The last conformations of the equilibration phase were used as the starting structures of ligand unbinding simulation. The distance-based Steered MD simulation (center-of-mass-pulling method) was used to pull the ligand away from the protein by approximately 30 Å over the course of 4 ns by using a 1000, kJ/(mol nm 2 ) force along the reaction coordinate (ξ), with a pulling speed (ν) set at 0.001, nm/ps. Snapshot intervals of 10 ps generated 400 configurations from these pulling simulations. Different ligands prompted the extraction of varying conformations, ranging from 22 to 28, along the reaction coordinate (ξ) at approximately 0.1 nm intervals. These distinct configurations were then employed as the initial points for individual Umbrella Sampling simulations, differing in quantity depending on the specific ligand under study. Each conformation underwent independent NPT equilibration for 5 ns, followed by a 20 ns MD run in triplicate for each ligand. The potential mean force (PMF) was determined via the weighted histogram analysis method (WHAM) , a component of Gromacs. The resultant PMF graphs depicted force in kcal/mol, representing the force required to dissociate the ligand from the binding pocket, against the corresponding distance. The computation of the binding free energy (∆G) for each ligand involved comparing the plateau region of the PMF curve to the energy minimum obtained from each simulation. In total, the Umbrella Sampling simulations spanned 400 to 560 ns, comprising 3 replicates, thereby accumulating simulation times ranging from 1.2 µs to 1.6 µs for each ligand.