The AMBER FF14SB force field was employed to describe the proteins and GAFF (version 1.8) was used for the ligands. Restrained electrostatic potential (RESP) charges for the ligands were calculated at the HF/6-31G(d) level of theory using the Gaussian 09 program and AMBERTools16. The parmchk utility from AMBERTools16 was used to generate the missing parameters for the ligands. The systems were solvated using the SPCE water model using cubic simulation cells. The resulting solvated protein/ligand systems were then charge neutralized by adding Na + or Cl -ions . The particle mesh Ewald (PME) method was used to treat the longrange electrostatic interactions. All bonds involving hydrogen atoms were constrained using SHAKE . The AMBER16 package was used to run the MD simulations. MD simulations for each protein-ligand system were performed to equilibrate the systems. The equilibration process involved 100000 steps of energy minimization, 1 ns of NVT followed by 5 ns of NPT equilibration using a 12.0 Å non-bonded interaction cutoff. The second ligand (the ligand to be mutated to) was then added and the dual topology for the TI simulation was then generated using the parmed.py utility.