Target protein conformation
The RdRp (RNA-dependent RNA Polymerase) Cryo-EM structure of the apo nsp12-nsp7-nsp8 complex protein (PDB ID 7BV1) with resolution 2.80Ǻ and the Crystal structure of SARS-COV-2 nucleocapsid protein N-terminal RNA binding domain (PDB ID 6M3M) with resolution 2.70Ǻ were retrieved from Protein Data Bank (PDB) database. The retrieved proteins checked structural conformations by ERRAT[30], PROCHEK[31] and PDBsum[32]. The validated 3d, secondary structural analysis and Ramachandran plot were shown in Fig.1, Fig.2.
The cavities (active sites) of the proteins RdRp and Nucleocapsid were obtained through MVD cavity detection algorithm and its place with volume illustrated in Fig.3.
Determination of ligand structures
The 56 structures of small molecules and natural plants compounds were used to find out their potential binding with the target proteins RdRp and N(Nucleocapsid). The compounds structural information and structural flexibility after single point energy and after geometrical optimization energy were shown in Table 1.
Validation of Docking Results
During the docking study, each compound is selected as best pose to determine the MolDock score, Rerank score, interactions energy, torsion angle and H-bonding against N and RdRp protein. The MolDock score, Rerank score, the total interaction energy between the pose and the target molecule, torsion angle and H-bonding energy of 56 compounds against N protein and RdRp protein are represented in Table 2 and Table 3 respectively. In our study we compared the binding efficacy of ligands against protein with and without energy minimization. We found that energy minimized compounds shows better binding results[33]. Among all docked 56 ligands, 9 compounds shows better binding energy with N protein and 7 compounds with RdRp protein. Among all these 56 compounds 9 and 7 compounds shows better inhibitory effect on N protein and RdRp protein respectively.
N protein results
Out of all 56 compounds, Cairicoside I, Ginsenoside rb1, polyphyllin I, Gambogic acid, Betanin, and Alpha solanine revealed the most lowest MolDock score on N protein which is -285.68kcal/mol, -172.65kcal/mol, -166.78kcal/mol, -164.94kcal/mol, -160.78kcal/mol, -160.01kcal/mol respectively and Naringin, Salvianolic acid A, Betanin, Paeoniflorin, Ginsenoside rb1, Polyphyllin I, Baicalin shows excellent H-Bonding on N protein which is -23.82 kcal/mol, -23.26 kcal/mol, -19.80 kcal/mol, -19.32 kcal/mol, -18.50kcal/mol, -18.28kcal/mol -18.27kcal/mol respectively. By comparing entire parameters out of 56, 9 compounds were selected for better inhibitory studies, which are Alpha solanine binds into the active site of N protein with MolDock score -160.01kcal/mol & binding site consists of amino acid residues like Asp129A, Lys62B, Glu68B, Lys128A, Asp64B, Arg89A, Glu119A, Pro118A, Asn154D, Ile131B, Ile132B, Trp132B, Trp133B, Ala126B, Gly125B, Asn127B, His146D, Ile147D, Trp53D, Asn78D, Asn151A, Asn49A, Asn155D, Thr50A. It forms hydrogen bonds on Arg89A, Asn127B, Asn155D, Gly125B, Asn78D, Asn49A, Ile131B amino acid residues with binding energy -17.11kcal/mol. Baicalin binds into the active site of N protein with MolDock score -105.37kcal/mol & binding site consists of amino acid residues like Trp109B, Lys66B, Lue65B, Asp64B, Ile132B, Arg90B, Gly130B, Ile131B, Trp133B, Asn49A, Asp129B,Lys128B, Asn154B, Asn151D, Asn127B, Asn155B, Arg150D, Trp53B, Thr149D. it forms hydrogen bonds on Asn151D, Thr149D, Asn127B, Asn155D, Asn154D, Gly130B, Asp129B, Asp64B With binding energy -18.27kcal/mol. Betanin binds into the active site of N protein with MolDock score -160.78kcal/mol & binding site consists of amino acid residues like Trp133B, Lys128B, Asn127B, Trp53D, Asn78D, Asn49A, Thr50A, Ala51A, Arg89A, Ala91A, Arg90A, Thr92A, Lys66B, Glu63B, Pro169B, Lys170B. it forms hydrogen bonds on Asn127B, Arg89A, Arg90A, Thr92A, Glu63B, Lys66B With binding energy -19.8kcal/mol. Cairicoside I binds into the active site of N protein with MolDock score -285.68kcal/mol & binding site consists of amino acid residues like Trp53D, Ile147D, Ile158D, Asn78D, Asn155D, Asn154D, Asn127B, Asn151A, Thr50A, Pro118A, Tyr112A, Ser52A, Ala51A, Gly125B, Ile131B, Trp133B, Ile132B, Arp69B, The67B, Pro68B, Val159C, Tyr110A, Asp64B, Trp109B, Lue65B, The67B. it forms hydrogen bonds on Asn49A, Asn127B, Asn154D, Asn78D, Thr50A, Pro68B, Lys66B With binding energy -16.8kcal/mol. Ginsenoside rb1 binds into the active site of N protein with MolDock score -172.65kcal/mol & binding site consists of amino acid residues like Arg108A, Arg93A, Thr92A, Ana91A, Tyr110A, Ser52A, Tyr112A, Ala51A, Arg89A, Thr50A, Tro118A, Asn49A, Lys66B, Asn155D, Asn154D, Asn151D, Asn127B, Gly125B, Trp133B, Ile132B, Ile131B, Lys128B, Ala126B, Thr149D, Asn151D, Arg150D, Trp53D. it forms hydrogen bonds on Asn155D, Asn151D, Thr149D, Asn127B, Asn49A, Lys128B, Ile131B, Thr50A, Tyr112A With binding energy -18.50kcal/mol. Naringin binds into the active site of N protein with MolDock score -145.45kcal/mol & binding site consists of amino acid residues like Arg90A, Arg89A, Thr92A, Tyr110A, Tyr112A, Ser52A, Pro118A, Glu63B, Ala51A, Thr50A, Asn49A, Lys66B, Trp153B, Ile132B, Phe67B. it forms hydrogen bonds on Trp133B, Thr50A, Thr92A, Glu63B, Lys66B, Arg89A, Ser52A, Tyr122A With binding energy -23.82kcal/mol. Paeoniflorin binds into the active site of N protein with MolDock score -114.76kcal/mol & binding site consists of amino acid residues like Trp53D, Asn155D, Thr50A, Asn49A, Asn127B, Asp129B, Lys128B, Ala126B, Gly125B, Gly130B, Tle131B, Trp133B, Lys66B. it forms hydrogen bonds on Asn127B,Thr50A, Ile131B, Ala126B, Lys128B, ASN 49A With binding energy -19.32kcal/mol. Polyphyllin I binds into the active site of N protein with MolDock score -166.78kcal/mol & binding site consists of amino acid residues like Thr50D, Thr149D, Gly148D, Tle147D, Trp53D, Asn51D, Asn155D, Asn154D, Asp129D, lys128D, Asn127B, gly130B, Thr50A, Ala51A, Lys66B, Pro152A, Asn49A, Ile132B, Trp133B, Phe67B, Pro68B, Arg69B, Gln161C. it forms hydrogen bonds on Arg69B, Trp133B, Phe67B With binding energy -18.28kcal/mol. Salvianolic acid A binds into the active site of N protein with MolDock score -143.71kcal/mol & binding site consists of amino acid residues like Tyr112A, Arg89A, Pro118A, Thr50A, Asn154D, Asn155D, Trp53D, Asn127B, Asn49A, Lys66B, Asp64B, Lys128B, Asp129B, Ile131B, Gly130B, Arg90B. It forms hydrogen bonds on Asn49A, Thr50A, Arg89A, Gly130B, Asp129B, Ile131B, Lys128B, Asn127B, and Tyr112A With binding energy -23.26kcal/mol. Fig.4 illustrates hydrogen bonding with amino acids of corona virus protein N.
Rdrp Protein Results
Out of all 56 compounds, Alpha solanine, Betanin, Cairicoside I, Gambogic acid, Ginsenoside rb I, Salvianolic acid A and Lycopene revealed the most lowest MolDock score on RdRp protein which is -151.07kcal.mol, -156.39kcal/mol, -201.55kcal/mol, -164.15kcal/mol, -147.04kcal/mol, -150.83kcal/mol, -159.57kcal/mol respectively and Polyphyllin I, Naringin, Ginsenoside rb 1, Gentiopicroside, Betanin and Alpha solanine shows excellent H-Bonding on RdRp protein which is -18.45kcal/mol, -18.25kcal/mol, -19.13kcal/mol, -20.08kcal/mol, -17.11kcal/mol and -16.47kcal/mol respectively. By comparing entire parameters out of 56 compounds, 7 compounds were selected for better inhibitory studies, which are Alpha solanine binds into the active site of RdRp protein with MolDock score -151.07kcal/mol & binding site consists of amino acid residues like Val557A, Ser682A, Lys545A, Thr556A, Arg555A, Asp62A, Asp760A, Asp452A, Arg624A, Cys622A, Arg553A, Tyr455A, Lys551A, Lys621A, Pro620A, Lys798A, Tyr619A and Ala554A. It forms hydrogen bonds on Lys621A, Cys622A, Tyr619A, Asp760A, and Asp623A amino acid residues with binding energy -16.47kcal/mol. Betanin binds into the active site of RdRp protein with MolDock score -156.39kcal/mol & binding site consists of amino acid residues like Lys545A, Lys500A, Val557A, Gly683A, Thr556A, Ser685A, Ala554A, Asp452A, Arg553A, Tyr455A, Lys621A, Arg624A, Asp623A, Asn691A, Ser759A, Thr687A, Ala688A, Ser759A and Lys545A. It forms hydrogen bonds on Ala688A, Thr556A, Arg624A, Arg555A, Asp452A, Lys621A, Asp623A and Lys545A amino acid residues with binding energy -17.11kcal/mol. Gentiopicroside binds into the active site of RdRp protein with MolDock score -89.50kcal/mol & binding site consists of amino acid residues like Tyr619A, Cys622A, Pro620A, Lys621A, Tyr458A, Asp623A, Arg624A, Tyr455A, Tyr458A, Arg553A, Asp452A, Ala554A, Arg555A and Tyr556A. It forms hydrogen bonds on Tyr619A, Cys622A, Asp623A, Lys621A, Arg555A and Ala554A amino acid residues with binding energy -20.08kcal/mol. Ginsenoside rb1 binds into the active site of RdRp protein with MolDock score -147.04kcal/mol & binding site consists of amino acid residues like Ala688A, Thr687A, Ser759A, Asn691A, Asp760A, Ser682A, Asp623A, Tyr619A, Cys622A, Arg555A, Thr556A, Asp618A, Pro620A, Lys621A, Asp452A, Ala554A, Tyr455A, Lys798A and Arg553A. It forms hydrogen bonds on Lys798A, Asp618A, Arg555A, Asp452A, Ala554A, Lys621A, Arg553A, Asp760A and Ser759A amino acid residues with binding energy -19.13kcal/mol. Naringin binds into the active site of RdRp protein with MolDock score -134.00kcal/mol & binding site consists of amino acid residues like Ala550A, Ser549A, Lys551A, Ala554A, Arg555A, Arg553A, Asp452A, Thr556A, Tyr455A, Arg624A, Lys621A, Ser682A, Asp623A, Cys622A, Thr687A and Asn691A. It forms hydrogen bonds on ASN 691A, Arg624 A, Thr556A, Lys621A, Cys622A and Asp623A amino acid residues with binding energy -18.25kcal/mol. Polyphyllin I binds into the active site of RdRp protein with MolDock score -140.21kcal/mol & binding site consists of amino acid residues like Thr556A, Ala554A, Asp452A, Ser682A, Thr687A, Arg553A, Tyr455A, Arg624A, Asp623A, Ser759A, Leu758A, Asp760A, Cys622A, Lys798A, Pro620A, Asp648A, Tyr619A, Lys798A and Lys551A. It forms hydrogen bonds on Asp623A, Thr556A, Arg555A, Ala554A, Asp452A, Ser759A and Lys798A amino acid residues with binding energy -18.45kcal/mol. Cairicoside I binds into the active site of RdRp protein with MolDock score -201.55kcal/mol & binding site consists of amino acid residues like Lys500A, Ala685A, Asp684A, Gly683A, Ala558A, Val557A, Ser682A, Thr687A, Ala688A, Ser759A, Leu758A, Asp761A, Cys813A, Ser814A, Asp760A, Tyr619A, Lys545A, Asp623A, Cys622A, Thr556A, Arg624A, Asp452A, Tyr455A, Arg553A, Lys621A and Pro620A. It forms hydrogen bonds on Asp684A, Asp761A, Asp760A, Lys545A, Lys621A, Arg624A and Ser628A amino acid residues with binding energy -7.29kcal/mol. Fig.5 illustrates hydrogen bonding with amino acids of corona virus protein RdRp.
Fig.6 and Fig.7 shows, steric interactions with residues of COVID-19 proteins N and RdRp respectively. Fig.8 and Fig.9 represents Ligand docked against the crystal protein structures of COVID-19 Nucleocapsid and RdRp Proteins respectively. The amino acid residues around active site and Docked against N and RdRp protein were illustrate in Table 4 and Table 5 with structures at Fig.10 and Fig.11 respectively.
ADMET results
In the docking study, more negative BE corresponded to the strong binding of selected compounds to the target proteins. It is a fact that weaker binding will ultimately have a rapid dissociation rate[34]. In this study, the selected compounds exhibited lower binding energy which means strong binding with the N and RdRp proteins, suggesting that these compounds could be utilize as an inhibitors of the RdRp and Nucleocapsid proteins to combat COVID-19. According to the Rule of Five, a molecule might be no longer orally active if it violates or greater of the 4 rules. But the rules are not suitable for natural products because they are complicated and they have been deduced from relatively simple small molecules[35]. However, we have filtered the compounds based on the binding efficacy on Both Proteins of COVID-19. The results of the pkCSM, SwissADME tool and molinspiration chemiinformatics tools shows that top scored compounds pharmacokinetic properties (N and RdRp protein inhibitory compounds) (Table 6).