human Aldehyde Oxidase

Aldehyde oxidase (AOX) is a cytosolic enzyme expressed across a wide range of species, including guinea pig and rhesus monkey. These species are believed to be the best preclinical models for studying human AOX-mediated metabolism. We compared AOX activity in rhesus monkeys, guinea pigs, and humans using phthalazine and N-[2-(dimethylamino)ethyl]acridone-4-carboxamide (DACA) as substrates and raloxifene as an inhibitor. Michaelis-Menten kinetics was observed for phthalazine oxidation in rhesus monkey, guinea pig, and human liver cytosol, whereas substrate inhibition was seen with DACA oxidase activity in all three livers. Raloxifene inhibited phthalazine and DACA oxidase activity uncompetitively in guinea pig, whereas mixed-mode inhibition was seen in rhesus monkey. Our analysis of the primary sequence alignment of rhesus monkey, guinea pig, and human aldehyde oxidase isoform 1 (AOX1) along with homology modeling has led to the identification of several amino acid residue differences within the active site and substrate entrance channel of AOX1. We speculate that some of these residues might be responsible for the differences observed in activity. Overall, our data indicate that rhesus monkeys and guinea pigs would overestimate intrinsic clearance in humans and would be unsuitable to use as animal models. Our study also showed that AOX metabolism in species is substrate-dependent and no single animal model can be reliably used to predict every drug response in humans.

Prediction of human metabolism of the sedative-hypnotic zaleplon using chimeric mice transplanted with human hepatocytes

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

Effect of Structural Variation on Aldehyde Oxidase-Catalyzed Oxidation of Zoniporide

Current studies explored the effect of structural changes on the aldehyde oxidase (AO)-mediated metabolism of zoniporide (1). Zoniporide analogs with modifications of the acylguanidine moiety, the cyclopropyl group on the pyrazole ring, and the quinoline ring were studied for their AO-catalyzed metabolism using the human S9 fraction. Analysis of the half-lives suggested that subtle changes in the structure of 1 influenced its metabolism and that the guanidine and the quinoline moieties were prerequisites for AO-catalyzed oxidation to 2-oxozoniporide (M1). In contrast, replacement of the cyclopropyl group with other alkyl groups was tolerated. The effect of structural variation on AO properties was rationalized by docking 1 and its analogs into the human AO homology model. These studies indicated the importance of electrostatic, π-π stacking and hydrophobic interactions of the three motifs with residues in the active site. Differences in substrate properties were also rationalized by comparing their half-lives with cLogD, electrophilicity parameters [electrostatic potential (ESP) charges and energy of lowest unoccupied molecular orbitals (E(LUMO))], and the energies of formation of tetrahedral intermediates (J Med Chem 50:4642-4647, 2007). Whereas the success of energetics in predicting the AO substrate properties of analogs was 87%, the predictive ability of other descriptors was none (cLogD) to 60% (ESP charges and E(LUMO)). Overall, the structure-metabolism relationship could be rationalized using a combination of both the energy calculations and docking studies. This combination method can be incorporated into a strategy for mitigating AO liabilities observed in the lead candidate or studying structure-metabolism relationships of other AO substrates.

Medicinal chemistry approaches to avoid aldehyde oxidase metabolism

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.

The Impact of Single Nucleotide Polymorphisms on Human Aldehyde Oxidase

Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N(1)-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95% and a yield of 50 μg/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs.

Urine metabolomics reveals novel physiologic functions of human aldehyde oxidase and provides biomarkers for typing xanthinuria

Classical xanthinuria is a rare inherited metabolic disorder caused by either isolated xanthine dehydrogenase (XDH) deficiency (type I) or combined XDH and aldehyde oxidase (AO) deficiency (type II). XDH and AO are evolutionary related enzymes that share a sulfurated molybdopterin cofactor. While the role of XDH in purine metabolism is well established, the physiologic functions of AO are mostly unknown. XDH and AO are important drug metabolizing enzymes. Urine metabolomic analysis by high pressure liquid chromatography and mass spectrometry of xanthinuric patients was performed to unveil physiologic functions of XDH and AO and provide biomarkers for typing xanthinuria. Novel endogenous products of AO, hydantoin propionic acid, N1-methyl-8-oxoguanine and N-(3-acetamidopropyl) pyrrolidin-2-one formed in the histidine, nucleic acid and spermidine metabolic pathways, respectively, were identified as being lowered in type II xanthinuria. Also lowered were the known AO products, N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-5-carboxamide in the nicotinamide degradation pathway. In contrast to the KEGG annotations, the results suggest minor role of human AO in the conversion of pyridoxal to pyridoxate and gentisaldehyde to gentisate in the vitamin B6 and tyrosine metabolic pathways, respectively. The perturbations in purine degradation due to XDH deficiency radiated further from the previously known metabolites, uric acid, xanthine and hypoxanthine to guanine, methyl guanine, xanthosine and inosine. Possible pathophysiological implications of the observed metabolic perturbations are discussed. The identified biomarkers have the potential to replace the allopurinol-loading test used in the past to type xanthinuria, thus facilitating appropriate pharmacogenetic counseling and gene directed search for causative mutations.

Inhibition of Human Liver Aldehyde Oxidase: Implications for Potential Drug-Drug Interactions

During the course of our research efforts to understand the kinetics of human aldehyde oxidase as a xenobiotic-clearing enzyme, we investigated the effect of eight different inhibitors on the oxidation of the probe substrate phthalazine. Saturation kinetic parameters for phthalazine oxidation in human liver cytosol were found to be the following: K(m) = 8.0 ± 0.4 μM and V(max) = 4.3 ± 0.1 nmol · min(-1) · mg protein(-1). Inhibitory potency of the inhibitors tested ranged from 0.1 to 5 μM. Of the eight different inhibitor compounds tested, seven were observed to inhibit through a mixed mode and one through a strictly competitive mode. A ratio of the K(ii) and K(is) values was used to assess the relative competitiveness of each inhibitor. For the mixed inhibitors, the mode of inhibition varied from mostly uncompetitive to predominantly competitive (K(ii)/K(is) values ranging from 0.1 to 15). The implications for potential drug-drug interactions and inhibition mechanism are discussed. We found two inhibitors, clozapine and chlorpromazine, that have a moderate predicted risk of drug-drug interactions based on the K(i) value relative to the inhibitor concentration in human plasma, having a calculated [I]/K(i) value of 0.4 and 0.8, respectively.

Increasing recognition of the importance of aldehyde oxidase in drug development and discovery

Aldehyde oxidases are molybdoflavoenzymes with broad substrate specificity, oxidizing different types of aldehydes, and heterocyclic rings. The physiological function of aldehyde oxidases is largely unknown, although the enzymes play an important role in the metabolism of numerous compounds of medicinal and toxicological interest, as they oxidize a wide range of aldehydes and heterocyclic compounds. In this article, we review the significance of aldehyde oxidases for the design and development of new drugs and discuss associated problems. These include species-specific differences in the complement of isoenzymes synthesized and general difficulties in studying the enzymatic characteristics of purified or recombinant aldehyde oxidases. We highlight the potential offered by human aldehyde oxidase targeting for the development of new pharmacological agents, limiting our attention to the realms of antiobesity and anti-cancer drugs. The last point is discussed in the context of the design of novel anticancer drugs, selectively activated or inactivated by this enzyme, with the final aim of achieving organ and/or tumor selectivity.