For comparison we also included in the plot the PMF obtained using HREM with acceptance ratio balancing. None of the approaches based on standard MD is apparently able to produce fully reliable Boltzmann weights for the two conformations of compound 1 whose binding is characterized by the induced fit mechanism. We recall that in alchemical free energy simulations, a correct sampling of the apo state, or of states with small alchemical coupling, is an essential requirement for delivering reliable binding free energies. Phenyl flipping in compound 1, as probed by α, never occur in a standard 16 ns MD simulations, while is severely undersampled by extending the simulation up to obtained using 16 ns replicates of standard MD simulations confirming the moderate effectiveness of this straightforward enhanced sampling approach. Ref.