When multiple binding poses were plausible (as determined by overlaying the ligand onto cocrystallized ligands of different PDB structures using a maximum common substructure overlay as well as docking compounds into the site), we ran SepTop between different binding modes to identify the more favorable pose. Especially for asymmetric phenyl substituents, the orientation of the ring in the binding site was unknown and sampling different rotamers was slow such that transitions did not occur during the length of the simulation. In the amide series, different rotamers of the pyrazine ring (see Figure ) affected computed relative free energies in the binding site by more than 6 kcal mol -1 . Running SepTop between the two poses differing in the orientation of the nitrogen atoms in the pyrazine ring, as depicted in Figure , resulted in a ∆∆G site = -6.6 ± 0.2 kcal mol -1 between the two poses in the binding site. This showed that one of the poses (Figure ) was predicted to be more favorable than the other pose, possibly due to the potential to form an intramolecular hydrogen bond between one of the nitrogen atoms in the pyrazine ring and the hydrogen on the amide nitrogen, or because the alternate pose places the lone pair of the nitrogen too close to the lone pair of the amide oxygen, resulting in strong repulsion. Transitions between the two poses were only observed in a few λ windows and were very slow. The binding mode with the best docking score was not always the same pose that was predicted to be more favorable in SepTop calculations. For ligands in the biaryl series with an unknown orientation of the asymmetrically substituted phenyl ring we also ran SepTop calculations between different poses, keeping the binding mode that was predicted to be more favorable for further calculations. In the spirocyclic series we chose the same orientation of the phenyl rings as given in the binding modes of the ligands in the PLBenchmark set. The coordinate files of the ligands in their binding modes that were predicted to be more favorable can be found in the SI. We adapted our alchemical protocol to try to enhance the sampling of different binding modes, however, we found that it was challenging to converge rotamer distributions at all λ windows. Here, our idea was to see whether, instead of running SepTop between different poses to identify the most favorable starting pose, we can start calculations with an unfavorable starting pose and adapt the alchemical protocol to sample the transition to the favorable binding mode. However, in the amide series, the torsion barrier around the rotatable bond between the pyrazine ring and the amide was so high, that even in the fully non-interacting state no transitions between different rotamers were observed. We adapted our enhanced sampling protocol and set the force constants to zero for all torsions that pass through the bond between the pyrazine ring and the carbonyl carbon of the amide in the non-interacting state, as well as further softening LJ-interactions of the atoms forming those torsions by scaling the LJ-ϵ by a factor of γ = 0.1. Starting SepTop from the two different poses using this adapted protocol gave a ∆∆G site = -0.6 ± 0.3 kcal mol -1 , which was much closer to zero which we would expect at sufficient sampling. The pyrazine ring now sampled different rotamers, however, sampling was not sufficient in all states along the alchemical path resulting in slow convergence. Thus, while we were eventually able to get this protocol to work, it involved considerable difficulty and manual tuning, and still exhibited signs of clear sampling problems, indicating that it would likely not work robustly for other, similar problems. Since we sought a general solution, we thus diverted our attention back towards protocols in which the preferred binding mode was an input (even if determined by an earlier SepTop calculation).