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Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites.After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite.Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450.When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2. Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites. After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite. Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450. When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2.

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.

Dendrobine is the main sesquiterpene alkaloid of Dendrobium nobile Lindl, which exhibits potent neuroprotective activity. However, its metabolism and disposition are little known. In this study, we investigated the metabolic characteristics of dendrobine in vitro and in rats. The metabolic stability and temporal profile of metabolites formation of dendrobine were assayed in human/rat liver microsomal and S9 fractions. Dendrobine metabolites were separated and identified mainly by UPLC-Q/Orbitrap MS. After oral administration of dendrobine (50 mg/kg) to rats, the accumulative excretion rate of dendrobine in feces, urine, and bile was 0.27%, 0.52%, and 0.031%, respectively, and low systematic exposure of dendrobine (AUC(0–∞) = 629.2 ± 56.4 ng·h/mL) was observed. We demonstrated that the elimination of dendrobine was very rapid in liver microsomal incubation (the in vitro elimination t(1/2) in rat and human liver microsomes was 1.35 and 5.61 min, respectively). Dendrobine underwent rapid and extensive metabolism; cytochrome P450, especially CYP3A4, CYP2B6, and CYP2C19, were mainly responsible for its metabolism. Aldehyde dehydrogenase, alcohol dehydrogenase and aldehyde oxidase were involved in the formation of carboxylic acid metabolites. By the aid of in-source fragmentation screening, hydrogen/deuterium exchange experiment, post-acquisition processing software, and available reference standards, 50 metabolites were identified and characterized in liver microsomal incubation and in rats. The major metabolic pathways of dendrobine were N-demethylation, N-oxidation, and dehydrogenation, followed by hydroxylation and glucuronidation. Collectively, the metabolic fate of dendrobine elucidated in this study not only yields benefits for its subsequent metabolism study but also facilitates to better understanding the mode of action of dendrobine and evaluating the pharmacologic efficiency of the high exposure metabolites.

Aldehyde oxidase (AOX) is a soluble, cytosolic enzyme that metabolizes various N-heterocyclic compounds and organic aldehydes. It has wide tissue distribution with highest levels found in liver, kidney, and lung. Human clearance projections of AOX substrates by in vitro assessments in isolated liver fractions (cytosol, S9) and even hepatocytes have been largely underpredictive of clinical outcomes. Various hypotheses have been suggested as to why this is the case. One explanation is that extrahepatic AOX expression contributes measurably to AOX clearance and is at least partially responsible for the often observed underpredictions. Although AOX expression has been confirmed in several extrahepatic tissues, activities therein and potential contribution to overall human clearance have not been thoroughly studied. In this work, the AOX enzyme activity using the S9 fractions of select extrahepatic human tissues (kidney, lung, vasculature, and intestine) were measured using carbazeran as a probe substrate. Measured activities were scaled to a whole-body clearance using best-available parameters and compared with liver S9 fractions. Here, the combined scaled AOX clearance obtained from the kidney, lung, vasculature, and intestine is very low and amounted to

Mycobacterial acid-resistant protease (MarP) is a membrane-associated serine protease involved in the survival of Mycobacterium tuberculosis in macrophages; here we produced MarP in the yeast Pichia pastoris and study its involvement in macrophage immune modulation. Pichia pastoris vectors, harboring a full-length or a partial sequence of MarP, were constructed. GS115 clones were selected, and homologous recombination at the AOX1 locus was assessed by PCR. Protein was purified by nickel affinity chromatography, and its effect on the cytokine profile was tested in human monocytes. Only the partial MarP protein (121–397 a.a.) lacking the transmembrane domain was successfully expressed as an N-glycosylated proteolytically active protease. In vitro stimulation of THP-1 cells with MarP promoted the release of TNF-α and IL-10. Mycobacterial MarP was successfully expressed in P. pastoris, and it is capable of cytokine release in vitro.

Favipiravir is an antiviral agent effective against several RNA viruses that is converted into an inactive oxidative metabolite (M1), mainly by aldehyde oxidase, in humans. In the present study, the biotransformation of favipiravir into M1 in male and female humans, monkeys, rats, and mice was examined in an in vitro system using liver cytosolic fractions. The kinetics for M1 formation followed the Michaelis-Menten model in all species. The Km , Vmax , and CLint values in humans were 602 µM, 466 pmol/min/mg protein, and 776 nl/min/mg protein in males, respectively, and 713 µM, 404 pmol/min/mg protein, and 567 nl/min/mg protein in females, respectively. Species differences in CLint values were monkeys > humans > mice > rats in both males and females, and the variations for males and females were 120- and 96-fold, respectively. Sex differences in CLint values were males > females in humans and mice, females > males in monkeys and rats, and marked variation (4.3-fold) was noted in mice. This suggests that the roles of aldehyde oxidase in the hepatic metabolism of favipiravir differ extensively depending on the species and sex, and this study will aid in the assessment of the antiviral activities of favipiravir against novel and/or variant viruses.

The cynomolgus macaque is a non-human primate species widely used in drug metabolism studies. Despite the importance of genetic polymorphisms in cytosolic aldehyde oxidase (AOX) 1 in humans, genetic variants have not been investigated in cynomolgus or rhesus macaques.Genetic variants in AOX1 were identified and allele frequencies were assessed using the genomes of 24 cynomolgus and 8 rhesus macaques. The analysis identified 38 non-synonymous variants, some of which were unique to cynomolgus macaques (bred in Cambodia, Indochina, or Indonesia) or rhesus macaques, whereas many variants were shared by the two lineages.Among the variants observed at relatively high frequencies, eight were selected for functional analysis. Recombinant P605L and V1338I AOX1 variants showed substantially lower phthalazine and carbazeran oxidation activities than the wild-type AOX1 protein.In liver cytosolic fractions from cynomolgus and rhesus macaques genotyped for P605L and V1338I AOX1, groups of cytosolic fractions with P605L and/or V1338I AOX1 variants showed significantly lower phthalazine and carbazeran oxidation activities than the wild type.These results indicate that AOX1 is polymorphic in cynomolgus and rhesus macaques, just as it is in humans. Further investigation is needed to reveal the functional significance of these AOX1 variants in drug metabolism.

To explore the interactive molecules of squid ink polysaccharides (SIP) for further understanding the action mechanisms of SIP bio-function, this study prepared SIP binding proteins from mouse liver using superparamagnetic nanometer beads. Michaelis–Menten constant (Km) was detected from a Lineweaver–Burk double reciprocal plot to assess effect of SIP on activity of aldehyde oxidase (AOX). Results showed that three proteins, AOX-3, regucalcin (RGN) and α1-antitrypsin (A1AT3) were separated from mouse liver by magnetic nanoparticles conjugated with SIP. Contents of AOX-3 were much more than RGN and A1AT3. SIP (0.5 mg mL−1) reduced Km value of aldehyde oxidase of mouse liver from 91.79 μmol L−1 to 43.70 μmol L−1.

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.

In plants, glucose (Glc) plays pivotal roles in development and stress responses mainly by supplying fuel for growth and regulating expression of genes essential for crosstalk with hormonal, oxidative, and defense signaling. However, the complicated relationship between Glc and plant hormones is still not very clear. In this study, gsm3 (glucose-sensitive mutant 3), an Arabidopsis mutant with Glc-sensitive phenotype, was identified. Compared to wild type, the cotyledon expansion rate of gsm3 was significantly decreased under the condition of 4.5% Glc. Fluridone was able to rescue the Glc-induced defects of gsm3 in cotyledon expansion. AAO3 and ABI4 are key genes involved in abscisic acid (ABA) biosynthesis and signaling transduction, respectively. We found that inactivation of AAO3 or ABI4 in gsm3 background led to reduced sensitivity to Glc. These results indicated that increased ABA synthesis resulted in the sensitivity of gsm3 to Glc. Moreover, our results indicated that gsm3 mutant accumulated more ROS, which made it more sensitive to the application of exogenous H2O2. Overall, GSM3 plays an important role in Glc-ABA signaling cascade during seed germination and early seedling growth.

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, O(6)-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.

The availability of assays that predict the contribution of cytochrome P450 (CYP) metabolism allows for the design of new chemical entities (NCEs) with minimal oxidative metabolism. These NCEs are often substrates of non-CYP drug-metabolizing enzymes (DMEs), such as UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), carboxylesterases (CESs), and aldehyde oxidase (AO). Nearly 30% of clinically approved drugs are metabolized by non-CYP enzymes. However, knowledge about the differential hepatic versus extrahepatic abundance of non-CYP DMEs is limited. In this study, we detected and quantified the protein abundance of eighteen non-CYP DMEs (AO, CES1 and 2, ten UGTs, and five SULTs) across five different human tissues. AO was most abundantly expressed in the liver and to a lesser extent in the kidney; however, it was not detected in the intestine, heart, or lung. CESs were ubiquitously expressed with CES1 being predominant in the liver, while CES2 was enriched in the small intestine. Consistent with the literature, UGT1A4, UGT2B4, and UGT2B15 demonstrated liver-specific expression, whereas UGT1A10 expression was specific to the intestine. UGT1A1 and UGT1A3 were expressed in both the liver and intestine; UGT1A9 was expressed in the liver and kidney; and UGT2B17 levels were significantly higher in the intestine than in the liver. All five SULTs were detected in the liver and intestine, and SULT1A1 and 1A3 were detected in the lung. Kidney abundance was the most variable among the studied tissues, and overall, high interindividual variability (15-fold) was observed for UGT2B17, CES2 (intestine), SULT1A1 (liver), UGT1A9, UGT2B7, and CES1 (kidney). These differential tissue abundance data can be integrated into physiologically based pharmacokinetic (PBPK) models for the prediction of non-CYP drug metabolism and toxicity in hepatic and extrahepatic tissues.

Early assessment of metabolism pathways of new chemical entities guides the understanding of drug-drug interactions. Selective enzyme inhibitors are indispensable in CYP reaction phenotyping. The most commonly applied CYP2C19 inhibitor, omeprazole, lacks selectivity. Two promising alternatives, (+)-N-3-benzylnirvanol and (−)-N-3-benzylphenobarbital, are already used as CYP2C19 inhibitors in some in vitro studies with suspended human hepatocytes. However, a full validation proving their suitability in terms of CYP and non-CYP selectivity has not been presented in literature. The present study provides a thorough comparison between omeprazole, (+)-N-3-benzylnirvanol, and (−)-N-3-benzylphenobarbital in terms of potency and selectivity and shows the superiority of (−)-N-3-benzylphenobarbital as a CYP2C19 inhibitor in suspended human hepatocytes. Furthermore, we evaluated the application of (−)-N-3-benzylphenobarbital to predict the in vivo contribution of CYP2C19 to drug metabolism [fraction metabolized (fm) of CYP2C19, fmCYP2C19]. A set of 10 clinically used CYP2C19 substrates with reported in vivo fmCYP2C19 data was evaluated. fmCYP2C19, which was predicted using data from suspended human hepatocyte incubations, underestimated the in vivo fmCYP2C19. The use of a different hepatocyte batch with a different CYP3A4/CYP2C19 activity ratio showed the impact of intrinsic CYP activities on the determination of fmCYP2C19. Overall, this study confirms the selective CYP2C19 inhibition by (−)-N-3-benzylphenobarbital over other CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4) and clinically relevant non-CYP enzymes [aldehyde oxidase, flavin-containing monooxygenase 3, N-acetyltransferase 2, uridine diphosphate glucuronosyltransferase (UGT) 1A1, UGT1A4, UGT2B7, UGT2B15] in suspended human hepatocytes. (−)-N-3-benzylphenobarbital is therefore the preferred CYP2C19 inhibitor to assess fmCYP2C19 in suspended human hepatocytes in comparison with omeprazole and (+)-N-3-benzylnirvanol. SIGNIFICANCE STATEMENT (−)-N-3-Benzylphenobarbital is a more potent and selective inhibitor of CYP2C19 in suspended human hepatocytes than omeprazole and (+)-N-3-benzylnirvanol. (−)-N-3-Benzylphenobarbital can be used to predict the fraction metabolized by CYP2C19 in suspended human hepatocytes.

BackgroundExposure of plants to different environmental insults instigates significant changes in the cellular redox tone driven in part by promoting the production of reactive nitrogen species. The key player, nitric oxide (NO) is a small gaseous diatomic molecule, well-known for its signaling role during stress. In this study, we focused on abscisic acid (ABA) metabolism-related genes that showed differential expression in response to the NO donorS-nitroso-l-cysteine (CySNO) by conducting RNA-seq-based transcriptomic analysis.ResultsCySNO-induced ABA-related genes were identified and further characterized. Gene ontology terms for biological processes showed most of the genes were associated with protein phosphorylation. Promoter analysis suggested that severalcis-regulatory elements were activated under biotic and/or abiotic stress conditions. The ABA biosynthetic geneAtAO3was selected for validation using functional genomics. The loss of function mutantatao3was found to differentially regulate oxidative and nitrosative stress. Further investigations for determining the role ofAtAO3in plant defense suggested a negative regulation of plant basal defense andR-gene-mediated resistance. Theatao3plants showed resistance to virulentPseudomonas syringaepv.tomatostrain DC3000 (PstDC3000) with gradual increase inPR1gene expression. Similarly,atao3plants showed increased hypersensitive response (HR) when challenged withPstDC3000 (avrB). Theatgsnor1–3andatsid2mutants showed a susceptible phenotype with reducedPR1transcript accumulation. Drought tolerance assay indicated thatatao3andatnced3ABA-deficient mutants showed early wilting, followed by plant death. The study of stomatal structure showed thatatao3andatnced3were unable to close stomata even at 7 days after drought stress. Further, they showed reduced ABA content and increased electrolyte leakage than the wild-type (WT) plants. The quantitative polymerase chain reaction analysis suggested that ABA biosynthesis genes were down-regulated, whereas expression of most of the drought-related genes were up-regulated inatao3than in WT.ConclusionsAtAO3negatively regulates pathogen-induced salicylic acid pathway, although it is required for drought tolerance, despite the fact that ABA production is not totally dependent onAtAO3, and that drought-related genes likeDREB2andABI2show response to drought irrespective of ABA content.

Simple Summary Insect sex pheromone biosynthesis has received widespread attention, while the terminal pathway related to aldehyde synthesis and metabolism is still poorly understood at a molecular level. Previous studies found that the silkworm, Bombyx mori (Lepidoptera, Bombycidae), has two pheromone compounds, bombykol and bombykal, with a ratio of 11:1, while its closest wild relative, B. mandarina, only uses bombykol as a pheromone. In this study, sex pheromone gland transcriptomes were compared between the domestic and wild silkworms. All the candidate gene families were identified. Then we used the differentially expressed information, tissue and developmental expression profiles, and phylogenetic analysis to identify the putative causal genes involved in the terminal pathway. Our findings provide insights into the aldehyde synthesis and metabolism pathways and evolutionary conservation in moths. Abstract Sex pheromones are vital to sexual communication and reproduction in insects. Although some key enzymes in pheromone production have been well studied, information on genes involved in the terminal pathway is limited. The domestic silkworm employs a pheromone blend containing (E,Z)-10,12-hexadecadienol (bombykol) and analogous (E,Z)-10,12-hexadecadienal (bombykal); whereas, its wild ancestor B. mandarina uses only bombykol. The two closely related moths might be a good model for exploring the genes involved in aldehyde pheromone synthesis and metabolism. By deep sequencing and analyzing the sex pheromone gland (PG) transcriptomes; we identified 116 candidate genes that may be related to pheromone biosynthesis, metabolism, and chemoreception. Spatiotemporal expression profiles and differentially expressed analysis revealed that four alcohol oxidases (BmorAO1; 2; 3; and 4); one aldehyde reductase (BmorAR1); and one aldehyde oxidase (BmorAOX5) might be involved in the terminal pathway. Phylogenetic analysis showed that, except for BmorAO3 and MsexAO3, AOs did not show a conversed orthologous relationship among moths; whereas, ARs and AOXs were phylogenetically conserved. This study provides crucial candidates for further functional elucidation, and which may be utilized as potential targets to disrupt sexual communication in other moth pests.

The Arabidopsis thaliana aldehyde oxidase 3 (AAO3) catalyzes the oxidation of abscisic aldehyde (ABal) to abscisic acid (ABA). Besides ABal, plants generate other aldehydes that can be toxic above a certain threshold. AAO3 knockout mutants (aao3) exhibited earlier senescence but equivalent relative water content compared with wild-type (WT) during normal growth or upon application of UV-C irradiation. Aldehyde profiling in leaves of 24-day-old plants revealed higher accumulation of acrolein, crotonaldehyde, 3Z-hexenal, hexanal and acetaldehyde in aao3 mutants compared with WT leaves. Similarly, higher levels of acrolein, benzaldehyde, crotonaldehyde, propionaldehyde, trans-2-hexenal and acetaldehyde were accumulated in aao3 mutants upon UV-C irradiation. Aldehydes application to plants hastened profuse senescence symptoms and higher accumulation of aldehydes, such as acrolein, benzaldehyde and 4-hydroxy-2-nonenal, in aao3 mutant leaves as compared with WT. The senescence symptoms included greater decrease in chlorophyll content and increase in transcript expression of the early senescence marker genes, Senescence-Related-Gene1, Stay-Green-Protein2 as well as NAC-LIKE, ACTIVATED-BY AP3/P1. Notably, although aao3 had lower ABA content than WT, members of the ABA-responding genes SnRKs were expressed at similar levels in aao3 and WT. Moreover, the other ABA-deficient mutants [aba2 and 9-cis-poxycarotenoid dioxygenase3-2 (nced3-2), that has functional AAO3] exhibited similar aldehydes accumulation and chlorophyll content like WT under normal growth conditions or UV-C irradiation. These results indicate that the absence of AAO3 oxidation activity and not the lower ABA and its associated function is responsible for the earlier senescence symptoms in aao3 mutant.

The influence of drought stress on barley (Hordeum vulgare) has been investigated. The experiments were conducted with seedlings of barley—by the application of combined drought and temperature stresses. Here we showed that combination of drought and high temperature inflicted more severe damage to plants than the drought and low temperature stress. The temperature stress triggered more drastic changes in plant morphology/physiology and biochemistry than the drought stress. Interestingly, plants exposed to high temperature exhibited significant reduction of shoot and root aldehyde oxidase (AO) activity. Moreover, increased temperature resulted in lower levels of reactive oxygen species (ROS), while drought stress had an opposite effect on ROS accumulation and AO activity. This is the first demonstration of inhibition of plant AO and ROS in response to heat stress. The combined heat and drought stresses resulted in increased activity of catalase (CAT) and superoxide dismutase (SOD) in roots but not in shoots. Our findings indicate that heat and drought stresses may induce activation of different antioxidant enzymatic defense mechanisms and heat stress significantly affects enzymes responsible for the ROS accumulation in plants.

Objective: According to the treatment records of Yang deficiency syndrome (YDS) with characteristic decoction pieces of lateral root of Aconitum carmichaelii—Yinfupian (YF) in traditional Chinese medicine prepare school, known as “Jianchangbang”. The aim of this study was to investigate differences in the composition and therapeutic mechanism of the unprocessed lateral root of Aconitum carmichaelii (ULRA) and its processed product (YF).Methods: Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and orthogonal partial least squares discriminant analysis method were used to determine and screen the main components of ULRA and YF. Changes in the histological structure and morphology of gonads in rats were observed using hematoxylin-eosin. Enzyme-linked immunosorbent assay was used to determine the contents of serum cyclic adenosine monophosphate and cyclic guanosine monophosphate in YDS rats treated with ULRA and YF. Tandem mass tag proteomics analysis was used to identify the differentially expressed proteins in YDS rats treated with ULRA and YF.Results: Both ULRA and YF exerted certain therapeutic effects on rats with YDS. They improved the gonadal morphology and increased the contents of serum cyclic adenosine monophosphate and cyclic guanosine monophosphate. After processing of ULRA into YF, the content of C19-diester-diterpenoid alkaloids decreased (converted into C19-monoester-diterpenoid alkaloids and C19-alkylol amine-diterpenoid alkaloids), whereas that of C20-diterpene alkaloids increased. Proteomics analysis showed that cytochrome P450 and aldehyde oxidase 3 (AOX3) were downregulated, whereas cathepsin G (CTSG) was upregulated in rats with YDS. Treatment with ULRA mainly downregulated the expression of α-actinin, fast skeletal troponin, creatine kinase, and myosin. Treatment with YF mainly upregulated the expression of mitochondrial ribosomal protein and mitochondrial inner membrane protein.Conclusion: ULRA and YF exerted good therapeutic effects on YDS; the main difference in components between these preparations was in C19-diterpenoid alkaloids. ULRA mainly acts on the muscle contraction-related proteins and is closely related to inflammation and myocardial injury. YF mainly acts on the mitochondrial proteins and is closely related to adenosine triphosphate energy metabolism.

Capmatinib, a mesenchymal-epithelial transition factor tyrosine kinase inhibitor, is metabolized by cytochrome P450 (CYP) 3A4 and aldehyde oxidase. In individuals with hepatic impairment, alterations in hepatobiliary excretion and metabolism could lead to higher capmatinib exposure. We compared the pharmacokinetics of a single oral dose of capmatinib 200 mg administered to participants with varying degrees of hepatic impairment vs. matched controls with normal hepatic function.This phase 1, multicentre, open-label, parallel-group study enrolled adult participants with normal hepatic function and mild, moderate and severe hepatic impairments. Eligible participants received a single oral dose of 200 mg capmatinib. The pharmacokinetic parameters of capmatinib were analysed and compared across participants with impaired and normal hepatic function.Of 31 enrolled participants, 29 had an evaluable pharmacokinetic profile: normal (n = 9); mild (n = 6); moderate (n = 8); severe (n = 6). Compared with the normal group, geometric mean (GM) maximum (peak) observed plasma drug concentration after single-dose administration decreased by 27.6% in the mild group (GM ratio [GMR] = 0.724; 90% confidence interval [CI]: 0.476-1.10), by 17.2% in the moderate group (GMR = 0.828; 90% CI: 0.563-1.22) and remained unchanged in the severe group (GMR = 1.02; 90% CI: 0.669-1.55). Compared with the normal group, GM area under the plasma concentration-time curve from time zero to infinity decreased by 23.3% in the mild group (GMR = 0.767; 90% CI: 0.532-1.11), by 8.6% in the moderate group (GMR = 0.914; 90% CI: 0.652-1.28) and increased by 24% in the severe group (GMR = 1.24; 90% CI: 0.858-1.78).Mild, moderate and severe hepatic impairment did not have a clinically relevant impact on capmatinib pharmacokinetics. No new safety findings are reported in this study.

As lead optimization efforts have successfully reduced metabolic liabilities due to cytochrome P450 (CYP)-mediated metabolism, there has been an increase in the frequency of involvement of non-CYP enzymes in the metabolism of investigational compounds. Although there have been numerous notable advancements in the characterization of non-CYP enzymes with respect to their localization, reaction mechanisms, species differences and identification of typical substrates, accurate prediction of non-CYP-mediated clearance, with a particular emphasis with the difficulties in accounting for any extrahepatic contributions, remains a challenge. The current manuscript comprehensively summarizes the recent advancements in the prediction of drug metabolism and the

Background: Molybden, as a vital and essential micronutrient is directly involved in the metabolism of other elements including carbon, sulfur, and nitrogen. Molybdenum alone is not biologically active unless it binds to specific cofactors. Except for the bacterial nitrogenase, which contains molybdenum-Iron complex, molybdenum cofactor (Moco) is considered as the bioactive component placed in active site regions of molybdenum-containing enzymes. This review aimed to discuss the biological mechanisms involved in molybdenum metabolism highlighting Molybdenum cofactor deficiencies. Methods: Articles indexed in Pubmed, Google Scholar, and Scopus databases were used to extract the required information. Results: Moco, as the cofactor of sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and nitrite reductase plays a substantial role in maintaining normal body homeostasis and reactive oxygen species (ROS) production. Lack of Moco is found to be associated with many inborn genetic disorders, such as mental retardation, brain immaturity, nervous shocks, and neurodegenerative diseases. Conclusion: Moco insufficiency compromises normal human body metabolism since it is reported to regulate the metabolic pathways of other elements. Although in recent years, substitution- and gene-therapies have been introduced to restore the metabolic pathways of patients with MoCD type A and B, the definitive treatment for this type of inborn disease has still remained ill-defined. More investigations are needed to completely understand the underlying pathophysiology of molybdenum-related diseases.

MET401 is a potent and selective c-Met inhibitor with a novel triazolopyrimidine scaffold. The aim of this study was to determine the pharmacokinetic profile of MET401 in preclinical species, and to identify the metabolic soft spot and enzyme involved, in order to help medicinal chemists to modify the compound to improve the pharmacokinetic profile. A metabolite identification study was performed in different liver fractions from various species. Chemical inhibition with selective cytochrome P450 (CYP) and molybdenum hydroxylase inhibitors was carried out to identify the enzyme involved. The deuterium substitution strategy was adopted to reduce metabolism. Pharmacokinetic studies were performed in rats to confirm the effect. Although M-2 is a minor metabolite in liver microsomal incubations, it became the predominant metabolite in incubations with liver S9, cytosol, hepatocytes and rat pharmacokinetic study. M-2 was synthesized enzymatically and the structure was identified as a mono-oxidation on the triazolopyrimidine moiety. The M-2 formation was ascribed to aldehyde oxidase (AO)-mediated metabolism based on the following evidence—M-2 production was NADPH independent, pan-CYP inhibitor 1-aminobenzotriazole and xanthine oxidase inhibitor allopurinol did not inhibit M-2 formation, and AO inhibitors menadione and raloxifene inhibited M-2 formation. The deuterated analog MET763 demonstrated an improved pharmacokinetic profile with lower clearance, longer terminal half-life and double oral exposure compared with MET401 in rats. These results indicate that the main metabolic pathway of MET401 is AO-mediated metabolism, which leads to poor in vivo pharmacokinetic profiles in rodents. The deuterium substitution strategy could be used to reduce AO-mediated metabolism liability.

The increase in demand for fish has necessitated the establishment of fish pond. The discharge of spent pond water pollutes the adjoining surroundings. The aim of this investigation was to determine the suitability of artificial pond wastewater (APWW) as source of manure for crop cultivation. Cowpea seeds were planted in polybags containing soil treated with varied amount of artificial pond wastewater. Thereafter, growth and various metabolic properties of cowpea seedlings were determined using standard procedures. Artificial pond wastewater treatment of soil increased significantly (P

The ricexip gene, which encodes the rice xylanase-inhibiting protein (riceXIP), was recombinantly expressed in Pichia pastoris GS115 under the control of AOX1 promoter. Recombinant riceXIP (rePriceXIP) was secreted into the supernatant and purified to homogeneity with the use of Ni-affinity resin. The molecular mass of rePriceXIP was approximately 44.0 kDa. RePriceXIP significantly inhibited the activity of GH11 xylanases in a concentration-dependent manner. The optimal inhibitory activity of rePriceXIP on GH11 xylanases (TfxA_CD214, reBaxA454, and TfxA_CD526) occurred at 40 °C for 30 min. The IC50 values of rePriceXIP inhibiting TfxA_CD214, reBaxA454, and TfxA_CD526 (0.5 U) were 45, 40, and 40 nM, respectively. The Ki of rePriceXIP on TfxA_CD214 xylanase was 12.2 nM. Increasing the concentration of rePriceXIP did not change Vmax, but increased Km, thereby suggesting that the inhibition was competitive. Analysis of the fluorescence intensities revealed that the Kq values for TfxA_CD214, reBaxA454, and TfxA_CD526 exceeded 2.0 × 1010 L mol−1•s−1, implying that static quenching occurred. Circular dichroism spectroscopy demonstrated that rePriceXIP bound to xylanase, thereby changing the secondary structure and reducing the catalytic activity of xylanase. Lower levels of hydrolytes are released from beechwood xylan by xylanases in the presence of rePriceXIP.

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

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.

Oxidative metabolism is one of the major biotransformation reactions that regulates the exposure of xenobiotics and their metabolites in the circulatory system and local tissues and organs, and influences their efficacy and toxicity. Although cytochrome (CY)P450s play critical roles in the oxidative reaction, extensive CYP450-independent oxidative metabolism also occurs in some xenobiotics, such as aldehyde oxidase, xanthine oxidoreductase, flavin-containing monooxygenase, monoamine oxidase, alcohol dehydrogenase, or aldehyde dehydrogenase-dependent oxidative metabolism. Drugs form a large portion of xenobiotics and are the primary target of this review. The common reaction mechanisms and roles of non-CYP450 enzymes in metabolism, factors affecting the expression and activity of non-CYP450 enzymes in terms of inhibition, induction, regulation, and species differences in pharmaceutical research and development have been summarized. These non-CYP450 enzymes are detoxifying enzymes, although sometimes they mediate severe toxicity. Synthetic or natural chemicals serve as inhibitors for these non-CYP450 enzymes. However, pharmacokinetic-based drug interactions through these inhibitors have rarely been reported in vivo. Although multiple mechanisms participate in the basal expression and regulation of non-CYP450 enzymes, only a limited number of inducers upregulate their expression. Therefore, these enzymes are considered non-inducible or less inducible. Overall, this review focuses on the potential xenobiotic factors that contribute to variations in gene expression levels and the activities of non-CYP450 enzymes.

Nonalcoholic fatty liver disease (NAFLD) has increased worldwide in recent years. NAFLD is classified into two types, nonalcoholic fatty liver (NAFL), with few complications, and nonalcoholic steatohepatitis (NASH), which leads to liver cirrhosis or cancer. This study was based on previous reports that N1-methylnicotinamide (MNA) can stabilise sirtuin 1 protein, leading to decreased lipid levels in the liver. We hypothesised that fatty liver improvement by MNA would be further enhanced by suppressing its rapid metabolism by aldehyde oxidase in the liver. To test this, hydralazine (HYD), a potent aldehyde oxidase inhibitor, was administered orally to NAFL model rats. Liver triglyceride (TG) levels in the model were nearly unchanged by administration of MNA alone. In contrast, TG levels were marked decreased in NAFL rats treated with a combination of MNA and HYD. In addition, TG levels were decreased even in NAFL rats treated with only HYD. These findings supported our hypothesis that maintaining MNA concentrations in the liver, by suppressing MNA metabolism, would at least partially ameliorate fatty liver.

BACKGROUND Age has a significant impact on activities of hepatic metabolizing enzymes in humans and animals. Flavin-containing Monooxygenase (FMO) and Aldehyde Oxidase (AO) are two important hepatic enzymes. Understanding of the impact of age on these two enzymes is still limited in pigs. OBJECTIVE The aim of this work was to assess hepatic FMO and AO activities of male domestic pigs at five different ages of 1 day, 2, 5, 10 and 20 weeks. METHODS Porcine liver microsomes and cytosol were prepared from the livers of male domestic pigs at ages of 1 day, 2, 5, 10 and 20 weeks. FMO activity was assessed using N-oxidation of benzydamine in porcine liver microsomes and AO activity was evaluated using oxidation of O6-benzylguanine in the porcine liver cytosol. RESULTS Porcine hepatic FMO activity was substantial at the age of 1 day, rapidly increased in 2 weeks, and remained high afterwards. Porcine hepatic AO activity was minimal at the age of 1 day and gradually increased to the maximum in 5 weeks and remained relatively constant to the age of 20 weeks. Porcine hepatic FMO activity is higher than other species, including humans. Age-dependent FMO developmental pattern in porcine liver is different from porcine hepatic CYP450 and human hepatic FMO. Porcine hepatic AO activity is much lower than humans although their developmental patterns are similar. CONCLUSION Age impact on hepatic activities of both FMO and AO is obvious in domestic male pigs although age patterns of both enzymes are different.

O-Dealkylation of the tyrosine kinase inhibitor lapatinib by cytochrome P450 3A enzymes is implicated in the development of lapatinib-induced hepatotoxicity. Conjugative metabolism of debenzylated lapatinib (M1) via glucuronidation and sulfation is thought to be a major detoxication pathway for lapatinib in preclinical species (rat and dog), limiting formation of the quinoneimine reactive metabolite. Glucuronidation of M1 by human recombinant UDP-glucuronosyltransferases (UGTs) has been reported in vitro; however, the relative UGT enzyme contributions are unknown, and the interspecies differences in the conjugation versus bioactivation pathways of M1 have not been fully elucidated. In the present study, reaction phenotyping experiments using human recombinant UGT enzymes and enzyme-selective chemical inhibitors demonstrated that UGT1A1 was the major hepatic UGT enzyme involved in lapatinib M1 glucuronidation. Formation of the M1-glucuronide by human liver microsomes from UGT1A1-genotyped donors was significantly correlated with UGT1A1 activity as measured by 17β-estradiol 3-glucuronidation (R(2) = 0.90). Interspecies differences were found in the biotransformation of M1 in human, rat, and dog liver microsomal and 9000g supernatant (S9) fractions via glucuronidation, sulfation, aldehyde oxidase–mediated oxidation, and bioactivation to the quinoneimine trapped as a glutathione (GSH) conjugate. Moreover, we demonstrated the sequential metabolism of lapatinib in primary human hepatocytes to the M1-glucuronide, M1-sulfate, and quinoneimine-GSH conjugate. M1 glucuronidation was highly correlated with the rates of M1 formation, suggesting that O-dealkylation may be the rate-limiting step in lapatinib biotransformation. Interindividual variability in the formation and clearance pathways of lapatinib M1 likely influences the hepatic exposure to reactive metabolites and may affect the risk for hepatotoxicity. SIGNIFICANCE STATEMENT: We used an integrated approach to examine the interindividual and interspecies differences in detoxication versus bioactivation pathways of lapatinib, which is associated with idiosyncratic hepatotoxicity. In addition to cytochrome P450 (P450)-mediated bioactivation, we report that multiple non-P450 pathways are involved in the biotransformation of the primary phenolic metabolite of lapatinib in vitro, including glucuronidation, sulfation, and aldehyde oxidase mediated oxidation. UGT1A1 was identified as the major hepatic enzyme involved in debenzylated lapatinib glucuronidation, which may limit hepatic exposure to the potentially toxic quinoneimine.

Abscisic acid (ABA) regulates many aspects of plant growth and development and the responses to abiotic stresses. Arabidopsis aldehyde oxidase 3 (AAO3) catalyzes the final step of ABA biosynthesis. We cloned and functionally characterized a novel aldehyde oxidase gene, OsAO3, the rice homolog of AAO3. OsAO3 was expressed in germinated seeds, roots, leaves, and floral organs, particularly in vascular tissues and guard cells, and its expression was significantly induced by exogenous ABA and mannitol. Mutation and overexpression of OsAO3 decreased and increased ABA levels, respectively, in seedling shoots and roots under both normal and drought stress conditions. The osao3 mutant exhibited earlier seed germination, increased seedling growth, and decreased drought tolerance compared to the wild-type, OsAO3-overexpressing lines exhibited the opposite phenotype. Mutation and overexpression of OsAO3 increased and decreased grain yield, respectively, by affecting panicle number per plant, spikelet number per panicle, and spikelet fertility. Thus, OsAO3 may participate in ABA biosynthesis, and is essential for regulation of seed germination, seedling growth, grain yield, and drought tolerance in rice.

Often it may be convenient and efficient to address multiple research questions with a single experiment. In many instances, however, the best approach is to design the experiment to address one question at a time. The design of enzyme mapping experiments is discussed in this chapter, focusing on considerations pertinent to the study of aldehyde oxidase (AO) vs. cytochrome P450 metabolism. Specifically, a case is presented in which reduced glutathione (GSH) was included in an experiment with human liver S9 fraction to trap reactive metabolites generated from cytochrome P450-mediated metabolism of lapatinib and its O-dealkylated metabolite, M1 (question 1). The AO inhibitor hydralazine was included in this experiment to investigate the involvement of AO-mediated metabolism of M1 (question 2). The presence of GSH was found to interfere with the inhibitory activity of hydralazine. Consideration of the time-dependent nature of hydralazine inhibitory activity toward AO when designing this experiment could have predicted the potential for GSH to interfere with hydralazine. This case underscores the importance of clearly identifying the research question, tailoring the experimental protocol to answer that question, and then meticulously considering how the experimental conditions could influence the results, particularly if attempting to address multiple questions with a single experiment.

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.

In this article, we report our efforts towards improving in vitro human clearance in a series of 5-azaquinazolines through a series of C4 truncations and C2 expansions. Extensive DMPK studies enabled us to tackle high Aldehyde Oxidase (AO) metabolism and unexpected discrepancies in human hepatocyte and liver microsomal intrinsic clearance. Our efforts culminated with the discovery of 5-azaquinazoline 35, which also displayed exquisite selectivity for IRAK4, and showed synergistic in vitro activity against MyD88/CD79 double mutant ABC-DLBCL in combination with the covalent BTK inhibitor acalabrutinib.

Background: Ovarian cancer causes high mortality rate worldwide, and despite numerous attempts, the outcome for patients with ovarian cancer are still not well improved. Microarray-based gene expressional analysis provides with valuable information for discriminating functional genes in ovarian cancer development and progression. However, due to the differences in experimental design, the results varied significantly across individual datasets. Methods: In the present study, the data of gene expression in ovarian cancer were downloaded from Gene Expression Omnibus (GEO) and 16 studies were included. A meta-analysis based gene expression analysis was performed to identify differentially expressed genes (DEGs). The most differentially expressed genes in our meta-analysis were selected for gene expression and gene function validation. Results: A total of 972 DEGs with P-value < 0.001 were identified in ovarian cancer, including 541 up-regulated genes and 431 down-regulated genes, among which 92 additional DEGs were found as gained DEGs. Top five up- and down-regulated genes were selected for the validation of gene expression profiling. Among these genes, up-regulated CD24 molecule (CD24), SRY (sex determining region Y)-box transcription factor 17 (SOX17), WFDC2, epithelial cell adhesion molecule (EPCAM), innate immunity activator (INAVA), and down-regulated aldehyde oxidase 1 (AOX1) were revealed to be with consistent expressional patterns in clinical patient samples of ovarian cancer. Gene functional analysis demonstrated that up-regulated WFDC2 and INAVA promoted ovarian cancer cell migration, WFDC2 enhanced cell proliferation, while down-regulated AOX1 was functional in inducing cell apoptosis of ovarian cancer. Conclusion: Our study shed light on the molecular mechanisms underlying the development of ovarian cancer, and facilitated the understanding of novel diagnostic and therapeutic targets in ovarian cancer.

Insect antennae play a fundamental role in perceiving and recognizing a broad spectrum of conventional semiochemicals and host plant-derived odors. As such, genes that are tightly associated with the antennae are thought to have olfactory-related roles related to signal transduction mechanisms. Several mechanisms suggest that enzymatic inactivation could contribute to the signal termination process, such as odorant-degrading enzymes (ODEs). To date, a few ODEs have been identified and characterized in detail in insect herbivores, but little is known about aldehyde oxidases (AOXs); moreover, direct in vivo experimental evidence is needed. AOXs are a major family of metabolic enzymes that oxidize a variety of aromatic aldehydes, and they may also play a significant role in detoxification and degradation of environmental chemical cues. Here, we report on the identification and characterization of a novel cDNA encoding the putative odorant-degrading enzyme, PxylAOX3, from the antennae of the diamondback moth, (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae). The purified recombinant protein showed a wide-range of substrate zymography oxidizing both sex pheromone compounds as well as plant-derived aldehydes with distinct activities. Our data suggest PxylAOX3 might be involved in the degradation of many structurally diverse aldehyde odorants. Furthermore, PxylAOX3 could participate in olfactory neuron protection by inactivation of redundant odorants and xenobiotic detoxification, making it a potential target for pesticide development as well.

The plant U-box E3 ubiquitin ligase-mediated ubiquitin/26S proteasome degradation system plays a key role in plant growth and development. Previously identified as a member of the grape PUB gene family, PUB38 was shown to participate in the berry-ripening progress. Here, we demonstrate that the E3 ligase VlPUB38 mediates abscisic acid (ABA) synthesis via 26S proteasome degradation and its involvement in regulating fruit-ripening processes. Strawberry-overexpressing VlPUB38 lines displayed obvious inhibition of mature phenotype, and this was rescued by exogenous ABA treatment and MG132. Post-ABA treatment, expression levels of ABA response-related genes in VlPUB38-overexpressed Arabidopsis significantly exceeded controls. Strawberry and Arabidopsis ectopic expression assays suggest that VlPUB38 negatively regulates fruit ripening in an ABA-dependent manner. Moreover, VlPUB38 has ubiquitin ligase activity, which depends on the U-box-conserved domain. VlPUB38 interacts with abscisic-aldehyde oxidase (VlAAO), targeting VlAAO proteolysis via the 26S proteasome system. These results indicate that VlPUB38 negatively regulates grape fruit ripening by mediating the degradation of key factor VlAAO in the ABA synthesis pathway.

This work was supported by Deutsche Forschungsgemeinschaft Grant Le1171/8-3 (to S. L.). FCT-MCTES, research unit UCIBIO (UID/Multi/04378/2019). project PTDC/BBB-BEP/1185/2014 (to M. J. R.). Fondazione Italo Monzino and the Associazione per la Ricerca sul Cancro (AIRC) (to E. G.). 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. publishersversion published

Affective disorders are a set of mental disorders and particularly disrupt the mental health of susceptible women during puberty, pregnancy, parturition and menopause transition, which are characterized by dramatic changes in reproductive hormone profiles. The serum FSH level changes significantly during these periods; yet, the role of FSH in mood regulation is poorly understood. In the current study, FSHR knockout (Fshr-/-) mice displayed enhanced affective disorder behaviors in an open field test and a forced swim test, accompanied by altered gene expression profiles. The differentially expressed genes between Fshr-/- mice and Fshr+/+ mice were enriched in multiple neuroendocrine metabolic pathways. FSHR deletion significantly increased/decreased the mRNA and/or protein expression levels of AOX1, RDH12, HTR3a and HTR4 in mood-mediating brain regions, including the hippocampus and prefrontal cortex. These results reveal that FSH signaling is involved in the development of affective disorders.

Pichia pastoris is able to metabolize methanol via a specific MUT (methanol utilization) pathway. Based on the powerful AOX1 (Alcohol Oxidase 1) promoter, the P. pastoris expression system has become one of the most widely used eukaryotic expression systems. The molecular mechanisms of methanol metabolic regulation remain unclearly understood, so it is important to identify and develop new transcriptional regulators. Our previous studies suggested that the expression of SUT2 could be induced by methanol but is repressed by glycerol, which indicates that SUT2 may be involved in methanol metabolism through an unknown mechanism. SUT2 encodes a putative transcription factor-like protein harboring a Gal4-like Zn2Cys6 DNA-binding domain in Pichia pastoris, and its homolog in Saccharomyces cerevisiae regulates sterol uptake and synthesis. This study shows that the overexpression of SUT2 promoted the expression of AOX1 and increases ergosterol content in cells. Furthermore, via truncation of the putative SUT2 promoter at diverse loci, the − 973 base pair (bp) to − 547 bp region to the ATG was shown to be the core element of the inducible promoter PSUT2, which strongly responds to the methanol signal. The transcriptional start site of SUT2, “A” at the 22nd bp upstream of ATG, was determined with 5′-rapid amplification of cDNA ends. A forward-loop cassette was constructed with MXR1 (Methanol Expression Regulator 1, a positive transcription factor of PAOX1) promoted by PSUT2, enabling moderate elevation in the expression level of Mxr1 and high activity of PAOX1 without damaging cellular robustness further boosting the production of heterologous proteins. The PAOX1-driven expression of enhanced green fluorescent protein in this novel system was improved by 18%, representing a promising method for extrinsic protein production. SUT2 may play roles in methanol metabolism by participating in sterol biosynthesis. PSUT2 was characterized as a novel inducible promoter in P. pastoris and a PSUT2-driven MXR1 forward-loop cassette was constructed to enhance the PAOX1 activity, laying a foundation for further development and application of P. pastoris expression system.

Currently, the salinization of soil one of the factors, which determine the productivity of crop plants. Under severe conditions, plants reveal the retardation in growth of shoots and roots,the decrease of productivity, because of biochemical and physiologicaleffects of salt high concentrations. To avoid any severe effect of biotic and abiotic stress is applied to the method of seed priming. Primed seed preserves the resistance signaling pathway for upcoming stress conditions. During biotic and biotic stress firstly accumulates reactive oxygen species which are the secondary messenger molecules in many signaling pathway of plant organisms. Excessive amounts of reactive oxygen species lead to macromolecules damage, such as lipids of cell walls, proteins, nucleic acids. To overcome the severe effect, plants evolved enzymatic and non-enzymatic systems of protection. The crucial enzymes are catalase, aldehyde oxidase, superoxide dismutase. The main components of the non-enzymatic antioxidant system are vitamins, low molecular mass substances such as glutathione. The phenomenon “cross-tolerance” suggests the resistance of plant organism to one stress by the exposure to another one. Early exposure salt stress to Nicotiana benthamiana leads to resistance to viral infection, change the activity of antioxidant enzymes (catalase, aldehyde oxidase). The analysis of viral particles revealed the decrease of viral particles content in salt primed plants.

The major pathway of xenobiotics metabolism are catalyzed by phase I enzymes (mainly cytochrome P450; CYPs) and phase II enzymes (mainly UDP-glucuronosyltransferases; UGTs). However, chemical modifications to diminish the CYP-mediated drug interaction have resulted in the synthesis of compounds with structural features that favor metabolism by non-CYP pathways. Non-CYP enzymes include UGT, flavin monooxygenase (FMO), monoamine oxidase (MAO), aldehyde oxidase (AOX1), xanthine oxidase (XO), carboxylesterases (CES), and aldo-keto reductases (AKR). Non-CYP enzymes can produce active or reactive/toxic metabolites, modulate the efficacy of therapeutically active drugs, or contribute to detoxification. Therefore, characterization of non-CYP metabolic pathways may enhance the understanding of in vitro-in vivo discrepancies and may help in the better prediction of human clearance and avoid expensive clinical failures. In this chapter, we will focus on the major non-CYP-mediated metabolism, their expression, mechanism, challenges, and clinical relevance.

The present study investigated the contribution of microsomal cytochrome P450 and cytosolic aldehyde oxidase-1 (AOX-1) to carbazeran 4-oxidation and O6-benzylguanine 8-oxidation in human liver microsomal, cytosolic, and S9 fractions. Incubations containing carbazeran and human liver microsomes with or without exogenously added NADPH yielded comparable levels of 4-oxo-carbazeran. O6-Benzylguanine 8-oxidation occurred in microsomal incubations, and the extent was increased by NADPH. Human recombinant CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 did not catalyze carbazeran 4-oxidation, whereas CYP1A2 was highly active in O6-benzylguanine 8-oxidation. 1-Aminobenzotriazole, a pan-cytochrome P450 inhibitor, decreased O6-benzylguanine 8-oxidation, but not carbazeran 4-oxidation, in microsomal incubations, whereas 1-aminobenzotriazole and furafylline (a CYP1A2-selective inhibitor) did not inhibit carbazeran 4-oxidation or O6-benzylguanine 8-oxidation in human liver S9 fraction. Carbazeran 4-oxidation in incubations containing human liver microsomes (from multiple donors and commercial suppliers) was attributed to microsomal preparations contaminated with AOX-1, as suggested by liver microsomal experiments indicating a decrease in carbazeran 4-oxidation by an AOX-1 inhibitor (hydralazine), and to detection of AOX-1 protein (at one-third the level of that in liver cytosol). Cytosolic contamination of liver microsomes was further demonstrated by the formation of dehydroepiandrosterone sulfate (catalyzed by cytosolic sulfotransferases) in liver microsomal incubations containing dehydroepiandrosterone. In conclusion, carbazeran 4-oxidation and O6-benzylguanine 8-oxidation are enzyme-selective catalytic markers of human AOX-1, as shown in human liver S9 fraction expressing cytochrome P450 and AOX-1. This study highlights the negative impact of cytosolic contamination of liver microsomes on the interpretation of reaction phenotyping data collected in an in vitro study performed in microsomal fractions.

Metabolic profiles of four drugs possessing diverse metabolic pathways (timolol, meloxicam, linezolid, and XK469) were compared following incubations in both suspended cryopreserved human hepatocytes and the HμREL hepatocyte coculture model. In general, minimal metabolism was observed following 4-hour incubations in both suspended hepatocytes and the HμREL model, whereas incubations conducted up to 7 days in the HμREL coculture model resulted in more robust metabolic turnover. In the case of timolol, in vivo human data suggest that 22% of the dose is transformed via multistep oxidative opening of the morpholine moiety. Only the first-step oxidation was detected in suspended hepatocytes, whereas the relevant downstream metabolites were produced in the HµREL model. For meloxicam, both the hydroxymethyl and subsequent carboxylic acid metabolites were abundant following incubation in the HμREL model, while only a trace amount of the hydroxymethyl metabolite was observed in suspension. Similar to timolol, linezolid generated substantially higher levels of morpholine ring-opened carboxylic acid metabolites in the HμREL model. Finally, while the major aldehyde oxidase–mediated mono-oxidative metabolite of XK469 was minimally produced in hepatocyte suspension, the HμREL model robustly produced this metabolite, consistent with a pathway reported to account for 54% of the total urinary excretion in human. In addition, low-level taurine and glycine conjugates were identified in the HµREL model. In summary, continuous metabolite production was observed for up to 7 days of incubation in the HµREL model, covering cytochrome P450, aldehyde oxidase, and numerous conjugative pathways, while predominant metabolites correlated with relevant metabolites reported in human in vivo studies.

Aldehyde oxidases are molybdenum and flavin dependent enzymes characterized by a very wide substrate specificity and performing diverse reactions that include oxidations (e.g., aldehydes and aza-heterocycles), hydrolysis of amide bonds, and reductions (e.g., nitro, S-oxides and N-oxides). Oxidation reactions and amide hydrolysis occur at the molybdenum site while the reductions are proposed to occur at the flavin site. AOX activity affects the metabolism of different drugs and xenobiotics, some of which designed to resist other liver metabolizing enzymes (e.g., cytochrome P450 monooxygenase isoenzymes), raising its importance in drug development. This work consists of a comprehensive overview on aldehyde oxidases, concerning the genetic evolution of AOX, its diversity among the human population, the crystal structures available, the known catalytic reactions and the consequences in pre-clinical pharmacokinetic and pharmacodynamic studies. Analysis of the different animal models generally used for pre-clinical trials and comparison between the human (hAOX1), mouse homologs as well as the related xanthine oxidase (XOR) are extensively considered. The data reviewed also include a systematic analysis of representative classes of molecules that are hAOX1 substrates as well as of typical and well characterized hAOX1 inhibitors. The considerations made on the basis of a structural and functional analysis are correlated with reported kinetic and metabolic data for typical classes of drugs, searching for potential structural determinants that may dictate substrate and/or inhibitor specificities.

Aldehyde Oxidase (hAOX1) is a cytosolic enzyme involved in the metabolism of drugs and xenobiotic compounds. The enzyme belongs to the xanthine oxidase (XO) family of Mo containing enzyme and is a homo‐dimer of two 150 kDa monomers. Nonsynonymous Single Nucleotide Polymorphisms (nsSNPs) of hAOX1 have been reported as affecting the ability of the enzyme to metabolize different substrates. Some of these nsSNPs have been biochemically and structurally characterized but the lack of a systematic and comprehensive study regarding all described and validated nsSNPs is urgent, due to the increasing importance of the enzyme in drug development, personalized medicine and therapy, as well as in pharmacogenetic studies. The objective of the present work was to collect all described nsSNPs of hAOX1 and utilize a series of bioinformatics tools to predict their effect on protein structure stability with putative implications on phenotypic functional consequences. Of 526 nsSNPs reported in NCBI‐dbSNP, 119 are identified as deleterious whereas 92 are identified as nondeleterious variants. The stability analysis was performed for 119 deleterious variants and the results suggest that 104 nsSNPs may be responsible for destabilizing the protein structure, whereas five variants may increase the protein stability. Four nsSNPs do not have any impact on protein structure (neutral nsSNPs) of hAOX1. The prediction results of the remaining six nsSNPs are nonconclusive. The in silico results were compared with available experimental data. This methodology can also be used to identify and prioritize the stabilizing and destabilizing variants in other enzymes involved in drug metabolism.

Basic amine substituents provide several pharmacokinetic benefits relative to acidic and neutral functional groups, and have been extensively utilized as substituents of choice in drug design. On occasions, basic amines have been associated with off-target pharmacology via interactions with aminergic G-protein coupled receptors, ion-channels, kinases, etc. Structural features associated with the promiscuous nature of basic amines have been well-studied, and can be mitigated in a preclinical drug discovery environment. In addition to the undesirable secondary pharmacology, α-carbon oxidation of certain secondary or tertiary cycloalkyl amines can generate electrophilic iminium and aldehyde metabolites, potentially capable of covalent adduction to proteins or DNA. Consequently, cycloalkyl amines have been viewed as structural alerts (SAs), analogous to functional groups such as anilines, furans, thiophenes, etc., which are oxidized to reactive metabolites that generate immunogenic haptens by covalently binding to host proteins. Detailed survey of the literature, however, suggests that cases where preclinical or clinical toxicity has been explicitly linked to the metabolic activation of a cycloalkyl amine group are extremely rare. Moreover, there is a distinct possibility for the formation of electrophilic iminium/amino-aldehyde metabolites with numerous cycloalkyl amine-containing marketed drugs, since stable ring cleavage products have been characterized as metabolites in human mass balance studies. In the present work, a critical analysis of the evidence for and against the role of iminium ions/aldehydes as mediators of toxicity is discussed with a special emphasis on often time overlooked detoxication pathways of these reactive species to innocuous metabolites.

Background Nitric oxide (NO) and abscisic acid (ABA) are important regulators of plant response to cold stress, and they interact in response to cold signals. The primary goal of this study was to determine the roles of exogenous NO and ABA on the synthesis of endogenous NO and ABA in cold-stored peach fruit. Results Exogenous NO and ABA maintained a relatively high content of NO, increased nitrate reductase (NR) activity, and inhibited the activity of NO synthase (NOS)-like and the levels of polyamine biosynthesis in peaches during cold storage. Treatments of potassium 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), NO, N-nitro-l-Arg-methyl ester (L -NAME), and sodium tungstate did not influence ABA content. Exogenous ABA increased the content of carotenoids and the activities of aldehyde oxidase (AO), 9-cis-epoxycarotenoid dioxygenase (NCED), and zeaxanthin epoxidase (ZEP) of ABA synthesis in peaches during cold storage, and upregulated the gene expression of PpAO1, PpNCED1, PpNCED2, and PpZEP. The production of endogenous NO was differentially inhibited by NO scavengers, ABA inhibitors, and NR inhibitors, but not affected by NOS-like inhibitors during cold storage. Conclusion Exogenous NO and ABA can induce endogenous NO synthesis in cold-stored peaches by the nitrate reductase pathway, and ABA can mediate endogenous ABA synthesis by the autocatalytic reaction. NO does not regulate ABA synthesis. © 2019 Society of Chemical Industry.

Atropisomerism is a type of axial chirality in which enantiomers or diastereoisomers arise due to hindered rotation around a bond axis. In this manuscript, we report a case in which torsional scan studies guided the thoughtful creation of a restricted axis of rotation between two heteroaromatic systems of a phosphoinositide 3-kinase (PI3K) β inhibitor, generating a pair of atropisomeric compounds with significantly different pharmacological and pharmacokinetic profiles. Emblematic of these differences, the metabolism of inactive ( M)-28 is primarily due to the cytosolic enzyme aldehyde oxidase, while active ( P)-28 has lower affinity for aldehyde oxidase, resulting in substantially better metabolic stability. Additionally, we report torsional scan and experimental studies used to determine the barriers of rotation of this novel PI3Kβ inhibitor.