Embedded ligands were subsequently docked into the receptor. For Glide workflows, calculations were performed using Schrodinger version 2021-4, with default settings used throughout, unless otherwise noted. A single conformer was generated for each ligand in all cases . In addition to the standard docking protocols, termed 'Vanilla' Figure : Overview of the steps comprising our NES RBFE workflow in this work, we investigated both fixed and maximum common substructure (MCS)-derived positional constraints available in Glide. Whilst equivalent functionality is not available in Vina, we implemented a post-hoc filtering step using RDKit functionality to identify the pose for each ligand that minimised RMSD compared to the reference pose .