2.3. MD simulations, MM-PBSA, and Gibbs free energy

The best technique for gaining protein-ligand equilibrium is the Molecular Dynamics (MD) Simulation. It is established upon the law of molecular motions by Newton (equation 2).

Fi = force exerted on particle i, mi = mass of particle i, and ai = acceleration of particle i. V = potential energy of the system. r = position. δ = change in velocity. Fi shows the force working on i given by the partial spatial derivative of V, which depends upon the positions rN (r1, r2,...., rN) of all N particles in the system [³⁵]. We have produced four systems of the Nsp1 complex with molecules BCH10, BCH15, BCH16, and BCH17 for 250 ns MD simulation in Gromacs 4.6.7 [^{36, 37, 38}]. The GROMOS 43a1 forcefield was adopted for protein topology creation, while the PRODRG 2.0 server was used to create ligand topology [³⁹]. The systems neutralization was done by adding 98888 water and 5Cl^- ions. The cubic box of 24.33 nm³ volume was used in systems solvation. Next, to eliminate the steric clashes during the energy minimization conjugant gradient and the steepest descent approach was adopted. After that, NPT and NVT simulation of 1 ns was performed to fix the systems temperature and pressure. The LINCS algorithm exercised to constrain the bond's length and the PME method to estimate absolute electrostatics [^{40, 41}]. The thermostat (V-rescale) method was applied to maintain the 300K temperature [⁴²]. The MD trajectories were kept for several structural interpretations like RMSD, RMSF, and H-bonds analysis.

The g_mmpbsa tool was utilized to determine binding free energy applying molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approach. The binding free energy gives the three dynamic terms. First is the standard molecular mechanics energy. Second is the entropic supplement to solvation and free energy in a vacuum [⁴³]. The low-energy state of selected complexes captured via free energy landscape analysis (FEL). The g_sham module was used for FEL calculations.