Full details of the methods used in this work can be found in the associated GitHub repository. If we have any updates to the tools/scripts, they will be made available on GitHub. We provide a brief overview of some of the key details of our methods here. 3.1. System preparation. We used four ligands to validate our MD/NCMC protocol (Figure ). Ligands 1-3 bind to JNK1 and have the same scaffold but with different substitutions in the terminal phenyl group. Their binding free energies were studied extensively by other groups both experimentally and computationally. In the previous computational study with a different force field than that employed here, the ligands exhibited two distinct binding modes with equal probability of occurrence. We modeled the binding mode for these three ligands based on the PDB 2gmx. The different aromatic ring substitutions were added to the ligand present in the co-crystal structure using Chimera 1.12. For ligand 4, PDB 2r3i 27 was used. Missing residues were modeled using Modeller 9.19. The partial charges of the ligand atoms were assigned based on the AM1-BCC model using Antechamber, included with the Amber 16 package and the protonation states of the protein residues using H++ 31 ().