3.5. Molecular Dynamics Simulation

The selected docking poses of compounds 1 and 2 in complex with HDAC3 (PDB ID: 4A69) were subjected to a 100 ns MD simulation in AMBER16 [70]. The Antechamber package was used to prepare the topologies, force field parameters, atom types, and bond types by applying the semi-empirical Austin Model1 with bond charge correction (AM1-BCC) [76,77]. Then, the tLEaP module was employed to prepare the protein–ligand complexes. General amber force field (GAFF), the Duan force field (ff03.r1), and 12-6-4LJ ionic model were used for the ligand, protein, and zinc, respectively [78,79,80,81]. The system was solvated by the TIP3P water model and a margin of 10 Å. Two minimization steps including the two sub-steps in each minimization were carried out. In the first step, 4000 iterations (2000 cycles of steepest descent and then 2000 of the conjugate gradient) were performed, while the protein residues, ligand, and zinc ion were restrained to their initial geometries (force constant of 10 kcal*mol⁻¹* Å⁻²) to relieve the bad contacts. In the second step, 4000 iterations (2000 cycles of steepest descent and then 2000 of the conjugate gradient) were performed to remove the steric clashes in the entire complex. The restraint on the protein, ligand, and zinc were removed during the second minimization. Then, the system was heated at 300 K through 100 ps of MD. The protein–ligand complex was restrained to prevent large structural deviations (force constant of 10 kcal*mol⁻¹* Å⁻²). The SHAKE algorithm was activated to constrain bonds involving hydrogens [82]. Finally, the system was equilibrated within a period of 200 ps. Langevin dynamics was applied to keep the temperature at 300 K with a collision frequency of 2 ps [83]. The pressure was kept at 1 bar using isotropic position scaling with a relaxation time of 2 ps. Afterwards, a 100 ns MD simulation was run with a time step of 2 fs using the same conditions as in the equilibration step. A non-bonded cut-off distance of 10 Å was used. The electrostatic interactions were calculated by applying the particle mesh Ewald (PME) method. After the MD simulation, CPPTRAJ module of AMBER was used to analyze the MD snapshots.