Enhanced sampling of molecular dynamics with approximation of continuum solvent (ES-MACS) calculations are fundamentally based on the Molecular Mechanics Poisson-Boltzmann/ Generalised-Born Surface Area (MMPB/GBSA) binding free energy calculation method. MMPB/GBSA calculations were conducted using AmberTools 20. For all MMPB/GBSA calculations, the 1-traj protocol was used, allowing the MMPB/GBSA ligand binding free energy (∆G M M P B/GBSA ) to be calculated from a single trajectory of the protein-ligand complex. Within the 1-traj protocol ∆G M M P B/GBSA is computed using the equation: