Molecular docking analysis and visualization

The PyRx virtual screening software integrated with the AutoDock Vina v1.2.0 platform was used to conduct a molecular docking analysis to assess the molecular binding energy of the requisite protein with the chosen phytochemicals^66,67. AutoDock Vina represents an open-source software for molecular docking and virtual screening, necessitating the 3D structures of receptor and ligand molecules in pdbqt file format to forecast their binding energy within studies focusing on receptor-ligand interactions⁶⁷. Docking was executed without selecting the binding pockets with the compounds serving as the ligands and the protein acting as the macromolecules. However AutoDock Vina can automatically calculate the active site (a specific region, often a pocket or groove, on the protein’s surface where substrate molecules attach and undergo a chemical reaction) with the help of algorithms that are integrated to predict and identify potential binding pockets based on the receptor's 3D structure⁶⁸. This analysis encompassed conducting rigid docking, wherein all rotatable bonds were converted into non-rotatable ones. The grid box position is a crucial factor in performing effective docking analysis, as it defines the specific area of the protein where the ligand docking will take place⁶⁹. Regions outside the grid box were not considered during the docking process. In our analysis, the center grid box size was set to 59.5132 Å along the x-axis, 50.9758 Å along the y-axis, and 62.3999 Å along the z-axis, covering the protein regions of our interest. The binding pocket regions were identified by DoGSiteScorer and the grid box size was set based on the output provided by DoGSiteScorer⁷⁰. Following the completion of the docking process, both the protein and the docked ligands were preserved in pdbqt format. This was undertaken to facilitate subsequent analysis of the docking score, non-bond interactions, and visualization. Additionally, BIOVIA Discovery Studio Visualizer v4.5 was utilized to visualize the interacting amino acid residues and the various types of bonds formed during these interactions⁵⁴.