3. RESULTS:
3.1 DOCKING:
The docking analysis of the compounds docked with Mpro and spike protein of SARS-CoV-2 yielded negative values for free energy respectively they were as follows. The grid box expressed negative values for free energies observed in Mpro they are -1.068 KJ/mol was observed for Eritadenine, -3.167 KJ/mol for Gallic acid, -0.420 KJ/mol for Ergosterol peroxide and -5.035 KJ/mol for Pleuran. Similarly, the values of free energies observed in Spike Protein were -0.783 KJ/mol, -2.140 KJ/mol, 1.027 KJ/mol and -5.241 KJ/mol respectively for the above four compounds. The binding confirmations of the docked protein of Mpro are hydrogen bonds and salt bridge formation for Eritadenine, hydrogen bonds were formed in Gallic acid, hydrogen bond and hydrophobic interactions were developed in Ergosterol peroxide and hydrogen bonds were formed with the molecule of Pleuran. Correspondingly, the binding confirmations of the docked protein of spike protein are hydrogen bonds were formed in the ligands of eritadenine, gallic acid and pleuran; the hydrogen bonds and hydrophobic interactions are developed in the compound Ergosterol peroxide.
3.1.1 ERITADENINE:
3.1.1.1 DOCKED POSE AND PROTEIN LIGAND INTERACTION:
The docked pose of the ligand eritadenine along with protein ligand interaction is given in the figures 2a, 2a1, 2b and 2b1. And protein ligand interactions are tabulated in Table-1.
3.1.2 GALLIC ACID:
3.1.2.1 DOCKED POSE AND PROTEIN LIGAND INTERACTION:
The docked pose of the ligand gallic acid along with protein ligand interaction is given in the figure 2c, 2c1, 2d and 2d1. And protein ligand interactions are tabulated in Table-2.
3.1.3 ERGOSTEROL PEROXIDE:
3.1.3.1 DOCKED POSE AND PROTEIN LIGAND INTERACTION:
The docked pose of the ligand ergosterol peroxide along with protein ligand interaction is given in the figure 2e, 2e1, 2f and 2f1. And protein ligand interactions are tabulated in Table-3.
3.1.4 PLEURAN
3.1.4.1 DOCKED POSE AND PROTEIN LIGAND INTERACTION:
The docked pose of the ligand pleuran along with protein ligand interaction is given in the figure 2g, 2g1, 2h and 2h1. And protein ligand interactions are tabulated in Table-4.
3.2 MOLECULAR DYNAMICS:
3.2.1 ROOT MEAN SQUARE DEVIATION (RMSD) AND ROOT MEAN SQUARE FLUCTUATION (RMSF):
The RMSD, RMSF produced from the docked compounds with targets Main Protease (MPro) and Spike Protein (S) are given as Figure 3a, 3b, 4a and 4b as follows:
3.2.2 HYDROGEN BONDING:
The hydrogen bonding which was formed during the molecular dynamics simulation was tabulated in Tables 5, 6, 7 and 8 for the ligand-protein bound complexes of Main Protease (MPro) and in Tables 9, 10, 11, and 12 for the docked complexes of Spike Protein (S).
3.3 ADME PREDICTION:
3.3.1 ERITADENINE:
The results which appeared after the submission of Eritadenine molecule in the SWISS-ADME database showed that the molecule, Eritadenine 18 heavy atoms, 7 hydrogen bond acceptors, 4 hydrogen bond donors and 147.38 Å2 of Topological Polar Surface Area (TPSA). The lipophilicity parameters are optimal in nature. The estimated solubility of Eritadenine is -0.36 which points it to be in the very soluble class, in silico prediction of aqueous solubility with response to TPSA is about -0.75 which categorizes to very soluble class as well, water solubility by fragmental method puts the compound to very soluble class with assigning the value of -0.29. The pharmacokinetics properties determined that Eritadenine has low gastrointestinal absorption, non-blood brain barrier permeant, does not act as P-gp substrate and does not inhibit factors such as CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4. The molecule is skin permeable with log Kp rate about -9.14cm/s. And the molecule obeys Lipinski’s rule of five and Muegge rules of druglikeness and also obeys Pan-Assay Interference Compounds (PAINS), Brenk, Leadlikeness rules of medicinal chemistry.
3.3.2 GALLIC ACID:
The results of Gallic acid contains 12 heavy atoms, 5 hydrogen bond acceptors, 4 hydrogen bond donors and TPSA is found to be 97.99 Å2. The lipophilicity parameters were very optimal for the ligand. And water solubility parameters were soluble in nature as predicted values are -1.64, -2.34 and -0.04 with respect to estimated solubility (Log S-ESOL), in silico prediction of solubility with reference to TPSA (Log S- Ali) and water solubility by fragmental method (Log S- SILICOS-IT) which gives an inference of very soluble, soluble and soluble classes according to the values projected. Estimated pharmacokinetic data projects that the Gallic acid is highly absorbable gastrointestinally, non- blood brain barrier permeant, non-P-gp substrate, does not inhibit potential cytochrome P450 enzymes as mentioned above, also has skin permeability rate of -6.84 cm/s. And the compound obeys druglikeness rules such as Lipinski’s, Veber and Egan. But the molecule doesn’t obey any rules of medicinal chemistry.
3.3.3 ERGOSTEROL PEROXIDE:
The ligand Ergosterol peroxide has 31 heavy atoms, 3 hydrogen bond acceptors and 1 hydrogen bond donor and TPSA is about 38.69 Å2. They are highly lipophilic in nature. The water solubility parameters are with predicted values of -6.46 for Log S- ESOL which is poorly soluble, -7.33 for Log S-Ali which is poorly soluble and -4.51 for Log S- SILICOS-IT which is moderately soluble. The pharmacokinetic estimated data exclaims that the molecule is highly absorbable gastrointestinally, non- blood brain barrier permeant, non-P-gp substrate, does not inhibit potential cytochrome P450 enzymes such as CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4. It also has skin permeability rate of -4.15 cm/s. Ergosterol peroxide obeys druglikeness rules of Lipinski and Veber. There’s no PAINS violation in medicinal chemistry rules.
3.3.4 PLEURAN:
Pleuran has 41 hydrogen bond acceptors, 26 hydrogen bond donors and it has TPSA about 664.43 Å2. They are highly lipophilic and less hydrophilic in nature. The pharmacokinetic data shows that the molecule is a non P-gp substrate, highly absorbable gastrointestinally, non- blood brain barrier permeant and does not inhibit potential cytochrome P450 enzymes such as CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4. The molecule obeys PAINS and Brenk medicinal chemistry rules.
3.4 TARGET PREDICTION:
After the analysis the target prediction of the ligands Eritadenine, Gallic acid, Ergosterol peroxide and Pleuran were given accordingly as the pie charts for top 15 hits for Eritadenine, all estimates for Gallic acid, top 15 hits for Ergosterol peroxide and all estimates for Pleuran (Figures 10-13) and the respective log output table with known protein complexes are given along with probability score with each binding to estimated protein complexes, the output table of results for the compounds are given in the supplementary file. As projected by the output file the compounds may reach the target with high accuracy to which it is targeted to. Whereas the target activities of Eritadenine, Gallic acid, Ergosterol peroxide and Pleuran are sought in literature and their validation is discussed in the discussion section to provide the fact of target accuracy.
3.5 TOXICITY PREDICTION:
3.5.1 SYSTEMIC TOXICITY:
The systemic toxicity properties of the ligands eritadenine, gallic acid, ergosterol peroxide and pleuran are tabulated in Table-13.
3.5.2 TOX21 PATHWAY:
The properties of the ligands which are studied for TOX21 pathway are given in the Table-14 as follows:
3.5.3 TOXICOPHORE RULES:
The toxicophore rules of the ligands of interest are tabulated in Table-15.