*4.8. 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.

#### **5. 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.

**Supplementary Materials:** All NMR spectra of compounds **1**–**6** are provided as Supplementary Information.

**Author Contributions:** B.L.T. and I.M. conceptualized the study. B.L.T. and N.D.C. planned the experiments for enzyme-inhibition assays. J.M. and I.M. authenticated and provided the natural products, R.N.B. carried out the chemistry work. N.D.C. performed the experiments on enzyme assays. B.L.T. and N.D.C. analyzed the results of enzyme inhibition. P.P. and R.J. planned the computational studies and analyzed the results. P.P. carried out computational studies. N.D.C., B.L.T., P.P., R.J.D., and I.M. prepared the original draft of the manuscript. All the authors contributed to the writing, review, and editing of the manuscript. All the authors have read and agreed to the published version of the manuscript.

**Funding:** The International Foundation of Uppsala University, Sweden is acknowledged for supporting the KEN; 02 project at the University of Nairobi, Kenya, where the compounds were isolated. This research was funded in part by grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS), a component of the National Institutes of Health (NIH), and was conducted in part in a facility constructed with support from the Research Facilities Improvements Program (C06RR14503) from the NIH National Center for Research Resources; its contents are solely the responsibility of the authors and do not necessarily represent the official view of University of Mississippi NIGMS, NIH or Southern Research.

**Acknowledgments:** The authors are thankful to the National Center for Natural Products Research (NCNPR), the University of Mississippi for the facilities to support this work.

**Conflicts of Interest:** The authors declare no conflict of interest.
