Inhibition of Human Monoamine Oxidase: Biological and Molecular

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Inhibition of Human Monoamine Oxidase: Biological and Molecular Modelling Studies on Selected Natural Flavonoids Simone de Carradori, Maria Concetta Gidaro, Anel Petzer, Giosuè Costa, Paolo Guglielmi, Paola Chimenti, Stefano Alcaro, and Jacobus Petrus Petzer J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03529 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 14, 2016

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Journal of Agricultural and Food Chemistry

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Inhibition of Human Monoamine Oxidase: Biological and

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Molecular Modelling Studies on Selected Natural Flavonoids

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Simone Carradori†,*, Maria Concetta Gidaro‡, Anél Petzer§, Giosuè Costa‡, Paolo Guglielmi⊥,

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Paola Chimenti⊥, Stefano Alcaro‡, Jacobus P. Petzer§

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†

Department of Pharmacy, “G. d’Annunzio” University of Chieti-Pescara, Via dei Vestini 31,

66100 Chieti, Italy ‡

Dipartimento di Scienze della Salute, “Magna Graecia” University of Catanzaro, Campus

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Universitario “S. Venuta”, Viale Europa Loc. Germaneto, 88100 Catanzaro, Italy

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§

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South Africa

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⊥

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00185 Rome, Italy

Centre of Excellence for Pharmaceutical Sciences, North-West University, Potchefstroom 2531,

Department of Drug Chemistry and Technologies, Sapienza University of Rome, P.le A.Moro 5,

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Corresponding author

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*

(S. C.) Tel./Fax: +39 0871 3554583. E-mail: [email protected].

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Abstract

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Naturally occurring flavonoids display a plethora of different biological activities, but emerging

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evidence suggests that this class of compounds may also act as antidepressant agents endowed with

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multiple mechanisms of action in the central nervous system; increasing central neurotransmission,

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limiting the reabsorption of bioamines by synaptosomes and modulating the neuroendocrine and

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GABAA systems. Due to their presence in foods, food-derived products and nutraceuticals, we

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established their role and structure-activity relationships as reversible and competitive human

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monoamine oxidase inhibitors. In addition, molecular modelling studies, which evaluated their

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modes of MAO inhibition, are presented. These findings could provide pivotal implications in the

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quest of novel drug-like compounds and for the establishment of harmful drug-dietary supplement

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interactions commonly reported in the therapy with antidepressant agents.

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Keywords: monoamine oxidase inhibitor, apigenin, taxifolin, diosmetin, naringin, eriocitrin,

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isorhamnetin, hesperidin, quercetin-3-O-glucoside.

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Introduction

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Human monoamine oxidases (hMAO-A and hMAO-B; EC 1.4.3.4), are mitochondrial enzymes

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which oxidatively deaminate monoaminergic neurotransmitters and (potentially harmful) dietary

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monoamines. In the brain, their main role is the regulation of important monoamines such as

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noradrenaline, dopamine, serotonin, and adrenaline. The hMAO-A isoform is present in

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catecholaminergic neurons and degrades sterically hindered amines like serotonin, adrenaline, and

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noradrenaline; hMAO-B is mainly abundant in the periventricular region of the hypothalamus and

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the substantia nigra, where it metabolizes small monoamines like β-phenethylamine and

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benzylamine. Both isoforms use dopamine and p-tyramine as common substrates.1

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Due to their crucial physiological roles, the hMAO-A isoform is an established pharmacological

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target for the therapy of (resistant and atypical) depression, anxiety disorders, dysthymia and major

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depressive disorder, whereas the hMAO-B isoform is a target for the treatment of

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neurodegenerative disorders including Parkinson’s disease and Alzheimer’s disease. Moreover, the

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selective hMAO-B inhibitors have been shown to protect neurons against degeneration by means of

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anti-apoptotic mechanisms, the reduction of reactive oxygen species and the stabilization of the

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mitochondria.2

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A plethora of molecules of natural origin have been shown to inhibit the hMAOs and, in addition,

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different synthetic scaffolds have often been inspired by natural products such as flavonoids,

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coumarins, purine derivatives and alkaloids.3-6 Secondary metabolites from plants are frequently

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used by medicinal chemists in an attempt to design simple small molecules that mimic the

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heterogeneity of scaffolds found in natural products. Moreover, there has been an interest in

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traditional flavonoid-enriched herbal medicines because of their application for the therapy of

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specific disorders.7-11 The exact knowledge of the molecular mechanism of action of several natural

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products, which could be ingested daily through the diet in gram amounts, could be important to

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avoid or limit harmful food–food and drug–food interactions.

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Flavonoids could be classified as plant polyphenolic metabolites responsible of specific effects in

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promoting human health and modulating physiological functions, which depend on the substitution

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pattern and related chemical-physical properties.12-14 Indeed, these compounds are characterized by

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a highly reactive hydroxyl moiety, which acts as a free radical scavenger by means of oxidation to

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less reactive molecule, thus preventing ailments associated to oxidative stress. These compounds

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also trigger important enzymes involved in mitochondrial respiration and chelate divalent metal

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ions. Emerging evidence suggests that flavonoids may possess antidepressant activity by acting via

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multiple mechanisms to increase monoamine neurotransmission in the brain. These mechanisms

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include inhibition of the reabsorption of bioamines by synaptosomes and modulation of the

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neuroendocrine and GABAA systems.15, 16

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Recently, we analyzed the structure-activity relationships of this class of natural compounds as

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MAO inhibitors and investigated their antidepressant-like effects in animal models.17, 18 Generally,

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the increasing number of -OH groups on the aromatic ring of this scaffold decreased the MAO

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inhibitory activity. Moreover, the introduction of a sugar or glucuronic acid at the C7 lowered this

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biological activity. MAO inhibition is strongly dependent on the presence of a (p-OH-

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substituted)phenyl at the C2, unsaturation at the C2-C3 positions of the structure, the possibility of

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establishing hydrophobic interactions, and ring planarity. These polar plant metabolites can cross

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the blood-brain barrier due to their cLogP values ranging from 1.5 to 3.5 and since they were shown

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to induce biological effects directly in the CNS.17

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Among natural flavonoid compounds, we selected the most abundant and representative derivatives

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and evaluated them as potential hMAO inhibitors in an attempt to improve and corroborate SARs

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for their target interaction (Figure 1). Apigenin (1) and diosmetin (2) are 2,5,7-trisubstituted

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flavones which are known inhibitors of human and rat liver MAO.8,

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isorhamnetin (4) possess an additional OH moiety on C3. Moreover, due to numerous reports of the

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MAO inhibition properties of quercetin, we also studied its 3-O-glucosidic derivative, isoquercitrin

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(5), which displayed an IC50 of 11.64 µM for the inhibition of bovine brain MAO-B.17 Lastly, 4 ACS Paragon Plus Environment

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Taxifolin (3) and

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naringin (6), eriocitrin (7), and hesperidin (8) are flavanones characterized by different bulky di-

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glycosidic substituents on C7. After the determination of their MAO inhibitory activity and kinetics

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in vitro, we also performed docking simulations and molecular dynamics studies in order to

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investigate the mechanism of action and ligand-target interactions for these widely distributed

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flavonoids.

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Materials and Methods

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All commercial samples of selected flavonoids (apigenin ≥99%, diosmetin ≥98%, taxifolin ≥98%,

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isorhamnetin ≥99%, isoquercitrin ≥98%, naringin ≥95%, eriocitrin ≥98%, hesperidin ≥97%) and

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reference drugs (harmine 98% and safinamide mesylate salt ≥98%) were purchased by Sigma-

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Aldrich (Milan, Italy).

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Microsomes from insect cells containing recombinant hMAOs (5 mg/mL) were used as sources of

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the MAO enzymes. These isoenzymes, kynuramine, (R)-deprenyl and pargyline were supplied by

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Sigma-Aldrich (St. Louis, MO, USA). The Prism 5 software package (GraphPad Software, La Jolla,

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CA, USA) was chosen for data analyses and for the construction of graphs. Fluorescence

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spectrophotometry was carried out with a Varian Cary Eclipse fluorescence spectrophotometer

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(Agilent Technologies, Santa Clara, CA, USA).

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For the computational studies the following commercial softwares were used: Maestro, version 9.7

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(Schrödinger, LLC, New York, NY); LigPrep, version 2.9 (Schrödinger, LLC, New York, NY);

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Glide, version 6.2 (Schrödinger, LLC, New York, NY); Desmond Molecular Dynamics System,

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version 3.7 (D. E. Shaw Research, New York, NY); Maestro-Desmond Interoperability Tools,

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version 3.7 (Schrödinger, New York, NY); The PyMOL Molecular Graphics System, version

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1.7.0.0 (Schrödinger, LLC, New York, NY).

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Biochemistry

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The measurement of IC50 and Ki values

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IC50 values for the inhibition of hMAO-A and hMAO-B were determined according to the

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published protocol.19 The experimental protocol was performed in 96-well microtiter plates (white) 5 ACS Paragon Plus Environment


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to a final volume of 200 µL. The reactions contained kynuramine (50 µM), different concentrations

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of the test inhibitors (0.003–100 µM) and DMSO (4%) as co-solvent. Potassium phosphate buffer

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(100 mM, pH 7.4, made isotonic with KCl) was used as reaction solvent. The enzymatic reactions

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were initiated with the addition of hMAO-A (0.0075 mg protein/mL) or hMAO-B (0.015 mg

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protein/mL) and, after 20 min incubation at 37 ºC, were terminated with the addition of sodium

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hydroxide (80 µL of 2 N). The MAO-generated metabolite, 4-hydroxyquinoline, was subsequently

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quantitated by fluorescence spectrophotometry (λex = 310; λem = 400 nm). For this purpose, linear

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calibration curves constructed with 4-hydroxyquinoline (0.047–1.56 µM) were employed. After

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fitting the inhibition data to the one site competition model incorporated into the Prism 5 software

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package, the IC50 values were calculated. These are expressed as the mean ± standard deviation

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(SD) of triplicate determinations (three replicates of one concentration range). To calculate Ki

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values for the inhibition of hMAOs, Lineweaver-Burk plots were accomplished. For each inhibitor,

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a set of six plots were constructed using the following inhibitor concentrations: 0, 0.25, 0.5, 0.75,

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1.0, and 1.25 x IC50, respectively. For each plot, eight different concentrations of kynuramine were

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employed (15–250 µM). The enzymatic reactions and fluorometric measurements were conducted

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as previously described with the exception that the concentration of both hMAO-A and hMAO-B

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was 0.015 mg protein/mL. Moreover, these experiments were performed in a final volume of 500

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µL and, after termination with NaOH (400 µL of 2 N), 1 mL water was added and the resulting

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samples were quantitated by fluorescence spectrophotometry (using a 3.5 mL quartz cuvette). The

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Ki values were estimated from replots of the slopes of the Lineweaver-Burk plots versus inhibitor

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concentration (–Ki = x-axis intercept) as well as by global fitting of the inhibition data to the

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Michaelis-Menten equation using the Prism 5 software package.

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Dialysis studies

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Dialysis of mixtures containing the hMAOs and test inhibitors were carried out using Slide-A-Lyzer

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dialysis cassettes (Thermo Scientific, Waltham, MA, USA) with a molecular weight cut-off of 10

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000 and a sample volume capacity of 0.5–3 mL. This protocol has been reported previously.20 The 6 ACS Paragon Plus Environment

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hMAOs (0.03 mg protein/mL) were incubated with the test inhibitors, apigenin (1) and diosmetin

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(2), as well as with the reference irreversible inhibitors, pargyline (IC50 = 13 µM) and (R)-deprenyl

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(IC50 = 0.079 µM), for 15 min at 37 °C. For these experiments, the inhibitor concentrations were 4

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× IC50 and the final volume, 0.8 mL. Control incubations were also evaluated in the absence of

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inhibitor. The incubation mixtures were dialyzed using potassium phosphate buffer (100 mM, pH

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7.4, containing 5% sucrose) as dialysis buffer (80 mL). The dialysis buffer was replaced with fresh

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buffer at 3 h and 7 h after the start of dialysis. Following dialysis, the mixtures were diluted twofold

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with the addition of kynuramine to obtain a substrate concentration equal to 50 µM and an inhibitor

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concentration of 2 × IC50. The final volume of these reactions was 500 µL and the residual hMAO

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activities were measured as described for the measurement of Ki values above. For comparison,

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non-dialyzed incubation mixtures containing the hMAOs and 1 or 2 were maintained at 4 °C for 24

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h, diluted twofold and the residual enzyme activity measured. The hMAO activities are reported as

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the mean ± SD of triplicate determinations. The Kruskal-Wallis test with Dunn’s post hoc test was

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used to determine the statistical differences among the means of the residual enzyme rates. A p

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value < 0.05 is judged as being statistical significantly different. These analyses were performed by

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the Prism 5 software package.

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Molecular modelling

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The X-ray structures of complexes of hMAO-A/harmine and hMAO-B/safinamide were

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downloaded from the Protein Data Bank (PDB)21 with the respective accession codes 2Z5X22 and

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2V5Z23. For each protein model, the “Protein Preparation Wizard” function in Maestro ver. 9.7

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(Schrödinger LLC, New York, NY) of Suite 2014 was applied in order to add hydrogens, assign

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partial charges, build side chains and loops with missing atoms. The co-crystallized inhibitors,

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harmine and safinamide, were used to generate the docking grid box and were then removed prior

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to grid generation in the next step. For each flavonoid (1-8), the 3D SDF file was imported from the

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PubChem24 site into the Maestro GUI 9.7 of the Scrodinger Suite 2014. Both tautomeric and

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protonated forms at pH 7.4 of each flavonoid were considered by the “LigPrep” module, ver. 2.9 7 ACS Paragon Plus Environment


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(Schrödinger). The lowest energy conformations, obtained using the OPLS-2005 force field, were

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used as starting points for the docking simulations carried out by Glide ver. 6.2 (Schrödinger). The

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Desmond Molecular Dynamics System, ver. 3.7 (Schrödinger) with OPLS-2005 force field, was

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used to perform the MD simulations of 100 ns with the NPT ensemble, temperature of 10 K and

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pressure of 1 atm. The Simulation Interactions Diagram utility of Desmond 3.7 was used to display

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the ligand-target interactions during the MD simulations. Finally, the PyMOL molecular graphics

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system, ver. 1.7.0.0 (Schrödinger) was used to visualize the molecules and generate all figures.

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Results and Discussion

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The hMAO inhibitory activities of the naturally occurring flavonoids 1-8 were evaluated using

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recombinant hMAO-A and hMAO-B (Table 1).25 To measure MAO activity the non-specific MAO-

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A and B substrate, kynuramine, was used. In a well-established protocol, the MAO-catalyzed

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oxidation product of kynuramine, 4-hydroxyquinoline, was quantitated by fluorescence

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spectrophotometry.26 By thus measuring MAO activity in the presence of several concentrations of

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each inhibitor, sigmoidal plots (residual MAO activity vs. logarithm of inhibitor concentration)

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were constructed from which IC50 values were extrapolated. All inhibition data were generated with

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the substrate concentration at ~1 × Km. The IC50 values for the inhibition of hMAO-A and hMAO-B

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by the selected flavonoids are collected in Table 1.

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Apigenin (1), the simplest flavonoid within our series, displayed promising hMAO inhibitory

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activity with IC50 values in the low micromolar range for the inhibition of both isoforms. However,

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it possessed slight selectivity for hMAO-A compared to the B isoform. The 3'-hydroxy-4'-methoxy

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derivative, diosmetin (2), inhibited the hMAOs with similar potencies compared to 1. Interestingly,

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the isoform selectivity of 2 is reversed compared to that of 1. Hydroxylation on C3 of the flavonoid

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moiety (4) significantly reduced hMAO-A and hMAO-B inhibition. This reduction of inhibition

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potency may also be attributed to the 4'-hydroxy-3'-methoxy substitution pattern, which differs from

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those of 1 and 2. hMAO inhibitory activity is completely abolished in 3, most likely due to the loss

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of planarity of the structure (absence of the C2-C3 double bond). Flavonoid 5 was inactive (>100 8 ACS Paragon Plus Environment

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µM) against both isoforms, whereas the di-glycosidic derivatives (6-8) exhibited relatively low

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hMAO inhibitory activity compared to the most potent flavonoids of the series, 1 and 2.

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Collectively, all selected natural flavonoids were less potent than the reference drugs, harmine and

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safinamide. As evident from the IC50 values, both safinamide (MAO-B inhibitor) and harmine

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(MAO-A inhibitor) are high potency inhibitors of the respective MAO isoforms. Also evident is the

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fact that these reference inhibitors are highly specific, with safinamide acting as a MAO-B inhibitor

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and harmine as a MAO-A inhibitor.

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To better characterize the inhibition mode of the two most potent flavonoid hMAO inhibitors, 1 and

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2 were incubated with hMAOs at concentrations equal to 4 × IC50 and subsequently dialyzed for 24

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h.27 The irreversible MAO-A and MAO-B inhibitors, pargyline and (R)-deprenyl, were similarly

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incubated and dialyzed, and served as positive controls. As negative control, the hMAOs were

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dialyzed in the absence of inhibitor. After dialysis, the mixtures were diluted twofold to yield

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inhibitor concentrations equal to 2 × IC50, and the residual MAO activities were measured. These

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activities were reported as percentage of the residual activity of the negative control (100%). The

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results of the dialysis experiments showed that 1 and 2 are both reversible hMAO-A and -B

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inhibitors (Figure 2) since dialysis restored the enzyme activity. In this respect, the enzyme

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activities were recovered to 104–130% of the negative control. Conversely, inhibition persists in

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enzyme-inhibitor mixtures that were not dialyzed, with the residual activities at 24–50% of the

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negative control, and similarly, after irreversible inhibition with pargyline and (R)-deprenyl, both

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hMAO-A and -B enzymatic activities were not recovered after dialysis and their residual activities

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remain at 3.2–3.9% of the negative control.

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We also focused our attention on the most active flavonoids of the current study in order to analyze

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their inhibition modes. 1 and 2 were shown to act as competitive hMAO-A and -B inhibitors, thus

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providing additional evidence for their reversible modes of inhibition. For this purpose,

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Lineweaver-Burk plots were constructed for each inhibitor by measuring hMAO-A and hMAO-B

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activities in the absence and presence of 1 and 2. As shown in Figure 3, the sets of Lineweaver9 ACS Paragon Plus Environment


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Burk plots were, in all instances, typical of competitive inhibition since the linear lines of each set

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intersect on the y-axis. Ki values for the inhibition of hMAOs by 1 and 2 were estimated from the

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Lineweaver-Burk plots as well as by global fitting of the inhibition data to the Michaelis-Menten

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equation. These values are given in Table 2.

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Natural and synthetic coumarin derivatives, which are structurally related to flavonoids, are known

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to inhibit the hMAOs28-31 and, in particular the MAO-B isoform. Two X-ray crystal structures of

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hMAO-B co-crystallized with coumarin derivatives are available in the Protein Data Bank21

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(accession codes 2V60 and 2V61). However, in order to perform the docking simulations we

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selected the X-ray crystal structures of the hMAOs in complex with the reference compounds used

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in this study. Therefore, hMAO-A/harmine and hMAO-B/safinamide complexes were obtained

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(accession codes 2Z5X22 and 2V5Z,23 respectively). The results of the modelling studies show that

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1 and 2 bind competitively with the best poses docked into the catalytic site of the enzymes (Figures

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4 and 5). The theoretical results confirmed that 1 is better accommodated in the hMAO-A active

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site than that of hMAO-B, with Gscore values of -9.09 and -8.73 Kcal/mol, respectively. Moreover,

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2 displayed a binding affinity for hMAO-B of -9.21 Kcal/mol, which is higher than the binding

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affinity to hMAO-A (-8.48 Kcal/mol). The reversal of isoform selectivity of 2 compared to that of 1

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may be explained by taking into account the different mechanism of recognition observed during

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the in silico studies. As often remarked, the amino acid residues in the area opposite to the FAD

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cofactor (hMAO-A, I180, N181, F208, S209 and I335 with the corresponding hMAO-B residues,

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L171, C172, I199, S200 and Y326 respectively) define the shapes of the active sites with the

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hMAO-A active binding site being smaller and broader than the elongated and narrower one of

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hMAO-B.32 Therefore, the docking study shows that in hMAO-A, both the flavonoids bind with the

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flavonoid portion oriented toward the FAD cofactor, while the phenyl substituents extend towards

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the active site entrance (Figure 4). In hMAO-B, 2 exhibits a similar positioning, while 1 binds in a

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reversed orientation (Figure 5). The different binding orientations in hMAO-B may explain the

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different isoform selectivity of 1 and 2. 10 ACS Paragon Plus Environment

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As reported in our previous work, bulky natural compounds such as crocin may inhibit the hMAOs

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non-competitively by possibly interacting with an allosteric site of the enzyme surface.33 In this

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study, we observed that the two glycosidic derivatives, 6 and 7, which are modest hMAO inhibitors,

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also bind to a site that is distant from the substrate binding cavity (Table 1 and Figure 6). Therefore,

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molecular dynamics studies were carried out for the complexes of hMAO-A/B with 1 and 2, in

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order to further investigate ligand-target interactions established with residues defining the binding

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pocket, and with 6 and 7 to explore target stabilization by a binding site that is distal from the

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catalytic site.

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The molecular dynamics results suggest that the glycosidic flavonoids do not fit into the binding

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pockets of the hMAOs due to the steric hindrance. Stabilization of complexes is mainly attributed to

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several H-bond contacts involving -OH groups of the sugar portions and the protein residues. In

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contrast, within the hMAOs catalytic sites which are defined by mostly hydrophobic amino acid

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residues, 1 and 2 establish a great number of hydrophobic contacts with key residues as well as

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several H-bond contacts. As a possible explanation for the higher selectivity of 1 for the hMAO-A

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compared to 2, the molecular dynamics results revealed that 1 establishes a great number of H-

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bond/hydrophobic contacts, whereas for 2, which is di-substituted on the phenyl, ligand-target

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interactions are limited due to intramolecular bonding between the ortho -OH and -OCH3 groups

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(Table 1).

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In conclusion, identification of (selective) hMAO inhibitors is of much interest for the therapy of

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central nervous system affecting diseases and to avoid possible interactions with other serotonergic

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drugs (serotonin syndrome) and foods rich in dietary-monoamines (cheese effect). Dietary

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restrictions are strictly mandatory for patients under therapy with MAO inhibitors. For this reason,

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we assessed the hMAO inhibitory properties of the most important and abundant flavone (1-5) and

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flavanone (6-8) derivatives and investigated the binding modes of selected derivatives to the

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hMAOs by molecular modelling studies for the first time. Flavonoids that are active MAO

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inhibitors are abundantly present in many foods. For example, 1 occurs in many common 11 ACS Paragon Plus Environment


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vegetables and fruits such as parsley, grapefruit, orange, and onions, and is also present in plant-

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derived beverages, such as chamomile tea.34 Due to their role as hMAO inhibitors, these natural

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compounds may be useful for the treatment of motor symptoms in the early stage of

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neurodegenerative diseases.35

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Abbreviations

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CNS, central nervous system; hMAO, human monoamine oxidase; SARs, structure-activity

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relationships; PDB, Protein Data Bank; SID, Simulation Interactions Diagram; Gscore, Glide

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scoring function.

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Acknowledgments

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This research was funded by “Progetto di Ateneo Ricerca 2013” (P. Chimenti) and the Interregional

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Research Center for Food Safety and Health at the Magna Græcia University of Catanzaro (MIUR

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PON a3_00359).

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Supporting Information

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Table 1S reports the theoretical affinities of the selected flavonoids for the hMAOs. Figures 1S-4S

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provides summaries of the MD simulations for the complexes of hMAO-A/B with apigenin (1),

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diosmetin (2), naringin (6) and eriocitrin (7). This material is available free of charge via the

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Internet at http://pubs.acs.org.”

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Conflict of interest

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The authors declare no conflicts of interest and that have received no payment for the preparation of

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this manuscript.

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References

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stages. J. Agric. Food Chem. 2010, 58, 3031-3036.

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387

Figure captions

388

Figure 1. Structures of the selected flavonoids (1-8) assayed as hMAO inhibitors.

389

Figure 2. Dialysis restores the activities of the hMAOs following inhibition by (A) apigenin (1) and

390

(B) diosmetin (2). Dialysis, however, does not restore the hMAO activities following inhibition by

391

the irreversible inhibitors, pargyline (parg) and (R)-deprenyl (depr). NI, no inhibitor. *Statistical

392

significantly different from the mean of inhibitor–dialyzed. The values are given as mean ± SD of

393

triplicate determinations.

394

Figure 3. Lineweaver-Burk plots for the inhibition of hMAO-A and hMAO-B by (A) apigenin (1)

395

and (B) diosmetin (2). Also shown as insets are replots of the slopes of the Lineweaver-Burk plots

396

versus inhibitor concentration.

397

Figure 4. Best orientations into the hMAO-A catalytic site of (A) apigenin (1) and (B) diosmetin

398

(2), displayed as orange and violet sticks, respectively. The FAD cofactor is shown grey sticks

399

while the amino acid residues that are involved in ligand-target interactions are shown as grey lines.

400

The hMAO proteins are represented as grey surfaces and cartoons. All non-carbon atoms are

401

colored according to atom types.

402

Figure 5. Best orientations into the hMAO-B catalytic site of (A) apigenin (1) and (B) diosmetin

403

(2), displayed as orange and violet sticks, respectively. The FAD cofactor is shown as grey sticks

404

while the amino acid residues that are involved in ligand-target interactions are shown as grey lines.

405

The hMAO proteins are represented as grey surfaces and cartoons. All non-carbon atoms are

406


407

Figure 6. Molecular recognition of (A) naringin (6) and (B) eriocitrin (7), shown as blue and yellow

408

sticks, respectively, by a binding site distal from the catalytic site of the hMAOs. The hMAO

409

proteins are displayed as transparent surfaces and the FAD cofactors are shown as grey sticks in

410

hMAO-A and cyan sticks in hMAO-B, respectively. All non-carbon atoms of the ligands are

411


412 17 ACS Paragon Plus Environment


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413

Table 1. IC50 Values for the Inhibition of hMAO-A and hMAO-B by Selected Flavonoids 1-8 and

414

Reference Drugs. Compound 1 2 3 4 5 6 7 8 Harmine Safinamide

hMAO-A inhibition (µM)* 1.55 ± 0.147 5.74 ± 0.571 > 100 64.2 ± 7.69 > 100 33.3 ± 7.05 86.5 ± 32.4 > 100 0.0029 ± 0.00042 112 ± 5.23

hMAO-B inhibition (µM)* 5.16 ± 0.410 1.58 ± 0.887 > 100 21.2 ± 4.99 > 100 µM 44.6 ± 11.2 164 ± 39.2 > 100 >100 0.048 ± 0.0047

*The values are given as mean ± SD of triplicate determinations.


Page 19 of 26


Table 2. Ki Values for the Inhibition of hMAOs by Apigenin (1) and Diosmetin (2).

1 2

Ki determined from Lineweaver-Burk plots (µM) hMAO-A hMAO-B 1.15 4.91 4.60 1.43

Ki determined by global fitting to MichaelisMenten equation (µM) hMAO-A hMAO-B 0.901 ± 0.040 (r2 = 0.99) 3.48 ± 0.266 (r2 = 0.99) 4.69 ± 0.259 (r2 = 0.99) 0.860 ± 0.056 (r2 = 0.99)



Page 20 of 26

OH

OH OH HO

OH

OCH3

O

O

HO

O

HO

OH OH O

OH O

OH O Diosmetin (2)

Apigenin (1)

Taxifolin (3)

OCH3 OH HO

OH OH HO

O

O OH O O HO

OH OH O

OH O

OH OH

Quercetin-3-O-ß-D-glucoside (isoquercitrin) (5)

Isorhamnetin (4)

OH OH OH

CH3 HO

O

HO

O

HO HO HO HO

O

OH O O

O

OH

O

O OH

O

HO

OH

O

O

H3C

OH O

OH OH

Naringin (6)

Eriocitrin (7) OH

HO HO H3C

O

OH

O HO HO

OCH3 O

O

OH OH O Hesperidin (8)

415

Figure 1 20 ACS Paragon Plus Environment

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A

MAO-B

MAO-A 150 125 100

100

Rate (%)

Rate (%)

125

75 50

50 25

25

*

0

NI

1

parg

dialysed

B

75

*

0

NI

1

1

depr

dialysed

undialysed

MAO-A

1 undialysed

MAO-B 125

100

Rate (%)

Rate (%)

100

75

50

25

75 50 25

*

0

NI

2

parg

dialysed

*

0

2

NI

undialysed

2

depr

dialysed

2 undialysed

416 Figure 2



A

1200

800

100

800 400

400

75 −2

−1

0 [I], µ M

1

50

25

0

0

0.00

0.02 0.04 1/[S]

−0.02

0.06

0 2 [I], µ M

4

6

50

25

−0.02

−4 −2

2

1/V (%)

1/V (%)

75

Slope

Slope

1200

100

Page 22 of 26

0.00

0.02 0.04 1/[S]

0.06

1 (MAO-B)

1 (MAO-A)

B

1/V (%)

75

800

100

400 −4

−1

2 [I], µ M

5

75

8

50

25

0

0 0.00

0.02 0.04 1/[S]

0.06

800 400

−2

−1

0 1 [I], µ M

2

50

25

−0.02

Slope

1200

1/V (%)

100

Slope

1200

−0.02

0.00

2 (MAO-A)

0.02 0.04 1/[S]

0.06

2 (MAO-B)

417 Figure 3 418


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419

Figure 4



Page 24 of 26

420

Figure 5


Page 25 of 26


Figure 6



Page 26 of 26

For Table of Contents Only SAR regarding MAO inhibition of the selected flavonoids

Substitution with sugars decreased or abolished hMAO inhibitory activity

Glycoside O

R1 R2

Mono-substitution led to hMAO-A selectivity; disubstitution led to hMAO-B selectivity

O OH OH O

Insaturation is important; introduction of -OH group decreased hMAO inhibitory activity


Inhibition of Human Monoamine Oxidase: Biological and Molecular

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