The edges between compounds from the ligand perturbation map were then taken through each step of the simulation protocol, each of which comprised a prepare_simulation stage to generate the tpr files, followed by a run_simulation stage to submit the gmx mdrun jobs. First, systems were subjected to a short minimisation job using the steepest descent algorithm, followed by a 10 ps equilibration in the NVT ensemble. Equilibrium simulations were then run for 6 ns in the NPT ensemble, after which transitions were prepared with a bespoke step to handle frame extraction and preparation. The first 2 ns of trajectory were discarded as additional equilibration time, and 80 equally spaced snapshots were then extracted from the last 4 ns of production trajectories, for both the ligand and complex simulations. Each extracted frame served as the start for performing non-equilibrium transitions. 80 such transitions were performed in each direction over 50 ps, and the work done was computed using thermodynamic integration. Finally, the analysis step was run to compute ΔΔG values and errors for each edge using maximum likelihood estimator based on Crooks Fluctuation Theorem , as implemented in the pmx analyse program, and generate summary files.