Tools

Molecular docking was undertaken with the help of AutoDock 4.0, Autogrid [33 (link)], and AutoDock Tools [34 ]. The standard AutoDock protocol was followed unless otherwise noted. Ligands were drawn and then processed with AutoDock Tools for charge and rotatable bonds assignment. 5-LO crystal structure chosen for docking is PDB ID: 3O8Y [34 ], which is a “stable-5-LOX.” To enable crystallization, several mutations are present in the noncatalytic domain and a small 3 residue sequence in the catalytic domain is replaced from KKK to ENL. The mutations maybe affect the structure, but “stable-5-LOX” catalytic activity was not affected [34 ]. The protein was prepared with AutoDock Tools. Water molecules were removed, polar hydrogens added, and charges assigned. The grid box used a default spacing of 0.375 with a bounding box of 60, 66, and 60 and a grid center of −2.24, 25.69, and −0.94, in both cases X, Y, and Z coordinates. As for the docking settings, 100 runs were completed per ligand and defaults were kept with exceptions for the following values: ga_pop_size 5000, ga_num_evals 100,000,000, ga_num_generations 500,000, and sw_max_its 5000. For analysis, AutoDock Tools (Schrödinger Release; Maestro, version 10.6) and LigPlot⁺ [35 (link)] were used. Results were clustered with a maximum of 2.00 Å RMSD, and the largest cluster with the lowest binding energy was chosen.

For the docking studies, the proteins were rigid, whereas the ligands were flexible. The *.pdb, ∗.pdbqt, ∗.gpf and ∗.dpf files were created in AutoDock Tools. After the docking simulations, the protein-ligand interactions were evaluated using AutoDock Tools. The grid box was of 60 ų with a grid spacing of 0.375 ų. For 4QPE the grid center was at X = −25.93, Y = 30.821, Z = −6.062. With this box, the residues H447, E334, S203, Y337 were included, whereas for 1Z0Q (alpha-helix) the grid center was X = 2.282, Y = 5.061, Z = −6.757; for 2BEG (beta-sheet) X = 2.937, Y = −4.619, Z = −1.241 and for 1Z0Q (RC) X = 9.387 Y = −4.642 Z = 1.805. The scoring sampling of docking study used the Lamarckian genetic algorithm as implemented in AutoDock Tools with an initial population of 100 individuals with 1 × 10⁷ evaluations. The ligand-protein complexes were analyzed to find the lowest free (ΔG) values by means of the AutoDock Tools program to then describe the ligand-protein interactions.

The crystal structure of STAT3 was obtained from the PDB database (http://www.pdb.org) and was then imported into Pymol software (version 2.4.0) for the removal of water molecules, co-crystallized ligands, and ions. Subsequently, hydrogens were added and gasteiger charges were computed by AutoDock Tools (version 1.5.6). The structures were saved as PDBQT format files after the AD4 type was assigned. 3D-structure of 4-OI was downloaded from PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and was converted into Mol2 format files by Open Babel GUI software (version 2.3.1) http://openbabel.org) [30 (link)]. Then the small molecule ligand file was imported into AutoDock Tools (version 1.5.6) and torsions were set automatically. The structures were then saved as PDBQT format files, too. Subsequently, PDBQT files of macromolecule receptors and corresponding small molecule ligands were imported into AutoDock Tools (version 1.5.6) for the construction of mating pockets. Further molecular docking simulation was conducted in AutoDock Tools (version 1.5.6) by using a Genetic algorithm. Autodock Vina (version 1.1.2) was used for docking the receptor protein with the small molecule ligands binding energy (DG in kcal/mol). Analysis and visualization were performed using PyMOL (version 2.4.0).