4.2. In Silico Study

Based on the existing literature, we selected two subunits (α2 and α3) of the GABA_A receptor liable for the anxiolytic activity to conduct molecular docking and ligand–receptor visualization [13,14,15]. Due to the unavailability of the 3D structures for the selected GABA_A receptor subunits in the RCSB Protein Data Bank [94], we developed a homology model to obtain the required 3D structures. The SWISS-MODEL was utilized to perform the homology model and obtain the desired receptors [95]. The sequences of the receptor subunits α2 (UniProt ID: {"type":"entrez-protein","attrs":{"text":"P47869","term_id":"126302547","term_text":"P47869"}}P47869) and α3 ((UniProt ID: {"type":"entrez-protein","attrs":{"text":"P34903","term_id":"462149","term_text":"P34903"}}P34903) were collected from the UniProt database (http://www.uniprot.org/ (accessed on 18 May 2023)) [96], then, a BLAST assessment was performed with the aid of the NCBI BLAST [97] tool to choose the template. The GABA_A homology modeling structures were assessed by GMQE [98] and a Ramachandran plot, via ProCheck [99,100,101]. After collection and developing homology, the receptors were fully optimized to eliminate docking interference by removing all unimportant molecules, and macromolecules, such as lipids, heteroatoms, and water molecules from the sequence of designated receptors using the PyMol software package (v2.4.1) [102,103,104]. Eventually, the receptors underwent energy minimization and geometry optimization using the SwissPDB Viewer software package. This process used the GROMOS96 force field, and the resulting PDB file was saved for subsequent molecular docking analysis.

The 3D conformers of diazepam (Compound CID: 3016) and trans-ferulic acid (Compound CID: 445858) were downloaded in SDF format from the PubChem chemical database (https://pubchem.ncbi.nlm.nih.gov/ (accessed on 18 May 2023)). Subsequently, the 3D conformers of the selected ligands underwent energy minimization via the Chem3D 16.0 program package. Then, the minimized conformers were saved as SDF files in preparation for the molecular docking process. In this respect, Gaussian View software (v5.0) was employed to optimize all the ligands. The 2D chemical structures of the ligands are depicted in Figure 6.

Chemical structures of standard and test compounds.

Molecular docking to estimate the active binding affinity of the selected ligands against the active sites of GABA_A receptors was conducted with the aid of the PyRx software package. To carry out molecular docking, the dimensions of the gird box were set as 80 × 80 × 80 Å along the x-, y- and z-axes, respectively, and the calculation was run at 200 steps [105]. The outcome of the docking potential is stored in ‘.csv’ format, while the ligand–protein complex is saved in PDB format. Additionally, the ligand is collected in PDBQT format for further analysis. We used the Discovery Studio Visualizer (v21.1.020298) and PyMol (v2.4.1) software applications to determine the interactions between ligands and receptors and the receptor’s active site. Table 1 lists the interacting amino acid (AA) residues and bond types between the ligand–receptor interactions.