2.5 | Docking of bupivacaine and ropivacaine
In order to study the binding site and mode of local anesthetics in Kv1.5 ion channel, we used a systematic pipeline that includes docking simulations in each binding site followed by molecular dynamics simulations (MDs) and clustering of conformers. Briefly, two binding sites were selected in the Kv1.5 homology model, the central cavity and one side pocket. We carry out the docking of local anesthetics into only one side pocket because the other three are integrated by the same residues due to the tetrameric nature of the functional channel. The local anesthetics (S-bupivacaine and ropivacaine (INN name implies that it is the S-enantiomer)) were sketched and prepared with using LigPrep (Schrödinger Release 2017-1, New York, USA) included in the Maestro suite (Schrödinger Release 2017-1, New York, USA), with the force field OPLS-AA (Kaminski, Friesner et al. , 2001). The nitrogen atom of the piperidine ring was protonated for both ligands. The charges were maintained during the parametrization process before docking and MDs. The docking simulations were performed with Glide (Friesner, Murphyet al. , 2006) using the standard precision scoring function, obtaining 10 poses per docking simulation. The centre of the grid boxes for each binding site was focused using the interacting residues identified by the functional alanine mutagenesis approach for each local anesthetic. All molecular docking runs were performed with the outer box edge of the grid setting as 30 Å, thus ensuring that the binding site residues were included in each grid box. The best poses according to the docking score were analyzed and the one that present the best correlation with the experimental findings were further studied by molecular dynamics simulations.