Results and Discussion Outline of the Computational strategy
In the present work a combined computational approach has been applied in order to clarify the effects produced, on the target macromolecule, by the binding of two structurally related ligands with different affinity toward MMP-2 as well as the structural and dynamical differences of the selected ligands. The ultimate goal of the computational approach illustrated in this study is to estimate the differential binding free energy of aqueous 1a, 1b and 2 toward MMP-2, and its comparison with available experimental data. The calculation of absolute binding free energy is a very complicated task [61] and TI, in this respect, is a widely employed approach [38], [55], [62] whose limitations mainly reside in its high sensitivity to the exhaustive sampling of configurational space (CS) hence requiring advanced computational strategies [63], [64] or, when possible, exhaustive or complete sampling of the CS. The described protocol, comprising the following steps, has been setup in order to reduce the systematic error associated to a poor sampling of the CS. ?Docking calculations of the studied ligands in the MMP-2 active site were carried out to obtain putative binding poses, that were evaluated and analyzed. ?MD simulations of the free enzyme, the three complexes and the free ligands were carried out and examined. ?The obtained trajectories were subsequently analyzed by means of Essential Dynamics (ED) in order to extract the significant starting configurations to be used for free energy calculations. ?Free energies calculations, initiated by the ED-based basins, were accomplished using TI method at 300 K. ?TI calculations were also performed at higher temperature (323 K) in order to qualitatively estimate the role of enthalpic and entropic factors. ?QM calculations of the stability of the two tautomers 1a and 1b were finally carried out in order to definitely assess their occurrence in aqueous solution.
Docking Calculations
The binding mode of compounds 1a, 1b and 2 into the MMP-2 catalytic domain was studied at first through docking calculations. The results show that all ligands, predicted as uncharged at neutral pH, bind MMP-2 adopting a conformation very similar to that experimentally observed for MMP-13 (PDB IDs: 3I7G and 3I7I), spanning from the S19 to the S39 sites and not coordinating the zinc ion [26]. On the basis of these data, this would be the first example of non-zinc-binding MMP-2 inhibitors. However, as the presence of the water molecule on the zinc would preclude the binding of the inhibitor to the catalytic zinc, to evaluate the ability of these compounds to coordinate this ion, docking runs were carried out also without the water molecule as the fourth zinc ligand. Poses showing the inhibitors binding the zinc, have much lower docking scores and an unfavourable positioning in the S19 site, which is widely recognized as the most relevant interaction of MMPIs (data not shown). These results, along with the previously described similarity to the experimental binding conformation in MMP-13, were considered as a validation of docking poses shown in Figure 3 and hereinafter described. The docking score assigned to the active ligands is higher (210.0 and 29.23 for ligand 1a and ligand 1b, respectively) in comparison with the inactive one (29.07). The active ligand establishes with the protein three hydrogen bonds, observed for both tautomers 1a and 1b, between: the methyl amide NH and the backbone Gly162 CO, the cyclohexyl amide CO and the Tyr223 NH, the furanyl amide CO and the Leu164 NH. The pyrazyl NH forms a H-bond with the Ile222 CO for the tautomer 1a and with Thr229 OH for the tautomer 1b. 1a establishes an additional H-bond between the furanyl amide NH and the Pro221 CO. The principal hydrophobic interaction in the S19 site consists of the p-p face to face stacking between the ligand pyridine and the His201 imidazole. The ligand 2 finds similar H-bonds between: the ligand methyl amide NH and the Gly162 CO, the cyclohexyl amide CO and the Tyr223 NH, the furanyl amide NH and the Pro221 CO, the furanyl amide CO and the Leu164 NH. Moreover the p- p stacking between the ligand phenyl and the His201 in the S19 subsite can be observed also for this ligand, that does not form any H-bond with the heterocycle ring at the bottom of the S19 site. It is clear that docking calculations do not provide an exhaustive rationalization of the different IC50 observed for studied inhibitors. In fact, a very similar binding mode for all ligands was obtained, with a comparable docking score. On the basis of these calculations ligand 1a results a slightly better binder of the enzyme, because it can establish more interactions and shows higher docking score with respect to the others. However these results do not help to explain the lack of activity of ligand 2, which binds the enzyme with similar interactions.
MD simulations
Dynamical-mechanical and structural features of the whole enzyme upon inhibitors binding. We initially focus our attention on the whole structural and mechanical-dynamical effects produced onto the MMP-2 by the presence of the inhibitors. For this purpose, MD simulations were performed for the free enzyme, the complexes starting from the docked poses, and the free inhibitors. Note that in the rest of this study the complexes of MMP-2 with the investigated inhibitors will be termed as MMP-2:1a, MMP2:1b and MMP-2:2. The stability of the simulations and the qualitative structural behavior of the protein were investigated by calculating the rootmean-square deviation (RMSD) of the Ca atoms for all models with respect to the corresponding starting structures. The results indicate that, within the simulated time, the obtained trajectories are rather stable. As a matter of fact, the Ca RMSD systematically increases during the simulation and, approximately after 1500 ps, it reaches a plateau at about 0.20 nm for all the systems. The RMSD, when computed for all atoms of the ligands, shows that in all of the cases the ligand roto-translational deviation does not exceed 0.2 nm from the docking pose. Another important aspect which deserves a careful inspection, is the analysis of the alteration of the internal enzyme framework mobility induced by the presence of the inhibitor.
