Nicotine inhalation and metabolism triggers AOX-mediated superoxide generation with oxidative lung injury.

With the increasing use of vaping devices that deliver high levels of nicotine (NIC) to the lungs, sporadic lung injury has been observed. Commercial vaping solutions can contain high NIC concentrations of 150 mM or more. With high NIC levels, its metabolic products may induce toxicity. NIC is primarily metabolized to form NIC iminium (NICI) which is further metabolized by aldehyde oxidase (AOX) to cotinine. We determine that NICI in the presence of AOX is a potent trigger of superoxide generation. NICI stimulated superoxide generation from AOX with Km = 2.7 µM and Vmax = 794 nmol/min/mg measured by cytochrome-c reduction. EPR spin-trapping confirmed that NICI in the presence of AOX is a potent source of superoxide. AOX is expressed in the lungs and chronic e-cigarette exposure in mice greatly increased AOX expression. NICI or NIC stimulated superoxide production in the lungs of control mice with an even greater increase after chronic e-cigarette exposure. This superoxide production was quenched by AOX inhibition. Furthermore, e-cigarette-mediated NIC delivery triggered oxidative lung damage that was blocked by AOX inhibition. Thus, NIC metabolism triggers AOX-mediated superoxide generation that can cause lung injury. Therefore, high uncontrolled levels of NIC inhalation, as occur with e-cigarette use, can induce oxidative lung damage.

AAO2 impairment enhances aldehyde detoxification by AAO3 in Arabidopsis leaves exposed to UV-C or Rose-Bengal.

Among the three active aldehyde oxidases in Arabidopsis thaliana leaves (AAO1-3), AAO3, which catalyzes the oxidation of abscisic-aldehyde to abscisic-acid, was shown recently to function as a reactive aldehyde detoxifier. Notably, aao2KO mutants exhibited less senescence symptoms and lower aldehyde accumulation, such as acrolein, benzaldehyde, and 4-hydroxyl-2-nonenal (HNE) than in wild-type leaves exposed to UV-C or Rose-Bengal. The effect of AAO2 expression absence on aldehyde detoxification by AAO3 and/or AAO1 was studied by comparing the response of wild-type plants to the response of single-functioning aao1 mutant (aao1S), aao2KO mutants, and single-functioning aao3 mutants (aao3Ss). Notably, aao3Ss exhibited similar aldehyde accumulation and chlorophyll content to aao2KO treated with UV-C or Rose-Bengal. In contrast, wild-type and aao1S exhibited higher aldehyde accumulation that resulted in lower remaining chlorophyll than in aao2KO leaves, indicating that the absence of active AAO2 enhanced AAO3 detoxification activity in aao2KO mutants. In support of this notion, employing abscisic-aldehyde as a specific substrate marker for AAO3 activity revealed enhanced AAO3 activity in aao2KO and aao3Ss leaves compared to wild-type treated with UV-C or Rose-Bengal. The similar abscisic-acid level accumulated in leaves of unstressed or stressed genotypes indicates that aldehyde detoxification by AAO3 is the cause for better stress resistance in aao2KO mutants. Employing the sulfuration process (known to activate aldehyde oxidases) in wild-type, aao2KO, and molybdenum-cofactor sulfurase (aba3-1) mutant plants revealed that the active AAO2 in WT employs sulfuration processes essential for AAO3 activity level, resulting in the lower AAO3 activity in WT than AAO3 activity in aao2KO.

Ontogeny of Human Liver Aldehyde Oxidase: Developmental Changes and Implications for Drug Metabolism.

Despite the increasing importance of aldehyde oxidase (AO) in the drug metabolism of clinical candidates, ontogeny data for AO are limited. The objective of our study was to characterize the age-dependent AO content and activity in the human liver cytosolic fraction (HLC) and human hepatocytes (HH). HLC (n = 121 donors) and HH (n = 50 donors) were analyzed for (1) AO protein content by quantitative proteomics and (2) enzyme activity using carbazeran as a probe substrate. AO activity showed high technical variability and poor correlation with the content in HLC samples, whereas hepatocyte samples showed a strong correlation between the content and activity. Similarly, AO content and activity showed no significant age-dependent differences in HLC samples, whereas the average AO content and activity in hepatocytes increased significantly (∼20-40-fold) from the neonatal levels (0-28 days). Based on the hepatocyte data, the age at which 50% of the adult AO content is reached (age50) was 3.15 years (0.32-13.97 years, 95% CI). Metabolite profiling of carbazeran revealed age-dependent metabolic switching and the role of non-AO mechanisms (glucuronidation and desmethylation) in carbazeran elimination. The content-activity correlation in hepatocytes improved significantly (R2 = 0.95; p < 0.0001) in samples showing <10% contribution of glucuronidation toward the overall metabolism, confirming that AO-mediated oxidation and glucuronidation are the key routes of carbazeran metabolism. Considering the confounding effect of glucuronidation on AO activity, AO content-based ontogeny data are a more direct reflection of developmental changes in protein expression. The comprehensive ontogeny data of AO in HH samples are more reliable than HLC data, which are important for developing robust physiologically based pharmacokinetic models for predicting AO-mediated metabolism in children.

Non-cytochrome P450 enzyme aldehyde oxidase is involved in the oxidative metabolic pathway of diquat and its detoxification effect.

Diquat (DQ) poisoning has garnered attention in recent years, primarily due to the rising incidence of cases worldwide, coupled with the absence of a viable antidote for its treatment. Despite the fact that diquat monopyridone (DQ-M) has been identified as a significant metabolite of DQ, the enzyme responsible for its formation remains unknown. In this study, we have identified aldehyde oxidase (AOX) as a vital enzyme involved in DQ oxidative metabolism. The metabolism of DQ to DQ-M was significantly inhibited by AOX inhibitors including raloxifene and hydralazine. The source of oxygen incorporated into DQ-M was proved to be from water through a H218O incubation experiment which further corroborated DQ-M formation via AOX metabolism. The product of DQ-M in vitro generated by fresh rat tissues co-incubation was consistent with its AOX expression. The result of the molecular docking analysis of DQ and AOX protein showed that DQ is capable of binding to AOX. Furthermore, the cytotoxicity of DQ was significantly higher than DQ-M at the same concentration tested in six cell types. This work is the first to uncover the involvement of aldehyde oxidase, a non-cytochrome P450 enzyme, in the oxidative metabolic pathway of diquat, thus providing a potential target for the development of detoxification treatment.

Cadmium Alters the Metabolism and Perception of Abscisic Acid in Pisum sativum Leaves in a Developmentally Specific Manner.

Abscisic acid (ABA) plays a crucial role in plant defense mechanisms under adverse environmental conditions, but its metabolism and perception in response to heavy metals are largely unknown. In Pisum sativum exposed to CdCl2, an accumulation of free ABA was detected in leaves at different developmental stages (A, youngest, unexpanded; B1, youngest, fully expanded; B2, mature; C, old), with the highest content found in A and B1 leaves. In turn, the content of ABA conjugates, which was highest in B2 and C leaves under control conditions, increased only in A leaves and decreased in leaves of later developmental stages after Cd treatment. Based on the expression of PsNCED2, PsNCED3 (9-cis-epoxycarotenoid dioxygenase), PsAO3 (aldehyde oxidase) and PsABAUGT1 (ABA-UDP-glucosyltransferase), and the activity of PsAOγ, B2 and C leaves were found to be the main sites of Cd-induced de novo synthesis of ABA from carotenoids and ABA conjugation with glucose. In turn, ß-glucosidase activity and the expression of genes encoding ABA receptors (PsPYL2, PsPYL4, PsPYL8, PsPYL9) suggest that in A and B1 leaves, Cd-induced release of ABA from inactive ABA-glucosyl esters and enhanced ABA perception comes to the forefront when dealing with Cd toxicity. The distinct role of leaves at different developmental stages in defense against the harmful effects of Cd is discussed.

Predicting routes of phase I and II metabolism based on quantum mechanics and machine learning.

1. Unexpected metabolism could lead to the failure of many late-stage drug candidates or even the withdrawal of approved drugs. Thus, it is critical to predict and study the dominant routes of metabolism in the early stages of research. In this study, we describe the development and validation of a 'WhichEnzyme' model that accurately predicts the enzyme families most likely to be responsible for a drug-like molecule's metabolism. Furthermore, we combine this model with our previously published regioselectivity models for Cytochromes P450, Aldehyde Oxidases, Flavin-containing Monooxygenases, UDP-glucuronosyltransferases and Sulfotransferases - the most important Phase I and Phase II drug metabolising enzymes - and a 'WhichP450' model that predicts the Cytochrome P450 isoform(s) responsible for a compound's metabolism. The regioselectivity models are based on a mechanistic understanding of these enzymes' actions, and use quantum mechanical simulations with machine learning methods to accurately predict sites of metabolism and the resulting metabolites. We train heuristic based on the outputs of the 'WhichEnzyme', 'WhichP450', and regioselectivity models to determine the most likely routes of metabolism and metabolites to be observed experimentally. Finally, we demonstrate that this combination delivers high sensitivity in identifying experimentally reported metabolites and higher precision than other methods for predicting in vivo metabolite profiles.

DNA Methylation-Mediated Lowly Expressed AOX1 Promotes Cell Migration and Invasion of Prostate Cancer.

INTRODUCTION: DNA methylation regulates gene transcriptional functions in the pathogenesis of malignant diseases. In prostate cancer, several tumor suppressors are known to be tumor specifically methylated. METHODS: In this study, 450K methylation data and mRNA expression data were accessed from The Cancer Genome Atlas-Prostate Adenocarcinoma database and analyzed bioinformatically. Methylation-specific PCR was used to examine the methylation condition in AOX1 promoter. qRT-PCR was applied to measure the mRNA expression of AOX1. Western blot was employed to detect the expressions of AOX1 and the EMT associated proteins. Transwell and scratch healing assays were used to examine the invasive and migratory abilities of the prostate cancer cells respectively. RESULTS: AOX1 was lowly expressed and hypermethylated in the prostate cancer tissues and cells. Also, AOX1 was downregulated at protein level in prostate cancer cells. Knocking down AOX1 could promote cell migration and invasion in the prostate cancer cells. By using a DNA methylation inhibitor, 5-AzadC was found to promote the expression of AOX1 and reverse the promoting effects of short interfering RNA against AOX1 on cell migration and invasion. CONCLUSION: This study suggested that DNA methylation and low AOX1 level might be biomarkers for prostate cancer.

Bringing Nitric Oxide to the Molybdenum World-A Personal Perspective.

Molybdenum-containing enzymes of the xanthine oxidase (XO) family are well known to catalyse oxygen atom transfer reactions, with the great majority of the characterised enzymes catalysing the insertion of an oxygen atom into the substrate. Although some family members are known to catalyse the "reverse" reaction, the capability to abstract an oxygen atom from the substrate molecule is not generally recognised for these enzymes. Hence, it was with surprise and scepticism that the "molybdenum community" noticed the reports on the mammalian XO capability to catalyse the oxygen atom abstraction of nitrite to form nitric oxide (NO). The lack of precedent for a molybdenum- (or tungsten) containing nitrite reductase on the nitrogen biogeochemical cycle contributed also to the scepticism. It took several kinetic, spectroscopic and mechanistic studies on enzymes of the XO family and also of sulfite oxidase and DMSO reductase families to finally have wide recognition of the molybdoenzymes' ability to form NO from nitrite. Herein, integrated in a collection of "personal views" edited by Professor Ralf Mendel, is an overview of my personal journey on the XO and aldehyde oxidase-catalysed nitrite reduction to NO. The main research findings and the path followed to establish XO and AO as competent nitrite reductases are reviewed. The evidence suggesting that these enzymes are probable players of the mammalian NO metabolism is also discussed.

Arabidopsis aldehyde oxidase 3, known to oxidize abscisic aldehyde to abscisic acid, protects leaves from aldehyde toxicity.

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 chemical compound 'Heatin' stimulates hypocotyl elongation and interferes with the Arabidopsis NIT1-subfamily of nitrilases.

Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.

Aldehyde oxidase mediated drug metabolism: an underpredicted obstacle in drug discovery and development.

Aldehyde oxidase (AO) has garnered curiosity as a non-CYP metabolizing enzyme in drug development due to unexpected consequences such as toxic metabolite generation and high metabolic clearance resulting in the clinical failure of new drugs. Therefore, poor AO mediated clearance prediction in preclinical nonhuman species remains a significant obstacle in developing novel drugs. Various isoforms of AO, such as AOX1, AOX3, AOX3L1, and AOX4 exist across species, and different AO activity among humans influences the AO mediated drug metabolism. Therefore, carefully considering the unique challenges is essential in developing successful AO substrate drugs. The in vitro to in vivo extrapolation underpredicts AO mediated drug clearance due to the lack of reliable representative animal models, substrate-specific activity, and the discrepancy between absolute concentration and activity. An in vitro tool to extrapolate in vivo clearance using a yard-stick approach is provided to address the underprediction of AO mediated drug clearance. This approach uses a range of well-known AO drug substrates as calibrators for qualitative scaling new drugs into low, medium, or high clearance category drugs. So far, in vivo investigations on chimeric mice with humanized livers (humanized mice) have predicted AO mediated metabolism to the best extent. This review addresses the critical aspects of the drug discovery stage for AO metabolism studies, challenges faced in drug development, approaches to tackle AO mediated drug clearance's underprediction, and strategies to decrease the AO metabolism of drugs.

GhAAO2 was observed responding to NaHCO3 stress in cotton compared to AAO family genes.

BACKGROUND: Abscisic acid (ABA) is an important stress hormone, the changes of abscisic acid content can alter plant tolerance to stress, abscisic acid is crucial for studying plant responses to abiotic stress. The abscisic acid aldehyde oxidase (AAO) plays a vital role in the final step in the synthesis of abscisic acid, therefore, understanding the function of AAO gene family is of great significance for plants to response to abiotic stresses. RESULT: In this study, 6, 8, 4 and 4 AAO genes were identified in four cotton species. According to the structural characteristics of genes and the traits of phylogenetic tree, we divided the AAO gene family into 4 clades. Gene structure analysis showed that the AAO gene family was relatively conservative. The analysis of cis-elements showed that most AAO genes contained cis-elements related to light response and plant hormones. Tissue specificity analysis under NaHCO3 stress showed that GhAAO2 gene was differentially expressed in both roots and leaves. After GhAAO2 gene silencing, the degree of wilting of seedlings was lighter than that of the control group, indicating that GhAAO2 could respond to NaHCO3 stress. CONCLUSIONS: In this study, the AAO gene family was analyzed by bioinformatics, the response of GhAAO gene to various abiotic stresses was preliminarily verified, and the function of the specifically expressed gene GhAAO2 was further verified. These findings provide valuable information for the study of potential candidate genes related to plant growth and stress.

Involvement of aldehyde oxidase in the metabolism of aromatic and aliphatic aldehyde-odorants in the mouse olfactory epithelium.

Xenobiotic-metabolizing enzymes (XMEs) expressed in the olfactory epithelium (OE) are known to metabolize odorants. Aldehyde oxidase (AOX) recognizes a wide range of substrates among which are substrates with aldehyde groups. Some of these AOX substrates are odorants, such as benzaldehyde and n-octanal. One of the mouse AOX isoforms, namely AOX2 (mAOX2), was shown to be specifically expressed in mouse OE but its role to metabolize odorants in this tissue remains unexplored. In this study, we investigated the involvement of mouse AOX isoforms in the oxidative metabolism of aldehyde-odorants in the OE. Mouse OE extracts effectively metabolized aromatic and aliphatic aldehyde-odorants. Gene expression analysis revealed that not only mAOX2 but also the mAOX3 isoform is expressed in the OE. Furthermore, evaluation of inhibitory effects using the purified recombinant enzymes led us to identify specific inhibitors of each isoform, namely chlorpromazine, 17ß-estradiol, menadione, norharmane, and raloxifene. Using these specific inhibitors, we defined the contribution of mAOX2 and mAOX3 to the metabolism of aldehyde-odorants in the mouse OE. Taken together, these findings demonstrate that mAOX2 and mAOX3 are responsible for the oxidation of aromatic and aliphatic aldehyde-odorants in the mouse OE, implying their involvement in odor perception.

Histamine elimination by a coupling reaction of fungal amine oxidase and bacterial aldehyde oxidase†.

Histamine (HIST) and other biogenic amines found in fish and fishery products accumulated by the action of bacterial amino acid decarboxylase cannot be decomposed and eliminated by heating or other chemical methods. A simple method for HIST elimination is proposed by a coupling reaction of the fungal amine oxidase (FAO) and bacterial aldehyde oxidase (ALOX) of acetic acid bacteria. As a model reaction, FAO oxidized benzylamine to benzaldehyde, which in turn was oxidized spontaneously to benzoic acid with ALOX. Likely, in HIST elimination, FAO coupled well with ALOX to produce imidazole 4-acetic acid from HIST with an apparent yield of 100%. Imidazole 4-acetaldehyde was not detected in the reaction mixture. In the absence of ALOX, the coupling reaction was incomplete given a number of unidentified substances in the reaction mixture. The proposed coupling enzymatic method may be highly effective to eliminate toxic amines from fish and fishery products.

Drug clearance by aldehyde oxidase: can we avoid clinical failure?

Despite increased awareness of aldehyde oxidase (AO) as a major drug-metabolising enzyme, predicting the pharmacokinetics of its substrates remains challenging. Several drug candidates have been terminated due to high clearance, which were subsequently discovered to be AO substrates. Even retrospective extrapolation of human clearance, from models more sensitive to AO activity, often resulted in underprediction.The questions of the current work thus were: Is there an acceptable degree of in vitro AO metabolism that does not result in high in vivo human clearance? And, if so, how can this be predicted?We built an in vitro/in vivo correlation using known AO substrates, combining multiple in vitro parameters to calculate the blood metabolic clearance mediated by AO (CLbAO). This value was compared with observed blood clearance (CLb-obs), establishing cut-off CLbAO values, to discriminate between low and high CLb-obs. The model was validated using additional literature compounds, and CLb-obs was predicted in the correct category.This simple, categorical, semi-quantitative yet multi-factorial model is readily applicable in drug discovery. Further, it is valuable for high-clearance compounds, as it predicts the CLb group, rather than an exact CLb value, for the substrates of this poorly-characterised enzyme.

COVID-19: Are We Facing Secondary Pellagra Which Cannot Simply Be Cured by Vitamin B3?

Immune response to SARS-CoV-2 and ensuing inflammation pose a huge challenge to the host's nicotinamide adenine dinucleotide (NAD+) metabolism. Humans depend on vitamin B3 for biosynthesis of NAD+, indispensable for many metabolic and NAD+-consuming signaling reactions. The balance between its utilization and resynthesis is vitally important. Many extra-pulmonary symptoms of COVID-19 strikingly resemble those of pellagra, vitamin B3 deficiency (e.g., diarrhoea, dermatitis, oral cavity and tongue manifestations, loss of smell and taste, mental confusion). In most developed countries, pellagra is successfully eradicated by vitamin B3 fortification programs. Thus, conceivably, it has not been suspected as a cause of COVID-19 symptoms. Here, the deregulation of the NAD+ metabolism in response to the SARS-CoV-2 infection is reviewed, with special emphasis on the differences in the NAD+ biosynthetic pathway's efficiency in conditions predisposing for the development of serious COVID-19. SARS-CoV-2 infection-induced NAD+ depletion and the elevated levels of its metabolites contribute to the development of a systemic disease. Acute liberation of nicotinamide (NAM) in antiviral NAD+-consuming reactions potentiates "NAM drain", cooperatively mediated by nicotinamide N-methyltransferase and aldehyde oxidase. "NAM drain" compromises the NAD+ salvage pathway's fail-safe function. The robustness of the host's NAD+ salvage pathway, prior to the SARS-CoV-2 infection, is an important determinant of COVID-19 severity and persistence of certain symptoms upon resolution of infection.

Evolution, expression, and substrate specificities of aldehyde oxidase enzymes in eukaryotes.

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.

The rice aldehyde oxidase OsAO3 gene regulates plant growth, grain yield, and drought tolerance by participating in ABA biosynthesis.

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.

Recent developments in predicting CYP-independent metabolism.

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 in vitro to in vitro extrapolation of clearance for substrates of non-CYP drug metabolizing enzymes.

Evaluation of species differences in the metabolism of the selective NaV1.7 inhibitor DS-1971a, a mixed substrate of cytochrome P450 and aldehyde oxidase.

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.