In particular, for non-zinc-binding MMPIs, it has been demonstrated the importance of the p-p stacking interaction with one of the His residues present in the conserved zinc-binding motif, to achieve binding potency. The aromatic groups of ligands, which are able to give the p-p stacking with the His201 of the enzyme, are the pyridine and the furan for the active ligands and the phenyl ring and the furan for the ligand 2. The interaction of His201 imidazole with these aromatic rings was analyzed measuring the distance between the CP-690550 477600-75-2 centre of mass, the shifting and the parallelism between the rings involved in the interaction. Any direct evaluation of binding free energy in large systems as the present one might be frustrated by its complexity. In fact, large amplitude motions revealed by previous analysis clearly indicate that quantitative free energy evaluation using standard TI approaches, if not extended for prohibitively long simulation times, might be severely affected by the choice of the initial conditions. For this reason we decided to carry out TI integration MK-1775 Wee1 inhibitor starting from different initial enzyme configurations, selected from the pre-viously described ED analysis. This, at least in principle, should reduce the systematic error due to the incompleteness of the phasespace sampling. The extracted structures were selected within the spots obtained from projection of the trajectories onto the related Ca essential plane. A first set of TI calculations were carried out at 300 K and a second set at 323 K in order to provide some information about the entropic and energetic factors affecting the ligand binding. In both sets we adopted, for each starting configuration, the computational scheme proposed by McCammon and coworkers. Details of the TI trajectories are reported in the Supporting Information. The results are collected in the Table 2 and indicate that at 300 K within the error, ligand 1b shows the highest affinity toward MMP-2, although quite similar to 1a. On the other hand ligand 2 shows the lowest affinity. These values are in line with equation is $15 kJ/mole at 300 K, not in disagreement with our data. A further important aspect concerns which of the two tautomers is actually more active. In principle this information might be derived by results in Table 2 from which it turns out that, although with a relatively high uncertainty, 1b seems more active than 1a. In order to rationalize the above results, the role of enthalpic and entropic factors in the stabilization of the MMP-2 complexes was evaluated. Lack of a relevant temperature dependence of relative binding free energies, allows us to consider that the main determinant for ligand affinity is not entropic but, rather, enthalpic. In this respect, however, analysis of the binding mode does not immediately reveal drastic differences among the three species.