2.3. Molecular Dynamics (MD) Simulation study

Using GROMACS v5.1.4 biomolecular simulation package, with CHARMM27 force field and TIP3P water model, all-atoms molecular dynamics (MD) simulations were carried out on the coordinates of hACE2-S protein complex docked with compounds: AP-NP, AP-3-OMe-Ph and AP-4-Me-Ph (Abraham et al., 2015; Mokaberi et al., 2019; Sharifi-Rad et al., 2020). The topology and parameter files for ligands parameters were repaired as define by Zoete et al. (2011), Mongre et al. (2019), and Sharifi-Rad et al. (2020). The simulation box was prepared with 10 Å buffer distance, and the protein–ligand complex was placed at the centre of box, padding around with water molecules. Along with counter ions for neutralizing the system, the salt concentration of 0.15 M (0.15 M NaCl) was used for the MD simulation (Joung & Cheatham, 2008). PBC condition was defined for all three x, y and z directions (Darden et al., 1993). All energy minimization and simulations were performed at physiological temperature, 300 K (Kumar et al., 2019; Mishra et al., 2018). Energy minimization was done estimating the steepest descent, followed by the conjugant gradients involving 50,000 steps for each. SHAKE algorithm was applied to constrain all the bonds involving hydrogen atom. Particle Mesh Ewald (PME) was applied for long-range electrostatic forces. The equilibration of system was done in two steps, NVT and NPT for the period of 500 ps. Pressure and temperature during the simulation were maintained by Berendsen thermostat (Berendsen et al., 1987) and Parrinello-Rahman pressure (Parrinello & Rahman, 1980), respectively. LINC algorithm was applied to constrain the bonds and angles (Hess et al., 1997). LJ potential with a cut off of 0.10 nm was defined for Van der Waals interactions. Finally, the production run was performed on NPT ensemble for a period of 100 ns, and the trajectory was updated at the time interval of 10 ps to record the energy and velocity. All production runs were performed on CUDA enabled Tesla GPU machine (DELL T640 with V100 GPU), and OS Centos 7 (Prakash et al., 2018; Singh et al., 2019) and the GROMACS utilities were used for the analyses of obtained MD trajectories.