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.