2.4. Docking Studies

To rationalize the experimentally determined inhibition potency of the selected compounds and suggest a binding mode, ligands were docked into the enzyme receptors using a flexible docking protocol described elsewhere [49], whereby selected residues outlining the active site gorge of AChE and BChE were allowed to rotate. Ligands to be docked in the enzyme structures were created with ChemBio3D Ultra 13.0 (PerkinElmer, Inc., Waltham, MA, USA) and minimized using the CHARMm force field and Smart Minimizer minimization method of the Minimize Ligands protocol implemented in Biovia Discovery Studio Client v18.1. (Dassault Systèmes, Vélizy-Villacoublay, France). Before the molecular docking was started, the ligands were prepared using the Prepare Ligands protocol with regard to possible different protonation states, isomers and tautomers at pH 7.4.

The enzyme structures were prepared starting from the crystal structures of free AChE (PDB ID: 4EY4) [50] and BChE (PDB ID: 1P0I) [12]. The binding site within AChE and BChE was defined by the sphere surrounding the residues that outline the active site gorge [14,51,52]. The representative pose of each of the docked ligands was chosen based on the highest Consensus score calculated from the scoring functions estimating binding affinity, as implemented in the Biovia Discovery Studio Client v18.1. Score Ligand Poses protocol. A more detailed description of parameters applied in the docking protocol is available in Supplementary Materials File S2.