Once the system converges, free energy F(s) (Eq. ( )) can be extracted by adding the deposited hills along the biased reaction coordinates. The centre of mass distance between the heavy atoms of the ligands and the protein backbone in the surrounding area of the binding pocket is considered as the reaction coordinates in the free energy sampling, since the aim of the calculation is taking into account the ligand dissociation from the binding site. In this study 5 independent simulations are done and an averaging is done for each combination to obtain better sampling and statistically reliable results.