The structures of the natural compounds are retrieved from the PubChem database 36. The structures of phytoestrogens (daidzein, genistein, formononetin and biochanin A, found in Cicer arietinum), palmitic acid (palm oil), linolenic acid (an essential omega-3 fatty acid found in vegetable oils like canola, soybean, flaxseed/linseed, and olive and some nuts), Chlorogenic acid (found in coffee) hydroxytyrosol (found in extra virgin olive oil), caffeic acid (found in many sources including berries, herbs, mushrooms, and coffee beans), caffeic acid phenethyl ester (CAPE, the bioactive component of honeybee hive propolis), p-Coumaric acid (found in fungi, peanuts, tomatoes, and garlic), cinnamaldehyde (found in Cinnamomum verum), and thymoquinone (found in the seeds of Nigella sativa), are retrieved using the following PubChem CIDs; 5281708, 5280961, 5280378, 5280373, 985, 5280934, 1794427, 82755, 689043, 5281787, 1549106, 637511, 10281, respectively. Additionally, the structures of physiological compounds like estrogens (estriol (5756), and β-estradiol (5757)), hydrocortisone (5754), cholesterol (5997), Progesterone (5994), and Testosterone (6013) are retrieved from PubChem database to be tested against HSPA5 SBDβ and compared to the natural compounds.
The only available solved structure in the Protein Data Bank (PDB) for the wild-type and full-length HSPA5 in the open configuration is 5E84 37,38. The coordinates of HSPA5 were downloaded and prepared for the docking study (water molecules and ligands are removed while missing Hydrogen atoms are added). National Center for Biotechnology Information (NCBI) nucleotide database was used to retrieve the gene (NC_045512.2) from which spike protein was translated (Expassy translate tool). A model was built with the aid of Swiss Model portal, where SARS HCoV (PDB ID: 6NUR, chain A) was used as a template in a previous study by the author 8,39,40. Structure analysis and verification server (SAVES) of UCLA was used to validate the model 41. The validated model of the SARS-CoV-2 spike was energy-optimized using the computational chemistry workspace SCIGRESS in order for the spike structure to be ready for the molecular docking experiments. The minimization of the model was performed using classical mechanics (MM3 force field) after Hydrogen atoms addition 42.
Docking experiments (AutoDock Vina software) are performed using the HSPA5 solved structure (PDB ID: 5E84) after 50 ns of classical molecular dynamics simulation (performed using NAMD software) 43-45. Four different conformations of HSPA5 representing the main four clusters (Chimera software) are used to test the ligands binding 46.
Thirteen different natural products-derived compounds are tested against the four different conformations of the host cell chaperone HSPA5 SBDβ, including; daidzein, genistein, formononetin, biochanin A, palmitic acid, linolenic acid, chlorogenic acid, hydroxytyrosol, caffeic acid, caffeic acid phenethyl ester, p-Coumaric acid, cinnamaldehyde, and thymoquinone. Additionally, six different physiological compounds are also docked to the HSPA5 SBDβ for comparison, including; estriol, estradiol, hydrocortisone, cholesterol, progesterone, and testosterone. All the dockings are done using flexible ligand into flexible active site protocol, where both the ligands and the active site residues (I426, T428, V429, V432, T434, F451, S452, V457, and I459) are treated as flexible during the search for a possible docking conformation using the vina scoring function of AutoDock Vina software 37,43. The grid boxes for the docking experiments were chosen to be of size 48 × 46 × 56 Å centered at (42.3, 54.9, -29.2) Å (with little differences between the different conformations of the HSPA5).
HADDOCK 2.4 web server is utilized to dock the spike model for SARS-CoV-2 against HSPA5 and the complex of HSPA5 with its docked ligands 47. The HADDOCK 2.4 easy interface was utilized in the study since there are no restraints to be defined 48. Again the HSPA5 active site (I426, T428, V429, V432, T434, F451, S452, V457, and I459) is treated as flexible. In contrast, the C480-C488 region of the SARS-CoV-2 spike is treated as the active residues (binding site) in HADDOCK 2.4, as reported in a previous study by the author 8.
After docking, the complexes are examined using the Protein-Ligand Interaction Profiler (PLIP) web server (Technical University of Dresden) 49.