Determining the complete set of ligands' binding/unbinding pathways is
important for drug discovery and to rationally interpret mutation data. Here we
have developed a metadynamics-based technique that addressed this issue and
allows estimating affinities in the presence of multiple escape pathways. Our
approach is shown on a Lysozyme T4 variant in complex with the benzene
molecule. The calculated binding free energy is in agreement with experimental
data. Remarkably, not only we were able to find all the previously identified
ligand binding pathways, but also we uncovered 3 new ones. This results were
obtained at a small computational cost, making this approach valuable for
practical applications, such as screening of small compounds libraries
