Monoamine Oxidase B

Two experiments were applied for each target:(a)

Self-docking of each ligand inside its own protein structural conformation.

(b)

Cross-docking of three selected ligands inside the remaining nine protein structures.

All the studied ligands in this work were prepared using LigPrep with the force field OPLS_2005 [61 (link)]. Docking tests were performed using the softwares Glide and Autodock. Glide offers a complete solution for ligand–receptor docking and is widely used for drug discovery [62 (link),63 (link)], virtual screening [64 (link),65 (link)], structure-activity relationship analysis [9 (link),66 (link)], etc. Grid boxes for self-docking and cross-docking experiments were centered in the ligand position coming from the crystal structures. The grid boxes’ dimensions were (20 × 20 × 20) ų in order to include the whole binding site for the three targets under study. High-throughput virtual screening (HTVS), standard precision (SP), and extra precision (XP) Glide modes were proved. Default docking parameters were used. Glide docking uses hierarchical filters to find the best ligand binding locations in the defined receptor grid space. The filters include positional, conformational, and orientational sampling of the ligand and subsequent energy evaluation of the interactions between the ligand and the protein [18 (link),19 (link),20 (link)]. Ligand minimization in the receptor field is carried out using the OPLS-AA force field [67 (link)] with a distance-dependent dielectric of 2.0. Afterward, the lowest energy poses are subjected to a Monte Carlo (MC) procedure that samples the nearby torsional minima. The best poses for a given ligand are determined by the GlideScore score [68 (link)], including terms for buried polar groups and steric clashes.
Autodock parameters were defined in a similar way as in Glide, with grid boxes dimensions of 20 × 20 × 20 ų. AutoDock uses a semi-empirical free energy force field to predict binding energies or ligands to macromolecular targets and Lamarckian genetic algorithm (GA) to search for docking solutions [69 (link)]. The force field is based on a comprehensive thermodynamic model that allows incorporation of intramolecular energies into the predicted free energy of binding [70 (link)]. Each ligand was located in the grid box for each docking run and torsional degrees of freedom were defined. The GA was applied with an initial population of 100 randomly placed individuals, a maximum number of 1 × 10⁶ energy evaluations, a maximum number of 3 × 10⁴ generations, a mutation rate of 0.02, a crossover rate of 0.80, and an elitism value of 2.
Each self-docking or cross-docking experiment between a target protein (MAO-B, thrombin or B-RAF) and a ligand was repeated three times, accounting for replicated instances. The effectiveness of the docking experiment (self-docking or cross-docking) in reproducing the crystallographic binding orientation of a ligand $\mathit{\alpha }$ was determined by comparing the docked pose with the orientation of the ligand in its native crystallographic structure ${\mathit{A}}_{\mathit{\alpha }}$ . In the case of MAO-B, the cofactor FAD was present in the binding site during docking experiments. The RMSD was employed as the term for performing such comparison, where ligand atoms were matched one to one and symmetrical atoms were considered equivalent. For comparing cross-docking poses, receptor structures were superimposed using Cα of binding site amino acids, by considering that amino acids that surround 10 Å the ligand in its native crystallographic structure form the binding site.

The binding of ligands to MAO-A and MAO-B could be quantitatively measured by using MM-GBSA combined with MD simulation.^{41 (link)} For each molecular species, apo and holo, the G_bind (binding free energy) was calculated by using the following equation:
The different components (G_R+L, G_R, and G_L) required for the free energy calculation of the apo and holo states are given in eqn (v). In the MM/GBSA and MM/PBSA methods, each free energy term in eqn (v) is calculated using the following equation:
In eqn (vi), E_bond, E_vdw, and E_elec are the bond energies, van der Waals, and electrostatic energy, including the dihedral bonds and angles, G_PB and G_SA. TS_S represents the solvation energy corresponding to the polar and non-polar contributions, including absolute energy and solute entropy. The optimized parameters and MIEC model, as proposed recently, work for calculating the free energies between protein–protein interfaces,^{42–45 (link)} but here we utilized the MM-PBSA.py method using interior solute and exterior solvent values as constant^{46 (link)} to calculate the free energy.

The n-BuOH fraction of the N. glandulifera extract was used for the ligand fishing test due to its moderate MAO-B inhibitory activity. First, the n-BuOH fraction was filtered with a 0.22 µm filtration membrane, concentrated to dryness, and diluted in PBS (50 mM, pH 7.4) to a concentration of 1 mg/mL, denoted as S0. A total of 3 mL of S0 was added to a 5 mL Eppendorf tube containing 20 mg of MNPs@MAO-B. The tube was oscillated for 30 min at room temperature, and the MNPs@MAO-B adsorbed with ligands of the enzyme were separated by an external magnet and washed three times using PBS (50 mM, pH 7.4). Then, 500 µL of 50% ACN was used to dissociate the ligands bound to the MNPs@MAO-B, denoted as S5. The S0 and S5 were analyzed by HPLC to determine the possible ligands of the enzyme.

Chemicals such as sodium hydroxide (NaOH), 3-aminopropyl-trimethoxysilane (APTMS), and dimethyl sulfoxide (DMSO) were obtained from the Chengdu Kelong chemical reagent factory (Chengdu, China). Monoamine oxidase B (MAO-B, 100.23 U/mL) was prepared in-house [12 (link)]. Kynuramine dihydrobromide was purchased from Sigma-Aldrich (St Louis, MO, USA). Safinamide mesylate and rasagiline were purchased from Meilunbio (Dalian, China). The MAO-B inhibition assay was carried out using Thermo Scientific Varioskan Flash equipped with a 96-well microplate (Thermo, Waltham, MA, USA). FT-IR spectra were recorded in KBr with a PerkinElmer FT-IR spectroscope (PerkinElmer, Waltham, MA, USA). Ultrapure water produced with a UP water purification (18.25 MΩ) system (Ultrapure, Chengdu, China) was used for HPLC. HPLC-grade methanol was obtained from JT Baker (Phillipsburg, NJ, USA). The HPLC system consisted of a Shimadzu LC-20 AD series equipped with a thermostatic column compartment, an SPD20A UV-vis detector (Shimadzu, Kyoto, Japan), and an Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm). The mobile phase was composed of solvent A (0.1%, v/v, formic acid/H₂O) and solvent B (100% MeOH) at a flow rate of 0.8 mL/min, and an injection volume of 20 µL. The eluting gradient was set to 30–100% MeOH for 0–30 min. The MS detection was in the positive ion mode using a capillary voltage of 3.0 kV; a source temperature of 180 °C; a desolvation temperature of 350 °C; and a desolvation gas flow of 800 L/h, while the nebulizer was set at 0.8 Bar and the sample flow rate was set at 0.3 mL/min for the ESI-MS (Bruker Compass Data Analysis 4.0; microTOF-Q11-10203). For the FT-IR, the samples were pretreated with a tablet press using potassium bromide (KBr) in a dry environment, and the wavenumbers of the FT-IR measurement were set in the range of 450 to 4500 cm⁻¹.