Fig. 3. Computational and experimental characterization of QUB-00006 binding within the Mpro binding pocket. (A) QUB-00006(R) (in light green) and QUB-00006(S) (in cyan) binding in a similar fashion at the interface of subpockets S2 and S4; the binding poses shown here were clustered and extracted from the trajectories of the binding free energy calculations performed on QUB-00006(R) and QUB-00006(S). (B) The analysis of our binding free energy trajectories showing that protons in groups A, B, E, and D have a low hydration ratio (less than 0.5), while the proton of group C has a high hydration ratio of 0.8. Hydration ratios calculated for the different proton groups of QUB-00006(R) correlate with those calculated for QUB-00006(S). (C) The WaterLOGSY spectra of QUB-00006 in the presence and absence of the Mpro. The assignment scheme is reported along with the 2D structure of the ligand. The strong negative intensity of the signals of the hydrogens of groups A, D, and E suggests that they are orientated towards the protein, while the hydrogen atom in C is solvent exposed. These experimental findings confirm the hydration ratio calculated during our binding free energy simulations and described in panel B.
