2. MATERIALS AND METHODS:
2.1 DOCKING OF ERITADENINE, GALLIC ACID, ERGOSTEROL PEROXIDE AND PLEURAN ON SARS-COV-2 MAIN PROTEASE AND SPIKE PROTEIN:
2.1.1 LIGAND PREPARATION:
The ligands of interest Eritadenine (PubChem CID 159961), Gallic Acid (PubChem CID 370), Ergosterol (PubChem CID 5351516) were obtained from PubChem database 18. Pleuran was designed using Glycam-Web webserver 19. Molecules in 2D formats were converted to 3D mol2 format and protonated using OpenBabel20.
2.1.2 RECEPTOR PREPARTION:
3D crystal structure of SARS-CoV-2 Main protease (PDB ID: 6LU7) and SARS-CoV-2 Spike protein (PDB ID: 6YOR) were obtained from RCSB PDB database 21. Protein chains with appropriate chain identifiers for the protein were selected and retained other ligands present in the molecule were removed. Webservers 3DLigandSite22 and RaptorX binding 23 was used to identify the active site in the protein given in the figure 1. The protein receptor was processed using the Dock Prep module of UCSF Chimera using AMBER parm99 partial charges.
2.1.3 DOCKING PARAMETERS:
All docking calculations were performed using Dock6 version (6.6). Active sites were identified and prepared by selecting spheres at a distance of 1–5 Å from the binding regions. Grid boxes with 5 Å and 15 Å margins were generated around the selected spheres for small and large ligands respectively. A standard flexible docking protocol with the maximum number of anchor orientations attempted was set to 1,000 and all 100,000 orientations was employed to sample internal degrees of freedom of the ligands. Grid scoring based on the intermolecular non-bonded terms (viz. van der Waals for steric and electrostatic interactions) was computed using AMBER force field ff99 to identify the best orientation of each ligand. Finally, the best-scored conformer was retained for scrutiny and further refinement and Amber-based scoring. All protein ligand interactions were detected and visualized using by the PLIP webserver 24 and LigPlot 25.
2.2 MOLECULAR DYNAMICS OF ERITADENINE, GALLIC ACID, ERGOSTEROL PEROXIDE AND PLEURAN ON SARS-COV-2 MAIN PROTEASE AND SPIKE PROTEIN:
The docked structures were used for molecular dynamics simulations using AMBER force field and TIP3P water model in GROMACS 2019. The parameters for docked ligands were prepared using Acypype. A vacuum energy minimization step was performed by employing the steepest descent algorithm for 50,000 steps and conjugate gradient minimization was performed for 5000 steps. Sodium (Na+) and chloride (Cl-) counter ions were added at a physiological concentration and the boundaries of the dodecahedron box were adjusted to 10Å in a unit cell. The complexes were again subjected to energy minimization for 50,000 steps for stabilization. Position restraint and unrestrained dynamics simulations and Parrinello- Rahman pressure- coupling bath was used for equilibration step of the system at temperature 300K using a Nose-Hoover thermostat under pressure of 1 atm for 100 ps. For simulations, all bonds were constrained using the LINCS algorithm. The Particle- Mesh Ewald method was used for electrostatic calculations by maintaining a cut-off distance of 1.4nm for coulomb and Van der Waals interactions, and finally, the production run for 10 ns was performed. GROMACS built-in functions were utilized to calculate RMSD, RMSF and Hydrogen bonds. Excel was used to produce graphs of the results.
2.3 ADME PREDICTION:
The prediction of Adsorption, Distribution, Metabolism and Excretion was done by obtaining the Simplified Molecular-Input Line-Entry System (SMILES) format of the ligands Eritadenine, Gallic acid, Ergosterol peroxide and Pleuran was taken from PubChem database and was provided as an input to the SWISS-ADMET website which analyses the lipophilicity, water solubility, druglikeness and medicinal chemistry of the compound submitted 26.
2.4 TARGET PREDICTION:
The target prediction of the compounds Eritadenine, Gallic acid, Ergosterol peroxide and Pleuran was analyzed by entering the SMILES and by choosing the Homo sapiens button to the Swiss target prediction website which in turn provides info about possible side effects or cross reactivity caused due to the effect of molecules in the physiological or genetic effect in the organs of the human body27.
2.5 TOXICITY PREDICTION:
The toxicity of the ligands eritadenine, gallic acid, ergosterol peroxide and pleuran was analyzed by incorporating the SMILES data of these compounds from PubChem database to the ADMET Lab2.0 website and Toxicity option was selected and analyzed. ADMET Lab2.0 examines the ligand’s toxicity among systemic organs in the human body, TOX21 Pathway and Toxicophore property rules ranging from acute toxicity, genotoxic carcinogenicity, non- genotoxic carcinogenicity, skin sensitization, aquatic toxicity, non-biodegradability and sureChEMBL Rule28.