3.6. Molecular Docking

The protein preparation, the molecule preparation, the grid generation, and the molecule-protein docking were performed using Autodock software [80]. For the protein preparation, the target proteins were initially pre-processed by removal of water molecules, addition of Kollman charges, and optimization of the hydrogen bond (H-bond). For the ligand preparation, the hydrogen atoms were added and Gasteiger charges. The coordinates of grid were obtained by CB-Dock online tool (http://cao.labshare.cn/cb-dock/, accessed on 7 May 2021) using the prepared ligand and protein. Discovery Studio Visualizer software (http://accelrys.com, accessed on 7 May 2021) was used to analyze ligand–receptor interactions to find the amino acids that interact with the molecules.

The 3D structure of the ligands and the complexes were realized with Chemdraw online version (https://chemdrawdirect.perkinelmer.cloud/js/sample/index.html, accessed on 7 May 2021). The structure was optimized using Avogadro version 1.2 (https://avogadro.cc/, accessed on 7 May 2021) with Universal Force Field (UFF) [81] in order to obtain PDB files for molecular docking.

The receptor proteins to be evaluated belong to the following organisms: Leishmania spp., T. cruzi, P. falciparum, and S. aureus. The proteins used as receptors for the ligands were leishmanin (1LML), cruzain (4PI3), tubulin Alpha (P. falciparum), and PBP2A of S. aureus (5M18). For P. falciparum tubulin Alpha, the GenBank sequence CAA34101 (453 aa) was used to build the 3D model using Swiss-model (https://swissmodel.expasy.org/, accessed on 7 May 2021); this was validated by PROSA (https://prosa.services.came.sbg.ac.at/prosa.php, accessed on 7 May 2021) and Molprobity (http://molprobity.biochem.duke.edu/, accessed on 7 May 2021).

In order to compare the affinity results of ligands and complexes, control molecules were used: amphotericin for leishmanin, vinylsulfone for cruzain, vinblastine for alpha tubulin, and finally cefepime, ceftobiprole, and benzopenicillin for PB2A. These controls were first obtained in a pubchem SDF format (https://pubchem.ncbi.nlm.nih.gov/, accessed on 7 May 2021). Then, with Avogadro, they were converted to a 3D structure, and the structure was optimized using the GAFF force field. Then, the PDB format for docking was obtained.

The validation of leishmanin with L2 ligand was performed through molecular dynamics. The model system was hydrated by means of Charmm-Gui solution builder. The simulation was performed with GROMACS version 2020.2. Minimization was run for 0.1 ns using the steep descent algorithm, with a Verlet cutoff scheme and coulombtype pme. Equilibration was performed for 2.5 ns, with a Verlet cutoff-scheme and coulombtype pme, using the Nosé–Hoover algorithm at 310.15 K. Finally, the molecular dynamics was run for 10 ns, also with the Nosé–Hoover algorithm at temperature 310.15 K, with a Verlet cutoff scheme and coulombtype pme. RMSD analysis, hydrogen bonds, affinity energy, and the distance between L2 ligand and leishmanin were performed. The visualization of the leishmanin surface with the L2 ligand was performed with pymol educative version (https://pymol.org/edu/?q=educational/, accessed on 7 May 2021).