The production runs of CF-SMD were performed from the final frame of the NPT equilibration step for each protein-ligand system. The CF-SMD runs were executed in the NVT ensemble using Langevin dynamics 37 at 300 K with a time constant of 2.0 ps and a time step of 2.0 fs. Covalent bonds with hydrogen atoms were constrained using LINCS ; non-bonded interactions were cut off at 10.0 Å; long-range interactions were calculated using the particle-mesh Ewald method . Trajectory frames were recorded every 10 ps during the simulation. Multiple CF-SMD simulations were performed under various strengths of the constant force (Table ). The CF-SMD simulations were automatically stopped if the reaction coordinate-the center-of-mass distance-was more than half the box size. In the CF-SMD simulation for the protein-ligand systems, a constant force was applied along the reaction coordinate between the center of mass of all protein atoms and that of all ligand atoms. The dissociation time τ(𝐹) under a constant force 𝐹 was defined as the first time that the distance between the center-of-mass points was greater than a threshold value at which molecules fully dissociated (Table ).