2.6. Molecular dynamics (MD) simulation

The highest scoring ligand-protein complexes, with high binding free energies (ΔG_Bind) and/or Glide docking scores, were submitted to a 100ns molecular dynamics (MD) simulation using Desmond (Schrödinger Release 2020-4: Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, USA, 2020) [⁵⁶]. The simulation was used to refine the docked poses and evaluate the stability of binding at the active sites on the target proteins. In order to compare the efficiency of these screened compounds, the native crystal complexes of Mpro, Nsp12 and Nsp15 were simulated using the same protocol. The simulation systems were prepared using the System Builder tool of the Desmond module. The single point charge (SPC) water [⁵⁷] was chosen as the explicit solvation model. Each system was neutralized using an appropriate number of Na+ or Cl-counter ions. An orthorhombic simulation box, with Periodic Boundary Conditions (PBC) and a 10 Å buffer space between the solute and the box edge, was used for each system. The simulation systems were minimized and equilibrated before the actual simulations and before production simulation in a stepwise manner. After system relaxation, the production simulation was performed using the NPT ensemble for 100 ns, using a reversible reference system propagator algorithms (RESPA) integrator [⁵⁶]. The temperature (300 K) was set using the Nosé–Hoover chain thermostat [^{[58], [59], [60]}], with a relaxation time of 1.0 ps. The pressure was set at 1.01325 bar with the Martyna–Tobias–Klein barostat [⁶¹], using isotropic coupling and a relaxation time of 2.0 ps. Long-range interactions were handled using the U-series method [⁶²] and for short range interactions, a cut-off radius of 9.0 Å was used. The MD trajectories were analyzed using the Maestro built-in Simulation Interactions Diagram tool and Microsoft Excel360.