Simulations. The binding affinity calculator (BAC) , an automated workflow tool for free energy calculations, was used to prepare and execute ESMACS and TIES simulations. The standard protocol was used, in which NAMD simulations with 25 and 5 replicas were performed for ESMACS and TIES, respectively. Each replica in the ensemble started with identical atomic coordinates, with different initial velocities generated independently from a Maxwell-Boltzmann distribution. a There was no activity at the highest concentration (250 µM) tested.