In Silico IC50 Calculation

The inhibition constant in this study was determined by AutoDock 4.0 tools. AutoDock software calculates and predicts the interactions between a ligand molecule and a protein molecule, based on predefined parameters. The interactions between the molecules can be calculated at a specified user-defined region of the protein. This region must be defined by the user through the Grip map option or GridBox. Thus, the use of the GridBox at the binding site or active site, or at other essential protein regions, is essential to perform AutoDock analysis. Before executing the AutoDock, the “.pdb” files of the protein and ligand have to be moved into one folder.

Analysis in AutoDock can be divided into the following categories: a) Initialising molecules; and b) Running AutoGrid.

Initialising the molecule involved the addition of hydrogen atoms. The ligand molecule required the addition of the Gasteiger charge, the identification of aromatic carbons, the detection of rotatable bonds, and the setting of the torsional degrees of freedom (TORSDOF) value. The protein must be initialised manually, whereas the ligand is automatically initialised when opened in the tool. The receptor and ligand files were saved in.pdbqt format. Next, the receptor was opened again using the “Grid” menu and the “Macromolecules” sub-menu, and “open” was selected. The ligand opens using the “Set Map Types” sub-menu and “Open Ligand.” Then, the GridBox is set in AutoDock to cover the identified binding sites. AutoDock only analyses the ligand molecule’s interactions and the amino acids that are present within the GridBox. The GridBox’s size can be increased or decreased using the number of points in the X/Y/Z dimension. The position of the GridBox can be adjusted to cover the binding site or binding residues, using the “Center GridBox” field that moves the GridBox in the X, Y, and Z-axis.

After setting up the GridBox, the AutoGrid file was saved. When in the “Grid” menu, select the “Output” and “Save GPF” sub-menu. The file saves in.gpf format. Meanwhile, the AutoDock file saving procedure is to choose the “Docking” menu and select “Lamarckian GA” in the “Output” menu. The file saves in.dpf format. The AutoGrid and AutoDock programs was run through a command prompt directed to the folder to be docked using the command: autogrid4 -p control.gpf -l control.glg. After the AutoGrid calculation was completed successfully, AutoDock was run using the command: autodock4 -p control.dpf -l control.dlg. The results of the docking calculations are obtained in a notepad file format, reporting the values for binding energy and the estimated inhibition constant.