Substrate For Monoamine Oxidase Research Articles

Why Does Monoamine Oxidase (MAO) Catalyze the Oxidation of Some Tetrahydropyridines?

Results pertaining to the mechanism of the oxidation of the tertiary amine 1-methyl-4-(1-methyl-1-H-pyrrol-2-yl)-1,2,3,6-tetrahydropyridine (MMTP, a close analog of the Parkinsonism inducing compound MPTP) by 3-methyllumiflavin (3MLF), a chemical model for the FAD cofactor of monoamine oxidase, are reported. MMTP and related compounds are among the few tertiary amines that are monoamine oxidase B (MAO-B) substrates. The MMTP/3MLF reaction is catalytic in the presence of O2 and the results under anaerobic conditions strongly suggest the involvement of radical intermediates, consistent with a single electron transfer mechanism. These observations support a new hypothesis to explain the MAO-catalyzed oxidations of amines. In general, electron transfer is thermodynamically unfavorable, and as a result, most 1° and 2° amines react via one of the currently accepted polar pathways. Steric constraints prevent 3° amines from reacting via a polar pathway. Those select 3° amines that are MAO substrates possess certain structural features (e. g., a C-H bond that is α- both to nitrogen and a C=C) that dramatically lower the pKa of the corresponding radical cation. Consequently, the thermodynamically unfavorable electron transfer equilibrium is driven towards products by an extremely favorable deprotonation step in the context of Le Chatelier's principle.

Catalysis & inhibition issues associated with Monoamine Oxidase (MAO). How unusually low α-C-H bond dissociation energies may open the door to single electron transfer

CH Bond dissociation energies for a unique selection of tertiary amines that are known substrates or inhibitors of monoamine oxidase have been calculated using density functional theory. These amines are unusual because they are the only tertiary amines that exhibit MAO substrate or inhibitor behavior. The unique structural feature common to these specific compounds is an sp3-hybridized CH2 moiety, which is α-both to nitrogen and an C=C or CC. The stabilization afforded the resulting radicals by extended delocalization dramatically lowers both the CH bond strength of the substrate (R-H → R· + H·) and pKa of the corresponding radical cation (RH·+ → R· + H+). This interplay of structure and thermodynamics may provide the driving force for an electron transfer mechanism for MAO catalysis and inhibition.

Synthesis and antidepressant-like effects of new 5-epi-incensole and 5-epi- incensole acetate in chronic unpredictable mild stress model of depression; behavioural and biochemical correlates

In the current investigation, 5-epi-incensole (3) and 5-epi-incensole acetate (5) were synthesized from the most potent anti-depressant constituents incensole (1) and incensole acetate (2) of Boswellia papyrifera Hochst. The resulting compounds were evaluated for their ability to ameliorate depressive symptoms in forced swim test (FST) and tail suspension test (TST) in chronic unpredictable mild stress (CUMS) induced depression paradigm. The results demonstrated that compounds 3 and 5 at the doses of 1 and 3 mg/kg administered for 28 days, significantly reduced the immobility time in FST and TST and were devoid of any effect on locomotor activity in the open field test (OFT). Both compounds 3 and 5 also reversed CUMS-induced reduction in the weight of animals and aversion in sucrose preference. The tested compounds also inhibited Monoamine oxidase-A (MAO) enzyme and increased the levels of brain noradrenaline (NA) and 5-Hydroxytryptamine (5-HT), decreased plasma corticosterone and pro-inflammatory cytokines including TNF-α, IL-6 in hippocampal homogenates. Compounds 3 and 5 also significantly reduced the increased lipid peroxidation and nitrite levels; decreased glutathione levels, and catalase activities in mice undergoing CUMS protocol. The binding mode of compounds 3 and 5 was predicted at the monoamine oxidase substrate binding site by molecular docking having docking scores of > −6 kcal/mol. Taken together these data revealed that compounds 3 and 5 exerted antidepressant-like effects in chronic unpredictable mild stress-induced depression paradigm and are likely mediated via modulating the central oxidative stress, MAO-A activity with a consequent increase in brain NA and 5-HT levels in inflammatory pathways.

Assay of MAO Inhibition by Chromatographic Techniques (HPLC/HPLC-MS).

Monoamine oxidase (MAO) enzymes (MAO A and B) catalyze the oxidative deamination of biogenic amines, neurotransmitters, and xenobiotic amines and contribute to the regulation of the contentof these activesubstances in mammalian organisms. The oxidation of biogenic amines by MAO produces hydrogen peroxide (H2O2) and aldehydes that represent risk factors for oxidative injury.The inhibitorsof MAO are useful as antidepressants and neuroprotective agents. Usually, the assays of MAO determine amine deamination products or measure the H2O2 released by using direct spectrophotometric or fluorimetric methods. Direct methods are more prone to interferences and can afford inaccurate results. Those limitations can beavoided by using chromatographic techniques. This work describesa chromatographic method to assay MAO A and MAOB activity by using kynuramine as a nonselective substrate and the subsequentanalysis of 4-hydroxyquinoline by RP-HPLC-DAD-fluorescence and mass spectrometry(MS). Alternatively, the assay uses the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin as a substrate of MAO that is oxidized (bioactivated) to neurotoxic pyridinium cations which areanalyzed by HPLC. These methods are applied to assess the inhibition of MAO by bioactive β-carboline alkaloids occurring in foods, plants, and biological systems.

High doses of tyramine stimulate glucose transport in human fat cells.

Among the dietary amines present in foods and beverages, tyramine has been widely studied since its excessive ingestion can cause catecholamine release and hypertensive crisis. However, tyramine exerts other actions than depleting nerve endings: it activates subtypes of trace amine associated receptors (TAARs) and is oxidized by monoamine oxidases (MAO). Although we have recently described that tyramine is antilipolytic in human adipocytes, no clear evidence has been reported about its effects on glucose transport in the same cell model, while tyramine mimics various insulin-like effects in rodent fat cells, such as activation of glucose transport, lipogenesis, and adipogenesis. Our aim was therefore to characterize the effects of tyramine on glucose transport in human adipocytes. The uptake of the non-metabolizable analogue 2-deoxyglucose (2-DG) was explored in adipocytes from human subcutaneous abdominal adipose tissue obtained from women undergoing reconstructive surgery. Human insulin used as reference agent multiplied by three times the basal 2-DG uptake. Tyramine was ineffective from 0.01 to 10µM and stimulatory at 100µM-1mM, without reaching the maximal effect of insulin. This partial insulin-like effect was not improved by vanadium and was impaired by MAO-A and MAO-B inhibitors. Contrarily to benzylamine, mainly oxidized by semicarbazide-sensitive amine oxidase (SSAO), tyramine activation of glucose transport was not inhibited by semicarbazide. Tyramine effect was not dependent on the Gi-coupled receptor activation but was impaired by antioxidants and reproduced by hydrogen peroxide. In all, the oxidation of high doses of tyramine, already reported to inhibit lipolysis in human fat cells, also partially mimic another effect of insulin in these cells, the glucose uptake activation. Thus, other MAO substrates are potentially able to modulate carbohydrate metabolism.

Phenethylamine is a substrate of monoamine oxidase B in the paraventricular thalamic nucleus

Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of neurotransmitters and trace amines. MAO has two subtypes (MAO-A and MAO-B) that are encoded by different genes. In the brain, MAO-B is highly expressed in the paraventricular thalamic nucleus (PVT); however, its substrate in PVT remains unclear. To identify the MAO-B substrate in PVT, we generated Maob knockout (KO) mice and measured five candidate substrates (i.e., noradrenaline, dopamine, 3-methoxytyramine, serotonin, and phenethylamine [PEA]) by liquid chromatography tandem mass spectrometry. We showed that only PEA levels were markedly elevated in the PVT of Maob KO mice. To exclude the influence of peripheral MAO-B deficiency, we developed brain-specific Maob KO mice, finding that PEA in the PVT was increased in brain-specific Maob KO mice, whereas the extent of PEA increase was less than that in global Maob KO mice. Given that plasma PEA levels were elevated in global KO mice, but not in brain–specific KO mice, and that PEA passes across the blood–brain barrier, the substantial accumulation of PEA in the PVT of Maob KO mice was likely due to the increase in plasma PEA. These data suggest that PEA is a substrate of MAO-B in the PVT as well as other tissues.

Vanadium-dependent activation of glucose transport in adipocytes by catecholamines is not mediated via adrenoceptor stimulation or monoamine oxidase activity.

BACKGROUNDBenzylamine and methylamine activate glucose uptake in adipocytes. For tyramine, this effect has even been extended to cardiomyocytes.AIMTo investigate the effects of catecholamines and other amines on glucose uptake.METHODSA screening compared 25 biogenic amines on 2-deoxyglucose (2-DG) uptake activation in rat adipocytes. Pharmacological approaches and transgenic mouse models were then used to decipher the mode of action of several hits.RESULTSIn rat adipocytes, insulin stimulation of 2-DG uptake was reproduced with catecholamines. 100 µmol/L or 1 mmol/L adrenaline, noradrenaline, dopamine and deoxyepinephrine, maximally activated hexose transport only when sodium orthovanadate was added at 100 µmol/L. Such activation was similar to that already reported for benzylamine, methylamine and tyramine, well-recognized substrates of semicarbazide-sensitive amine oxidase (SSAO) and monoamine oxidase (MAO). Several, but not all, tested agonists of β-adrenoreceptors (β-ARs) also activated glucose transport while α-AR agonists were inactive. Lack of blockade by α- and β-AR antagonists indicated that catecholamine-induced 2-DG uptake was not mediated by AR stimulation. Adipocytes from mice lacking β1-, β2- and β3-ARs (triple KO) also responded to millimolar doses of adrenaline or noradrenaline by activating hexose transport in the presence of 100 µmol/L vanadate. The MAO blocker pargyline, and SSAO inhibitors did not block the effects of adrenaline or noradrenaline plus vanadate, which were blunted by antioxidants.CONCLUSIONCatecholamines exert unexpected insulin-like actions in adipocytes when combined with vanadium. For limiting insulin resistance by activating glucose consumption at least in fat stores, we propose that catecholamine derivatives combined with vanadium can generate novel complexes that may have low toxicity and promising anti-diabetic properties.

Methylxanthines Inhibit Primary Amine Oxidase and Monoamine Oxidase Activities of Human Adipose Tissue.

Background: Methylxanthines including caffeine and theobromine are widely consumed compounds and were recently shown to interact with bovine copper-containing amine oxidase. To the best of our knowledge, no direct demonstration of any interplay between these phytochemicals and human primary amine oxidase (PrAO) has been reported to date. We took advantage of the coexistence of PrAO and monoamine oxidase (MAO) activities in human subcutaneous adipose tissue (hScAT) to test the interaction between several methylxanthines and these enzymes, which are involved in many key pathophysiological processes. Methods: Benzylamine, methylamine, and tyramine were used as substrates for PrAO and MAO in homogenates of subcutaneous adipose depots obtained from overweight women undergoing plastic surgery. Methylxanthines were tested as substrates or inhibitors by fluorimetric determination of hydrogen peroxide, an end-product of amine oxidation. Results: Semicarbazide-sensitive PrAO activity was inhibited by theobromine, caffeine, and isobutylmethylxanthine (IBMX) while theophylline, paraxanthine, and 7-methylxanthine had little effect. Theobromine inhibited PrAO activity by 54% at 2.5 mM. Overall, the relationship between methylxanthine structure and the degree of inhibition was similar to that seen with bovine PrAO, although higher concentrations (mM) were required for inhibition. Theobromine also inhibited oxidation of tyramine by MAO, at the limits of its solubility in a DMSO vehicle. At doses higher than 12 % v/v, DMSO impaired MAO activity. MAO was also inhibited by millimolar doses of IBMX, caffeine and by other methylxanthines to a lesser extent. Conclusions: This preclinical study extrapolates previous findings with bovine PrAO to human tissues. Given that PrAO is a potential target for anti-inflammatory drugs, it indicates that alongside phosphodiesterase inhibition and adenosine receptor antagonism, PrAO and MAO inhibition could contribute to the health benefits of methylxanthines, especially their anti-inflammatory effects.

Highly Variable Pharmacokinetics of Tyramine in Humans and Polymorphisms in OCT1, CYP2D6, and MAO-A.

Tyramine, formed by the decarboxylation of tyrosine, is a natural constituent of numerous food products. As an indirect sympathomimetic, it can have potentially dangerous hypertensive effects. In vitro data indicated that the pharmacokinetics of tyramine possibly depend on the organic cation transporter OCT1 genotype and on the CYP2D6 genotype. Since tyramine is a prototypic substrate of monoamine oxidase A (MAO-A), genetic polymorphisms in MAO-A may also be relevant. The aims of this study were to identify to what extent the interindividual variation in pharmacokinetics and pharmacodynamics of tyramine is determined by genetic polymorphisms in OCT1, CYP2D6, and MAO-A. Beyond that, we wanted to evaluate tyramine as probe drug for the in vivo activity of MAO-A and OCT1. Therefore, the pharmacokinetics, pharmacodynamics, and pharmacogenetics of tyramine were studied in 88 healthy volunteers after oral administration of a 400 mg dose. We observed a strong interindividual variation in systemic tyramine exposure, with a mean AUC of 3.74 min*µg/ml and a high mean CL/F ratio of 107 l/min. On average, as much as 76.8% of the dose was recovered in urine in form of the MAO-catalysed metabolite 4-hydroxyphenylacetic acid (4-HPAA), confirming that oxidative deamination by MAO-A is the quantitatively most relevant metabolic pathway. Systemic exposure of 4-HPAA varied only up to 3-fold, indicating no strong heritable variation in peripheral MAO-A activity. Systolic blood pressure increased by more than 10 mmHg in 71% of the volunteers and correlated strongly with systemic tyramine concentration. In less than 10% of participants, individually variable blood pressure peaks by >40 mmHg above baseline were observed at tyramine concentrations of >60 µg/l. Unexpectedly, the functionally relevant polymorphisms in OCT1 and CYP2D6, including the CYP2D6 poor and ultra-rapid metaboliser genotypes, did not significantly affect tyramine pharmacokinetics or pharmacodynamics. Also, the MOA-A genotypes, which had been associated in several earlier studies with neuropsychiatric phenotypes, had no significant effects on tyramine pharmacokinetics or its metabolism to 4-HPAA. Thus, variation in tyramine pharmacokinetics and pharmacodynamics is not explained by obvious genomic variation, and human tyramine metabolism did not indicate the existence of ultra-low or -high MAO-A activity.

The pharmacodynamic profile of “Blackadder” blackcurrant juice effects upon the monoamine axis in humans: A randomised controlled trial

Emerging evidence from human intervention trials indicates health benefits of consuming blackcurrant fruit, including improvements to cognitive performance, modulation of blood flow, regulation of blood glucose and inhibition of enzymes underpinning normal cognitive function. Of particular relevance is our previous demonstration of monoamine oxidase (MAO)-A and B inhibition after the consumption of a New Zealand “Blackadder” blackcurrant juice in humans. The current study uses a double-blind, placebo-controlled, randomised cross- over design to assess the pharmacodynamics of the effects on platelet MAO-B inhibition and associated substrates, plasma prolactin levels and blood glucose levels after consumption of a single serve of “Blackadder” blackcurrant juice standardised to 500 mg polyphenols. Eight healthy male (20-–35 years) participants completed the trial. Measurements were obtained at baseline 15, 30, 45, 60, 100, 120, 150, 180, 240 mins and 24 h post dose. A fast, absolute and reversible inhibition of blood platelet MAO-B (P < 0.001) and a significant but delayed reduction in plasma prolactin (P < 0.001) were observed following the consumption of “Blackadder” blackcurrant juice when compared to a placebo control. No interpretable changes in substrates of MAO or associated metabolites were seen. These data provide a clear time course of the reversible inhibition of MAO-B after the single consumption of a of New Zealand “Blackadder” blackcurrant juice standardised at 500 mg of polyphenols and, therefore, provide a therapeutic window on which to base future nutritional interventions.

Drug Repurposing for Duchenne Muscular Dystrophy: The Monoamine Oxidase B Inhibitor Safinamide Ameliorates the Pathological Phenotype in mdx Mice and in Myogenic Cultures From DMD Patients.

Oxidative stress and mitochondrial dysfunction play a crucial role in the pathophysiology of muscular dystrophies. We previously reported that the mitochondrial enzyme monoamine oxidase (MAO) is a relevant source of reactive oxygen species (ROS) not only in murine models of muscular dystrophy, in which it directly contributes to contractile impairment, but also in muscle cells from collagen VI-deficient patients. Here, we now assessed the efficacy of a novel MAO-B inhibitor, safinamide, using in vivo and in vitro models of Duchenne muscular dystrophy (DMD). Specifically, we found that administration of safinamide in 3-month-old mdx mice reduced myofiber damage and oxidative stress and improved muscle functionality. In vitro studies with myogenic cultures from mdx mice and DMD patients showed that even cultured dystrophic myoblasts were more susceptible to oxidative stress than matching cells from healthy donors. Indeed, upon exposure to the MAO substrate tyramine or to hydrogen peroxide, DMD muscle cells displayed a rise in ROS levels and a consequent mitochondrial depolarization. Remarkably, both phenotypes normalized when cultures were treated with safinamide. Given that safinamide is already in clinical use for neurological disorders, our findings could pave the way toward a promising translation into clinical trials for DMD patients as a classic case of drug repurposing.

Utilization of Liver Microsomes to Estimate Hepatic Intrinsic Clearance of Monoamine Oxidase Substrate Drugs in Humans.

Monoamine oxidases (MAOs) are non-CYP enzymes that contribute to systemic elimination of therapeutic agents, and localized on mitochondrial membranes. The aim of the present study was to validate quantitative estimation of metabolic clearance of MAO substrate drugs using human liver microsomes (HLMs). Three MAO substrate drugs, sumatriptan, rizatriptan and phenylephrine, as well as four CYP substrates were selected, and their disappearance during incubation with HLMs or mitochondria (HLMt) was measured. Metabolic clearance (CL) was then calculated from the disappearance curve. CL obtained in HLMs for sumatriptan and a typical MAO substrate serotonin was correlated with that obtained in HLMt among ten human individual livers. Hepatic intrinsic clearance (CLint,vitro) estimated from CL in HLMs was 14-20 and 2-5 times lower than in vivo hepatic intrinsic clearance (CLint,vivo) obtained from literature for MAO and CYP substrates, respectively. Utilization of HLMs for quantitatively assessing metabolic clearance of MAO substrates was further validated by proteomics approach which has revealed that numerous proteins localized on inner and outer membranes of mitochondria were detected in both HLMs and HLMt. CLint,vitro values of MAO substrate drugs can be quantitatively estimated with HLMs and could be used for semi-quantitative prediction of CLint,vivo values.

In vitro monoamine oxidase inhibition potential of alpha-methyltryptamine analog new psychoactive substances for assessing possible toxic risks

Tryptamines have emerged as new psychoactive substances (NPS), which are distributed and consumed recreationally without preclinical studies or safety tests. Within the alpha-methylated tryptamines, some of the psychoactive effects of the prototypical alpha-methyltryptamine (AMT) have been described decades ago and a contributing factor of its acute toxicity appears to involve the inhibition of monoamine oxidase (MAO). However, detailed information about analogs is scarce. Therefore, thirteen AMT analogs were investigated for their potential to inhibit MAO. An in vitro assay analyzed using hydrophilic interaction liquid chromatography–high resolution-tandem mass spectrometry was developed and validated. The AMT analogs were incubated with recombinant human MAO-A or B and kynuramine, a non-selective MAO substrate to determine the IC50 values. The known MAO-A inhibitors 5-(2-aminopropyl)indole (5-IT), harmine, harmaline, yohimbine, and the MAO-B inhibitor selegiline were tested for comparison. AMT and all analogs showed MAO-A inhibition properties with IC50 values between 0.049 and 166μM, whereas four analogs inhibited also MAO-B with IC50 values between 82 and 376μM. 7-Me-AMT provided the lowest IC50 value against MAO-A comparable to harmine and harmaline and was identified as a competitive MAO-A inhibitor. Furthermore, AMT, 7-Me-AMT, and nine further analogs inhibited MAO activity in human hepatic S9 fraction used as model for the human liver which expresses both isoforms. The obtained results suggested that MAO inhibition induced by alpha-methylated tryptamines might be clinically relevant concerning possible serotonergic and adrenergic effects and interactions with drugs (of abuse) particularly acting as monoamine reuptake inhibitors. However, as in vitro assays have only limited conclusiveness, further studies are needed.

Characterization of 1-Aminobenzotriazole and Ketoconazole as Novel Inhibitors of Monoamine Oxidase (MAO): An In Vitro Investigation.

1-Aminobenzotriazole, a known time-dependent inhibitor of cytochrome P450 (CYP) enzymes, and ketoconazole, a strong inhibitor of the human CYP3A4 isozyme, are used as standard probe inhibitors to characterize the CYP and/or non-CYP-mediated metabolism of xenobiotics. In the present investigation, 1-Aminobenzotriazole and ketoconazole are characterized as potent monoamine oxidase (MAO) inhibitors in vitro using mouse, rat and human liver microsomes and S9 fractions. Inhibition potential of 1-aminobenzotriazole and ketoconazole was studied in mice, rat and human liver microsomes, S9 fractions, MAO-A and MAO-B expressed enzymes by monitoring the formation of 4-hydroxyquinoline (4-HQ) from kynuramine, a specific substrate of MAO by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mechanism of MAO inhibition was studied by incubating varying concentration of kynuramine with mouse, rat and human S9 fractions at varying concentration of 1-aminobenzatriazole and ketoconazole and monitoring the formation of 4-HQ. 1-aminobenzotriazole and ketoconazole inhibited both MAO isozymes (MAO-A and MAO-B) with more specificity towards MAO-B. Kynuramine substrate kinetics in mouse, rat and human S9 fractions with varying 1-aminobenzotriazole and ketoconazole concentrations showed decreased maximum rate (V max) for 4-HQ formation without affecting theMichaelis-Menten constant (K m). A non-competitive inhibition model was constructed and inhibition constants (K i) for 1-aminobenzotriazole (7.87±0.61, 8.61±0.92, 65.2±1.61µM for mice, rat and humans, respectively) and ketoconazole (0.12±0.01, 2.04±0.08, 5.52±0.47µM for mice, rat and humans, respectively) were determined. 1-Aminobenzotriazole and ketoconazole are characterized as non-competitive inhibitors of mice, rat and human MAO in vitro and the extent of their MAO inhibition potential is species specific. 1-Aminobenzotriazole or ketoconazole can be used as a probe inhibitor in vitro for screening the involvement of MAO-dependent metabolism of new chemical entities (NCE) in early drug discovery.

MAOA-VNTR polymorphism modulates context-dependent dopamine release and aggressive behavior in males

A recent [18F]FDOPA-PET study reports negative correlations between dopamine synthesis rates and aggressive behavior. Since dopamine is among the substrates for monoamine oxidase A (MAOA), this investigation examines whether functional allelic variants of the MAOA tandem repeat (VNTR) promotor polymorphism, which is known to modulate aggressive behavior, influences dopamine release and aggression in response to violent visual stimuli.We selected from a genetic prescreening sample, strictly case-matched groups of 2×12 healthy male subjects with VNTRs predictive of high (MAOA-High) and low (MAOA-Low) MAOA expression. Subjects underwent pairs of PET sessions (dopamine D2/3 ligand [18F]DMFP) while viewing a movie of neutral content, versus violent content. Directly afterwards, aggressive behavior was assessed by the Point Subtraction Aggression Paradigm (PSAP). Finally, PET data of 23 participants and behavioral data of 22 participants were analyzed due to post hoc exclusion criteria.In the genetic prescreening sample MAOA-Low carriers had significantly increased scores on the Buss–Perry Aggression Questionnaire. In the PET-study-group, aggressive behavior under the emotional neutral condition was significantly higher in the MAOA-Low group. Interestingly, the two MAOA-groups showed inverse dopaminergic and behavioral reactions to the violent movie: The MAOA-High group showed higher dopamine release and increased aggression after the violent movie; MAOA-Low subjects showed decreases in aggressive behavior and no consistent dopamine release.These results indicate a possible impact of the MAOA-promotor polymorphism on the neurobiological modulation of aggressive behavior. However, the data do not support approaches stating that MAOA-Low fosters aggression by a simple pro-dopaminergic mechanism.

Catalytic properties of liver monoamine oxidase in the chum salmon Oncorhynchus keta

The substrate and inhibitory specificity of mitochondrial monoamine oxidase (MAO) in the liver of males of the summer form of the chum salmon Oncorhynchus keta was studied. As to the spectrum of deaminated substrates, the hepatic MAO of the chum salmon is similar to MAO of most terrestrial mammals, for eight classical MAO substrates similarity in their substrate characteristics were found. Analysis of the antimonoamine oxidase activity of two derivaties of 2-propinilamine, five derivatives of acridine as well as of pyronine G revealed significant qualitative and quantitative differences as compared to the hepatic enzyme of tuna and whitefish. The compounds tested manifested themselves as irreversible inhibitors of chum salmon's hepatic MAO possessing various efficacy, but lacking the selectivity of action as dependent on the deaminated substrate. The obtained data on the substrate and inhibitory analysis provide an indirect evidence for the presence of a single molecular form of MAO in the chum salmon liver.

High throughput screening of monoamine oxidase (MAO-N-D5) substrate selectivity and rapid kinetic model generation

Full kinetic models provide insight into enzyme mechanism and kinetics and also support bioconversion process design and feasibility assessment. Previously we have established automated microwell methods for rapid data collection and hybrid kinetic modelling techniques for quantification of kinetic constants. In this work these methods are applied to explore the substrate selectivity and kinetics of monoamine oxidase, MAO-N-D5, from Aspergillus niger. In particular we examine the MAO-N-D5 variant Ile246Met/Asn336Ser/Met348Lys/Thr384Asn to allow the oxidation of secondary amines Initial screening showed that MAO-N-D5 enabled the selective oxidation of secondary amines in 8 and 9 carbon rings, as well as primary ethyl and propyl amines attached to secondary amines of indolines and pyrrolidines. Subsequently we developed a first kinetic model for the MAO-N-D5 enzyme based on the ping-pong bi-bi mechanism (similar to that for the human MAO-A enzyme). The full set of kinetic parameters were then established for three MAO-N-D5 substrates namely; 3-azabicyclo[3,3,0]octane, 1-(2 amino ethyl) pyrrolidine and 3-(2,3-dihydro-1H-indole-1-yl)propan-1-amine. The models for each amine substrate showed excellent agreement with experimentally determined progress curves over a range of operating conditions. They indicated that in each case amine inhibition was the main determinant of overall reaction rate rather than oxygen or imine (product) inhibition. From the perspective of larger scale bioconversion process design, the models indicated the need for fed-batch addition of the amine substrate and to increase the dissolved oxygen levels in order to maximize bioconversion process productivity.

Inhibition of monoamine oxidase (MAO) by α-ethylphenethylamine and N,α-diethylphenethylamine, two compounds related to dietary supplements

Phenethylamines can interact with the metabolic enzyme monoamine oxidase (MAO), which can cause neurochemical dysfunction or changes in drug potency. A methamphetamine analog, N,α-diethylphenethylamine (N,α-DEPEA), was recently discovered in athletic performance-enhancing supplements, along with discovery of its metabolite, α-ethylphenethylamine (AEPEA). In vitro inhibition of human recombinant MAO by AEPEA and N,α-DEPEA was evaluated by measuring the fluorescence of 4-hydroxyquinoline produced from MAO substrate, kynuramine. AEPEA competitively inhibited human recombinant MAO A (Ki = 14.0 µM), which was 17-fold stronger compared to MAO B (Ki = 234 µM). Furthermore, N,α-DEPEA was a weak inhibitor of both MAO A (Ki = 251 µM) and MAO B (Ki = 159 µM). Trends regarding MAO A inhibition were explored among structural analogs, yielding the following ranking: amphetamine (Ki = 5.3 µM), AEPEA (Ki = 14.0 µM), methamphetamine (Ki = 17.2 µM), phentermine (Ki = 196 µM), and N,α-DEPEA (Ki = 251 µM). This study provides important data relating chemical structures and biochemical effects for two emerging compounds associated with dietary supplements.

Determination of Antidepressants Using Monoamine Oxidase Amperometric Biosensors Based on Screen-Printed Graphite Electrodes Modified with Multi-Walled Carbon Nanotubes

Novel amperometric biosensors based on screen-printed graphite electrodes modified with multi-walled carbon nanotubes (MWCNTs) dispersed in various solvents (chitosan, DMF) and immobilized monoamine oxidase (MAO) were developed for the determination of the antidepressants imipramine and afobazole. It was found that afobazole and imipramine inhibited MAO, which allowed these pharmaceuticals to be determined within the concentration range from 0.1 mM to 1 nM by recording the H2O2 oxidation wave when dopamine and epinephrine were used as MAO substrates. The best analytical performance was observed for MAO biosensors modified by MWCNTs in chitosan solution, for which the lower detection limit was 3 – 9 nM. The main active ingredients in melipramine and afobazole preparations were quantified with an RSD of 0.08 using the MAO-based amperometric biosensors.

Определение антидепрессантов амперометрическими моноаминоксидазными биосенсорами на основе графитовых печатных электродов, модифицированных многостенными углеродными нанотрубками

Для определения лекарственных соединений имипрамина и афобазола, обладающих антидепрессивным действием, разработаны амперометрические биосенсоры на основе графитовых печатных электродов модифицированных многостенными углеродными нанотрубками (МУНТ) в различных растворителях (хитозан, ДМФА) и иммобилизованной моноаминооксидазы (МАО). Установлено, что имипрамин и афобазол проявляют свойства ингибиторов МАО, что позволило определять их в концентрационном диапазоне от 0,1 мМ до 1 нМ по волне окисления пероксида водорода при использовании в качестве субстратов дофамина и адреналина. Лучшие аналитические характеристики наблюдались в случае использования МАО-биосенсоров, модифицированных раствором МУНТ в хитозане (нижняя граница определяемых концентраций (3 – 9) нМ). Показана возможность определения основного активного компонента с помощью амперометрических МАО-биосенсоров в лекарственных препаратах «Мелипрамин» и «Афобазол» с погрешностью определения (Sr) на уровне 0.08.