*3.3. Molecular Docking Studies*

Molecular docking studies were carried out to characterize the interaction between drug substance and CD. The RM-β-CD structure used in this work was generated from the curated coordinates of ligand 2QKH (X-ray diffraction, resolution 1.9 Å) downloaded from the Protein Data Bank database [59]. Methyl groups were manually added on free hydroxyl groups in order to obtain a degree of substitution equal with 12 (GaussView 5, Semichem Inc). Substituents were added on the β-cyclodextrin natural core, namely, 4-CH<sup>3</sup> on the O2- position for the 2, 3, 4, and 6 glucopyranose units, 5 groups on the O3- for the 1, 2, 4, 5, and 7 glucose residues, and finally, 3-CH<sup>3</sup> on 1, 5, and 7 glucopyranose units on the O6- position. All dihedral angles of the methoxy groups were homogenized, the resulting

conformations being compatible with an unhindered CD cavity. CD was optimized in the same manner with RSP (DFT/B3LYP/6-311G). Three-dimensional coordinates of RSP was generated using the Gaussian program suite at DFT/B3LYP/6-311G optimization.

The molecular docking analysis was carried out using the Autodock 4.2.6 software together with the AutoDockTools [41]. The docking between RSP and RM-β-CD involves adding all the polar hydrogens, computing the Gasteiger charge; a grid box was created using Autogrid 4 with 50 × 50 × 50 Å in x, y and z directions with 0.375 Å spacing from CD center. All the calculations were performed in vacuum. For the docking process, we chose the Lamarckian genetic algorithm (Genetic Algorithm combined with a local search), with a population size of 150 and a number of 50 runs. In order to generate the molecular modeling figures, we exported all Autodock results in the PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC, New York, NY, USA) [43]. To validate the repeatability and reproducibility of the docking method, we performed redocking and then expressed the results as RMSD in Å using Discovery Studio software. All the calculations were performed in triplicate and expressed as an average.
