In this work we consider only relative binding free energy (RBFE) calculations. Several existing software applications can facilitate these calculations such as PMX based on GRO-MACS , FEP+ proprietary software produced by Schrödinger 36 or FESetup . Our group has recently publicly released the comprehensive TIES toolkit 38 to automatically setup, execute and analyse such calculations; this software was used to set up and perform all calculations for this study. The specifics of RBFE methodologies vary between implementations, being based on user choices about how to carry out calculations. Some key areas where this variation could significantly influence the results include the topology of the transformed moieties, the thermodynamic path followed between end states and how much sampling is performed in each state. These factors introduce some uncertainty in the results but this is generally controlled by probing them on a case by case basis.