Selective Interactions of O-Methylated Flavonoid Natural Products with Human Monoamine Oxidase-A and -B

A set of structurally related O-methylated flavonoid natural products isolated from Senecio roseiflorus (1), Polygonum senegalense (2 and 3), Bhaphia macrocalyx (4), Gardenia ternifolia (5), and Psiadia punctulata (6) plant species were characterized for their interaction with human monoamine oxidases (MAO-A and -B) in vitro. Compounds 1, 2, and 5 showed selective inhibition of MAO-A, while 4 and 6 showed selective inhibition of MAO-B. Compound 3 showed ~2-fold selectivity towards inhibition of MAO-A. Binding of compounds 1–3 and 5 with MAO-A, and compounds 3 and 6 with MAO-B was reversible and not time-independent. The analysis of enzyme-inhibition kinetics suggested a reversible-competitive mechanism for inhibition of MAO-A by 1 and 3, while a partially-reversible mixed-type inhibition by 5. Similarly, enzyme inhibition-kinetics analysis with compounds 3, 4, and 6, suggested a competitive reversible inhibition of MAO-B. The molecular docking study suggested that 1 selectively interacts with the active-site of human MAO-A near N5 of FAD. The calculated binding free energies of the O-methylated flavonoids (1 and 4–6) and chalcones (2 and 3) to MAO-A matched closely with the trend in the experimental IC50′s. Analysis of the binding free-energies suggested better interaction of 4 and 6 with MAO-B than with MAO-A. The natural O-methylated flavonoid (1) with highly potent inhibition (IC50 33 nM; Ki 37.9 nM) and >292 fold selectivity against human MAO-A (vs. MAO-B) provides a new drug lead for the treatment of neurological disorders.


Introduction
Monoamine oxidases (EC. 1.4.3.4; MAO-A and -B) are FAD-dependent enzymes that are responsible for the metabolism of neurotransmitters such as dopamine, adrenaline, serotonin, and noradrenaline, and also for the inactivation of exogenous arylalkyl amines [1][2][3]. Due to their vital role in neurotransmitter metabolism, these enzymes signify attractive drug targets for the pharmacological therapy of neurodegenerative diseases and neurological disorders [4][5][6][7]. Recent efforts toward the

Isolation, Purification, and Characterization of O-Methylated Flavonoids
The O-methylated flavonoids reported in this paper were isolated from various plants, using general methods reported earlier [25,27]. Aerial parts (leaves and branches) were dipped in a non-polar solvent for short periods to wash off the exudates into the solvent (without affecting cell vacuole compounds). The solvents used were normally medium polarity solvents such as acetone or ethyl acetate. The solvent was removed using a rotary evaporator and the remaining solid materials were subjected to column chromatography using silica gel as a stationary phase and eluting with hexane/dichloromethane in a gradient fashion continuously increasing polarity followed by dichloromethane/methanol. Compounds 1-6 were isolated from different plants, namely, S. roseiflorus (1) [24], P. senegalense (2 and 3) [25], G. ternifolia (5) [27], P. punctulata (6) [28], and B. macrocalyx (4). Compound 4 is the first report from the genus Baphia. The isolation of compound 4 was not reported in the literature by us, therefore, its structure was determined using 1D and 2D NMR spectral data and TOF-MS (see Material and Methods section for details).

Enzyme Inhibition and Kinetics Mechanism of MAO-A and -B with Compounds 1-6
The inhibition (IC 50 ) of the MAO-A and -B enzymes by compounds 1-6 are shown in Table 1. Compounds 1, 2, and 5 showed selective potent inhibition of MAO-A compared to compound 3, which was potent at MAO-A but only slightly selective for MAO-A over -B. Compounds 4 and 6 were more potent than 3 at MAO-B and were selective for MAO-B over -A.  Furthermore, the MAO-A inhibition mechanisms of compounds 1-3 and 5 were studied, using varying concentrations of kynuramine, a nonselective substrate, to investigate the nature of inhibition of the enzymes. Based on dose-response inhibition results, at least two concentrations of 1-3 and 5 were selected for the inhibition kinetics assay-one below and another above the IC 50 value. Three sets of assays were completed at varying concentrations of the substrate for each experiment, one control without inhibitor and the others with two different concentrations of the inhibitor. The data were evaluated by double reciprocal Lineweaver-Burk plots for determination of the Ki (i.e., inhibition/binding affinity) values. Binding of compounds 1-3 and 5, with human MAO-A, yielded the Km value (i.e., the affinity of the substrate for the enzyme) as well as Vmax (maximum enzyme activity) (Figure 2A-D). Ki values were computed from the double reciprocal plots ( Table 2). Binding of compounds 3, 4, and 6 to human MAO-B yielded the Km value (i.e., the affinity of the substrate for the enzyme) as well as Vmax (maximum enzyme activity) ( Figure 3A-C). Ki values were computed from the double reciprocal plots (Table 2). Compounds 3, 4, and 6 showed inhibitory activity of MAO-B with substantially high affinity (Ki = 1.242, 0.809, and 0.874 µM, respectively) ( Table 2).
The results are presented as the mean ± SD of three observations; b Clorgyline and L-deprenyl were sed as positive controls for MAO-A and -B, respectively.

Binding and Time-Dependent Assays of MAO-A and -B with Compounds 1-6
The characteristics of binding of compounds 1-3 and 5 with MAO-A were investigated by the equilibrium-dialysis assay. High concentrations of the compounds 1-3 and 5 (10.0, 25.0, 25.0, and 100.0 μM, respectively) were incubated with the MAO-A enzyme for 20 min and the resulting enzyme-inhibitor-complex preparation was dialyzed overnight against the 0.025 M phosphate buffer (pH 7.4). The activities of the enzyme were analyzed before and after the dialysis ( Figure 4). The binding of compounds 1-3 with MAO-A was reversible and compound 5 showed partial reversibility ( Table 2). Incubation of MAO-B with compounds 3, 4, and 6 (50.0, 50.0, and 50.0 μM, respectively) produced more than 60% inhibition of activity, and 80% of the activity of the enzyme was recovered after dialysis ( Figure 5). Thus, the binding of compounds 3, 4, and 6 with MAO-B was reversible ( Table 2). The selective MAO-B inhibitor deprenyl was confirmed to bind irreversibly with the enzyme ( Table 2).   Table 2). Incubation of MAO-B with compounds 3, 4, and 6 (50.0, 50.0, and 50.0 µM, respectively) produced more than 60% inhibition of activity, and 80% of the activity of the enzyme was recovered after dialysis ( Figure 5). Thus, the binding of compounds 3, 4, and 6 with MAO-B was reversible ( Table 2). The selective MAO-B inhibitor deprenyl was confirmed to bind irreversibly with the enzyme ( Table 2).

Binding and Time-Dependent Assays of MAO-A and -B with Compounds 1-6
The characteristics of binding of compounds 1-3 and 5 with MAO-A were investigated by the equilibrium-dialysis assay. High concentrations of the compounds 1-3 and 5 (10.0, 25.0, 25.0, and 100.0 μM, respectively) were incubated with the MAO-A enzyme for 20 min and the resulting enzyme-inhibitor-complex preparation was dialyzed overnight against the 0.025 M phosphate buffer (pH 7.4). The activities of the enzyme were analyzed before and after the dialysis ( Figure 4). The binding of compounds 1-3 with MAO-A was reversible and compound 5 showed partial reversibility ( Table 2). Incubation of MAO-B with compounds 3, 4, and 6 (50.0, 50.0, and 50.0 μM, respectively) produced more than 60% inhibition of activity, and 80% of the activity of the enzyme was recovered after dialysis ( Figure 5). Thus, the binding of compounds 3, 4, and 6 with MAO-B was reversible ( Table 2). The selective MAO-B inhibitor deprenyl was confirmed to bind irreversibly with the enzyme ( Table 2).

Computational Analysis of Enzyme-Inhibitor Interactions
A molecular docking study was performed to understand the binding pose and interaction profiles of compounds 1-6 to MAO-A and -B. Schrӧdinger's Induced-Fit docking protocol was adopted to consider the optimal geometry of the protein-ligand complex after conformational changes induced by the bound ligand. The GlideScores and binding free-energies of compounds 1-6 in the active sites of the hMAO-A and hMAO-B X-ray crystal structures are presented in Table 3. The docking protocol used in this study was validated by self-or native-docking. The native ligands, harmine and pioglitazone, were extracted from the X-ray structures of MAO-A and -B, respectively, and docked into their corresponding protein models. The calculated RMSD between the docked and experimental poses were found to be identical <0.6 Å , which verified the suitability of the docking method for the current study. The putative binding mode and interactions of the best compounds with the X-ray crystal structures of MAO-A and -B are presented in Figure 7. The calculated binding free energies vary between 28-76 kcal/mol against MAO-A and -B. Since some of the measured Ki values are in the micromolar range, the binding affinities should be somewhere around 6-10

Computational Analysis of Enzyme-Inhibitor Interactions
A molecular docking study was performed to understand the binding pose and interaction profiles of compounds 1-6 to MAO-A and -B. Schrӧdinger's Induced-Fit docking protocol was adopted to consider the optimal geometry of the protein-ligand complex after conformational changes induced by the bound ligand. The GlideScores and binding free-energies of compounds 1-6 in the active sites of the hMAO-A and hMAO-B X-ray crystal structures are presented in Table 3. The docking protocol used in this study was validated by self-or native-docking. The native ligands, harmine and pioglitazone, were extracted from the X-ray structures of MAO-A and -B, respectively, and docked into their corresponding protein models. The calculated RMSD between the docked and experimental poses were found to be identical <0.6 Å , which verified the suitability of the docking method for the current study. The putative binding mode and interactions of the best compounds with the X-ray crystal structures of MAO-A and -B are presented in Figure 7. The calculated binding free energies vary between 28-76 kcal/mol against MAO-A and -B. Since some of the measured Ki values are in the micromolar range, the binding affinities should be somewhere around 6-10

Computational Analysis of Enzyme-Inhibitor Interactions
A molecular docking study was performed to understand the binding pose and interaction profiles of compounds 1-6 to MAO-A and -B. Schrödinger's Induced-Fit docking protocol was adopted to consider the optimal geometry of the protein-ligand complex after conformational changes induced by the bound ligand. The GlideScores and binding free-energies of compounds 1-6 in the active sites of the hMAO-A and hMAO-B X-ray crystal structures are presented in Table 3. The docking protocol used in this study was validated by self-or native-docking. The native ligands, harmine and pioglitazone, were extracted from the X-ray structures of MAO-A and -B, respectively, and docked into their corresponding protein models. The calculated RMSD between the docked and experimental poses were found to be identical <0.6 Å, which verified the suitability of the docking method for the current study. The putative binding mode and interactions of the best compounds with the X-ray crystal structures of MAO-A and -B are presented in Figure 7. The calculated binding free energies vary between 28-76 kcal/mol against MAO-A and -B. Since some of the measured Ki values are in the micromolar range, the binding affinities should be somewhere around 6-10 kcal/mol. This is a known limitation of the employed computations, which are useful not on an absolute scale but in relative terms among structurally similar ligands, which is the focus here Compound 1 exhibited a strong binding affinity to the MAO-A receptor in terms of GlideScore and binding free energy (∆G = −57.522 kcal/mol) compared to the other compounds. The p-methoxy phenyl at the C-2 position (Ring-B) of 1 showed π-π stacking with Phe208 and was surrounded by an array of hydrophobic residues, including Leu97, Phe108, Ala111, Ile180, and Ile325. The hydroxyl at C-5 of ring A formed H-bonding with N5 and C=O of FAD and the hydroxyl at C-7 of ring A exhibited water-mediated H-bonding with Tyr444. In addition, Ring A was surrounded by strong hydrophobic residues Tyr69, Tyr197, Tyr407, and Tyr444. The best GlideScore and binding free energy matched well with the experimental binding affinity of 1. Interestingly, 1 and 5 had the difference of -OCH 3 and -OH, respectively, at the C-3 position of ring C but had significant differences in their MAO-A binding affinity. Our docking results also predicted relatively poor GlideScores and binding free energy for compound 5 compared to compound 1. After careful observation, we have found that the methoxy group at C-3 of 1 exhibited strong hydrophobic interactions with Ile335 and Leu337 compared to the hydroxyl group at C-3 of 5. Compounds 1 and 5 have very similar poses; however, compound 1 slightly shifted towards FAD (~1.5 Å) compared to 5. In addition, the hydroxyl at Ring A of 5 did not show any H-bonding with FAD, further helping to explain the poorer binding affinity of compound 5 for MAO-A. Interestingly, compound 1 showed better GlideScore and binding free energy for MAO-B than for MAO-A (see Table 3); however, its best-ranked docking pose left it 15 Å away from the N5 of FAD, which apparently is an unrealistic docking prediction. In the search for an alternative pose for 1 to MAO-B, we found a pose in which 1 fit into the active site of MAO-B (near N5 of FAD) with a GlideScore of −9.705 kcal/mol, and that is the one reported in Table 3. The substitutions of acetyl (-CH 3 CO) and methylsulfone (-SO 2 CH 3 ) at the C-3 and C-4 of Ring B of 1 are predicted to enhance the affinity towards MAO-A. The poly-substituted methoxy group at Ring B caused a loss of binding affinity towards MAO-A but submicromolar activity towards MAO-B.  a The data are mean ± SD of three observations.; b The best pose is 15 Å away from the N5 of FAD (substrate active site); the numbers given here are for an alternate pose (see text); c ND = Not determined.

Discussion
The molecules with reversible selective inhibition of MAO-A or MAO-B have therapeutic potential for the treatment of neurological and psychiatric disorders, especially caused due to The next structural category, chalcone, represented by compounds 2 and 3, was analyzed. Compound 2 showed the more negative binding free energy (∆G = −47.724 kcal/mol) compared to compound 3 (∆G = −37.683 kcal/mol) for binding to MAO-A, and these data match closely with the experimental binding affinities (cf. Table 1). The only structural difference between compounds 2 and 3 involves C-4 carrying methoxy and hydroxyl moieties, respectively. The docked pose of 2 in the MAO-A receptor showed H-bonding of its hydroxyl moiety at C-6 and its C-1 carbonyl (water-mediated H-bonding) with Asn181. In addition, the oxygen of the methoxy at C-4 exhibited water-mediated H-bonding with Gln215 and Tyr444. The major difference of binding free energy between 2 and 3 was because of an additional strong hydrophobic interaction (CH . . . C, C . . . C, and CH . . . π) of the C-4 methoxy group of 2 with Tyr69, Phe352, and Tyr407, respectively.
The GlideScores and binding free energies of the flavonoids 4 and 6 showed a better binding affinity for interaction with MAO-B (4: GlideScore = −10.225 kcal/mol, ∆G = −53.574 kcal/mol and 6: GlideScore = −11.191 kcal/mol, ∆G = −68.053 kcal/mol) than with MAO-A. Compounds 4 and 6 docked in a very similar orientation and showed H-bonding interactions between their C-4 carbonyl and Cys172. In addition, the C-5 hydroxyl of 4 and 6 showed water-mediated H-bonding with Tyr188, and Gln206 and the C-4 hydroxyl had direct hydrogen-bonding with the backbone carbonyl of Cys172. Ring A had an orientation towards the isoalloxazine ring of FAD and was surrounded by an array of hydrophobic residues, including Tyr60, Phe343, Tyr398, and Tyr435. Furthermore, Ring B was surrounded by hydrophobic residues Leu164, Leu167, Phe168, Ile199, Ile316, and Tyr326 (including π-π stacking for Tyr326). Overall, the docking results of compounds 1-6 were in good agreement with the experimental binding data for MAO-A and -B.

Discussion
The molecules with reversible selective inhibition of MAO-A or MAO-B have therapeutic potential for the treatment of neurological and psychiatric disorders, especially caused due to depletion of neurotransmitter biogenic amines [9,29,30]. Previous studies from our lab have reported selective inhibition of human MAO-B with flavonoid natural products [18][19][20]. A recent study has also reported MAO-A and MAO-B inhibition activity by acacetin 7-O-(6-O-malonylglucoside), a derivative of acacetin isolated from Agastache rugosa plant leaves [31]. The follow-up studies presented here with a select set of O-methylated flavonoids (1-6) identified MAO inhibitors selective against both MAO-A (1-3 and 5) and MAO-B (4 and 6). Compounds 1-3 interact with MAO-A through reversible binding as assessed by the enzyme-inhibitor complex equilibrium dialysis assay, while the binding of compound 5 with MAO-A was partially reversible. The inhibition of MAO-A by compounds 1-3 and 5 was not time-dependent. A recent paper reported MAO-A and -B inhibition activity by natural constituent acacetin 7-O-(6-O-malonylglucoside) that was isolated and purified from Agastache rugosa plant leaves [32]. Compounds 3, 4, and 6 also interact with MAO-B reversibly, as assessed by the enzyme-inhibitor complex equilibrium dialysis assay, and the inhibition was not time-dependent.
Computational analysis of the binding of 1-6 with human MAO-A and -B revealed the putative binding mode and interaction profiles of the compounds with MAO-A and -B. Among all the O-methylated flavonoids, 1 showed the strongest computed interaction with MAO-A and exhibited H-bonding with N5 and C=O of FAD through the hydroxyl at C-5 of Ring A. In addition, the hydroxyl at C-7 of Ring A also exhibited water-mediated H-bonding with Tyr444. 1 also showed π-π interactions with Phe208 and was surrounded by an array of hydrophobic residues. On the other hand, 4 and 6 showed strong interactions with MAO-B and shared an identical binding mode with MAO-B. This study suggests that it would be worthwhile to perform further evaluation of compounds 1-6 including considering the effects of their MAO-A and -B inhibitory actions in experimental animal models of neurological and/or neurodegenerative disorders.
The O-methylated flavonoids are predominant bioactive secondary metabolites present in several plants [32]. The O-methylated flavonoids are generated in plants through the action of specific O-methyltransferase (OMT) enzymes [33]. O-methylation changes the solubility of flavonoids and improves bioactive properties compared to their non-methylated counterparts [34]. The natural product O-methylated flavonoid 3,4 -di-O-methylkaempferol (1) isolated from S. roseiflorus was identified as a highly potent inhibitor of human MAO-A with IC 50 and Ki values of 33 nM and 37.8 nM, respectively. The metabolite 1 was more than 292-fold selective for MAO-A over MAO-B. The compound formed a reversible enzyme-inhibitor complex and was had a very low Ki for MAO-A. With its highly potent MAO-A inhibition and extraordinary selectivity for human MAO-A over MAO-B, 1 is worth optimizing further as a new-drug lead and merits advancement to preclinical evaluations regarding utility for the treatment of neurological and psychiatric disorders.

MAO Inhibition Assay
In this study, we have investigated the effect of the isolated constituents (1-6) on human recombinant MAO-A and -B. The kynuramine oxidation deamination assay was performed in 96-well plates as previously reported, with modification [18,35]. A fixed concentration of kynuramine substrate and varying concentrations of test compounds or inhibitor were used to determine the IC 50 values. Kynuramine concentrations for MAO-A and -B were 80 µM and 50 µM, respectively. The concentrations of compounds 1-6 varied from 0.001 µM to 100 µM for the rhMAO-A and -B enzyme activity inhibition. The test compounds 1-6 were dissolved in DMSO, diluted in the buffer solution just before the assay, and pre-incubated with the enzyme for 10 min at 37 • C. The final concentration of DMSO in the enzyme-assay reaction mixtures did not exceed 1%. The enzymatic reactions were initiated by the addition of MAO-A (5 µg/mL) or -B (12.5 µg/mL), and incubated for 20 min at 37 • C. The enzyme reactions were terminated by the addition of 78 µL of 2N NaOH. The formation of 4-hydroxyquinoline (the enzyme reaction end product) was recorded fluorometrically on a SpectraMax M5 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA) with an excitation (320 nm) and emission (380 nm) wavelength, using the Soft MaxPro-6 program. The inhibition effects of enzyme activity were calculated as the percent of product formation compared to the corresponding controls (enzyme-substrate reaction) without inhibitors. The assay controls, to define the interference of the test compounds with the fluorescence measurements, were set up simultaneously, and the enzyme or the substrate was added after stopping the reaction.

Determination of IC 50 Values
The enzyme assays were performed at a fixed concentration of the substrate kynuramine (80 µM for MAO-A and 50 µM for MAO-B) and different concentrations of the test compounds (1)(2)(3)(4)(5)(6). The dose-response enzyme-inhibition curves were generated using Microsoft ® Excel and the IC 50 values were computed with XLfit ® .

Enzyme Kinetics and Mechanism Studies
For determination of the binding affinity of the inhibitor (Ki) to MAO-A and -B, the enzyme assays were carried out at different concentrations of kynuramine substrate (1.90 µM to 500 µM) and varying concentrations of the inhibitors/compound. The flavonoids (1)(2)(3)(4)(5)(6) were tested at 0.030-0.100 µM for MAO-A and 0.100-0.500 µM for -B. The controls without inhibitor were also run simultaneously. The results were analyzed by SigmaPlot version 10 using standard double reciprocal Lineweaver-Burk plots for computing Km and Vmax values, which were further analyzed to determine the Ki values [18,36,37].

Analysis of Binding of Inhibitor with The Enzymes
Enzyme-inhibitors mostly produce inhibition of the target enzyme through the formation of an enzyme-inhibitor complex. The formation of the enzyme-inhibitor complex may be accelerated in the presence of a high concentration of the test inhibitor. The property of binding of test compounds to MAO-A or -B was determined by the formation of the enzyme-inhibitor complex by incubation of the enzyme with a high concentration of the test compound. This was followed by extensive equilibrium dialysis of the enzyme-inhibitor complex. Recovery of catalytic activity of MAO-A and -B was determined before and after the dialysis. The MAO-A enzyme (0.2 mg/mL protein) was incubated with each test compound: 1 (10.0 µM), 2 (25.0 µM), 3 (25.0 µM), 5 (100.0 µM) and clorgyline (0.100 µM), in 1 mL of potassium phosphate buffer (100 mM, pH 7.4). After 20 min incubation at 37 • C, the reaction was stopped by chilling the tubes in an ice bath. Similarly, the MAO-B enzyme (0.2 mg/mL protein) was incubated with each test compound: 3 (50.0 µM), 4 (50.0 µM), 6 (50.0 µM), and deprenyl (0.500 µM), in 1.0 mL potassium phosphate buffer (100 mM, pH 7.4). After 20 min incubation at 37 • C, the reaction was stopped by chilling the tubes in an ice bath. All the samples with enzyme-inhibitor complex were individually dialyzed against potassium phosphate buffer (25 mM; pH 7.4) at 4 • C for 16-18 h (including three buffer changes). The control enzyme (without inhibitor) was also run through the same procedure and the activity of the enzyme was determined before and after the dialysis [36].

Time-Dependent Inhibition of the Enzyme
To investigate if the binding of the inhibitor with MAO-A and -B followed time-dependent inhibition kinetics, the enzyme was pre-incubated with the inhibitor for different time periods (0-15 min). The compound concentrations used to test time-dependent inhibition were: 1 (0.20 µM), 2 (1.6 µM), 3 (3.0 µM) and 4 (16.0 µM) and clorgyline (0.010 µM), with MAO-A (5.0 µg/mL). The inhibitor concentrations used to test time-dependent inhibition were: 3 (3.0 µM), 4 (1.0 µM), 6 (2.0 µM), and deprenyl (0.070 µM), with MAO-B (12.5 µg/mL). The controls without inhibitors were also run simultaneously. The activities of the MAO-A and -B enzymes were determined as described above and the percentage of enzyme activity remaining was plotted against the pre-incubation time to determine time-dependent inhibition.

Computational Analysis of The Interaction of Test Compounds with MAO-A or -B
The X-ray crystal structures of MAO-A (PDB ID: 2Z5X) and MAO-B (PDB ID: 4A79 [38] were directly imported from the Protein Data Bank website (https://www.rcsb.org) to Maestro [39] using the Protein Preparation Wizard module of the Schrödinger software (Cambridge, MA, USA) [40]. We followed a similar method and protocol for docking as previously described [19]. The protein structures of MAO-A and -B were each used as monomers in the docking study. In brief, these proteins were prepared by adding hydrogens, adjusting bond orders, adding missing side chains, setting the proper ionization states at pH 7.4, refining overlapping atoms, and making H-bond assignments using PROPKA at pH 7.0. The water molecules beyond 5 Å from the co-crystallized ligands were deleted and the protonation states of the co-crystallized ligands were generated using Epik at pH 7.4. During the refinement process, water molecules with fewer than two H-bonds to non-waters were also removed and, finally, restrained minimization of hydrogens only was performed using the Optimized Potentials for Liquid Simulations 3 (OPLS3) force field [41]. The cofactor FAD was not removed during protein preparation and docking. The 2D structures of compounds 1-6 were sketched in the 2D sketcher module of Maestro, prepared, and energy-minimized at a physiological pH of 7.4 using the LigPrep module [42] of the Schrödinger software. The compounds were docked as neutral molecules. The OPLS3 force field was used for protein and ligand preparation, and docking. The active sites of the MAO-A and -B proteins were generated using the centroid of the co-crystallized ligands of 2Z5X and 4A79, respectively. The Induced Fit docking [43] protocol was used for the docking of compounds 1-6. The standard precision (SP) docking method was applied during the initial docking stages. In the initial Glide docking, the receptor and the ligand were "softened" by van der Waals radii scaling. The scaling factor was chosen to be 0.50 for both the ligand and the receptor to permit enough flexibility for the ligand to dock in the best poses. The "trim-side chains" option was not used in this study. The maximum number of poses was chosen to be 20. In the next step, residues that are within 5 Å of the active site (ligand) were refined using the "Prime Refinement" Table In the final step, a threshold of 30 kcal/mol was used to redock the best structure, for eliminating high-energy structures from the Prime refinement step. The top 20 poses were kept for analysis and the best poses were selected based on IFD scores and visual inspection of protein-ligand interactions. The best docking poses were subjected to binding free-energy calculations using the Prime MM-GBSA module of the Schrödinger software allowing protein flexibility within 5 Å of the ligand. Only protein side-chains were minimized during the calculations. Finally, the Maestro Version 11.5 molecular graphics system was used to create all the computationally derived figures.

Conclusions
Screening of a selected set of O-methylated flavonoid constituents isolated from Senecio roseiflorus, Bhaphia macrocalyx, Polygonum sengalense, Psiadia punctulata, and Gardenia ternifolia identified compounds 1-3 and 5 as potent and selective inhibitors of human MAO-A, relative to MAO-B, and compounds 4 and 6 as selective inhibitors of human MAO-B. Further investigations suggested compounds 1-3 as reversible and competitive inhibitors and compound 5 as a partially reversible mixed-type inhibitor of MAO-A and compounds 3, 4, and 6 as reversible and competitive inhibitors of MAO-B. The computational results for compounds 1-6 were in good agreement with the experimental binding data for MAO-A and -B. The compounds 1 and 6 with high potency and selectivity of inhibition against MAO-A and MAO-B, respectively, may be promising new drug leads for further development as therapeutic treatment of neurological disorders, depression, Alzheimer's disease, and Parkinson's disease. It is important to mention that the flavonoid scaffold possesses promiscuous biological activity that may be due to inherent structural features. For this reason, they should be treated with caution as lead compounds for drug development.