*4.9. MD Simulation*

To conduct MD simulations, GROMACS 5.1.2 Bio-Simulation package was used. To clarify the molecular dynamic characteristics and different computations of proteins and ligands employed in this in silico study, the force field GROMOS96 43a2 was used [73]. The receptor-ligand docked complexes files were retrieved using the gmx grep module. To create ligand topology and force-field conditions, PRODRG server was used [74]. To solvate the protein, the water model SPC216 was employed. A 50 ns MD simulation in water at 298 K was used as a control. All protein and ligand atoms were equilibrized in a three-dimensional box with a range of almost 10.5 Å from all side. The protein was thoroughly equilibrated in water, and redundant molecules were removed. To eliminate all poor contacts, energy was minimized for each system with the steepest decline up to a forbearance of 1000 kJ mol−1nm−<sup>1</sup> and the overall charge was neutralized in the system by adding ionic concentrations of NaCl. To perform the simulation, the sizes (x, y, and z) of the simulation frame were established depending on the size and 3D positioning of the protein. All the systems were produced in a specified box, with the protein in the center and water and co-solvents padded around it. The energy minimization method was carried out using the steepest-descent algorithm and conjugate gradient. Two troupe methods, NVT and NPT, were used to equilibrize the system. Before beginning the MD run, environments, such as pH and temperature, were pre-defined. All this evidence was contained in the NVT, NPT, and MD criteria files. The binary trajectory file was generated after the production run for additional examination [75].
