Ethanopharmacological Approach to Control the Replication of 2019-nCov in Host-An Insilico Study.

2019-nCoV viral disease threatening every individual throughout the world. It is a highly challenging task to control the spread everywhere. There are certain antiviral drugs, steroids that are currently being prescribed to infected patients for faster recovery. But there is no proper cure today for this pandemic. In this present study, we have focused on controlling the replication and other possibilities of interaction of the 2019-nCoV virus inside the host cells. There are various herbs prescribed to us to improve our immunity and prepare our bodies to �ght against this pathogen. In this study, herb compounds that are selected are Andrographolide (AP 1 ), 14-deoxy-11,12-didehydroandrographolide(AP 3 ), Ascorbic acid, Cinnamaldehyde, Curcumin, Diallyl sul�de, Eugenol. Gingerol, Kaempferol, Deacetylnimbin, Piperine, Quercetin, thymol, thymoquinone, Vasicine based on the literature survey. Selected ligands are most of them to treat respiratory tract infections and are also related to improving humoral immunity. Selected Ligand was allowed to dock against viral proteins which Crystal Structure of the SARS COV-2 Papain-like protease (Figure 1a) (PDB ID: 6wx4), RNA dependent RNA polymerase (Figure 1b) (PDB ID: 7c2k), Crystal structure of SARS COV-2 ORF7A encoded accessory protein (Figure 1c) (PDB ID: 6w37) Crystal structure of SARS COV-2 ADP-Ribose phosphatase NSP3 Proteins (PDB ID: 6w6y), PDB ID 6zsl Crystal structure of SARS COV-2 helicase NSP13 (PDB ID: 6ZSL), Crystal structure of NSP10-NSP16 Complex (PDB ID: 7bq7), PDB ID 6xdc Crystal structure of SARS COV-2 ORF3a Protein (PDB ID: 6xdc) to study their e�cacy to control the replication and possible interactions in the human system using computational docking study. A Protein-Protein interaction study was also performed to study the e�cacy of papain enzyme inhibitory e�cacy of selected target proteins. Molecular dynamics studies were also performed to ensure the ligand e�cacy for the selected target proteins.

Introduction 2019-nCoV viral strain is a new edition of the coronavirus family.Coronovirade is a family of novel corona viral strains.It belongs to RNA viruses.Nowadays many anti-viral drugs, steroids are practicing to treat them to control the disease but it works differently with patients some may get cured others may continue to suffer (Yu et al., 2020).This viral genome expresses many proteins and it happens to interact with human host cells.There are various docking studies to carried out on envelop proteins.This present study focuses on three target proteins which are as follows Crystal Structure of the SARS COV-2 Papain-like protease (Figure 1a) (PDB ID: 6wx4), RNA dependent RNA polymerase (Figure 1b) (PDB ID: 7c2k), Crystal structure of SARS COV-2 ORF7A encoded accessory protein (Figure 1c) (PDB ID: 6w37) Crystal structure of SARS COV-2 ADP-Ribose phosphatase NSP3 Proteins (PDB ID: 6w6y), PDB ID 6zsl Crystal structure of SARS COV-2 helicase NSP13 (PDB ID: 6ZSL), Crystal structure of NSP10-NSP16 Complex (PDB ID: 7bq7), PDB ID 6xdc Crystal structure of SARS COV-2 ORF3a Protein (PDB ID: 6xdc) helped the virus to replicate in the host system (Barik et al., 2020).papain-like protease is one of the two important proteases in the viral genome.papain-like protease helps in the virus replication process and helps to destroy the viral response in the deubiquitination of host cell factors.The papain-like protease one of the targets for antiviral development processes (Rut et al., 2020).
RNA-dependent RNA polymerase is one of the important proteins in viral replication.It is a central compartment of viral replication and transcription machinery.It is one of the targets for antiviral drugs (Ishihama&Barbier 1994; Wang et al., 2020).
Orf7A is one of the open reading frame proteins in nCov19.ORF7A inhibits the activity of Bone Marrow Stromal Protein-2 (BST-2) which plays an important role in viral infections.It controls the release of virions into the system.Inactivation of BST-2 leads to the down-regulation in the in ammatory response.Inhibiting ORF7A helps to retain the BST-2 activity (Taylo et al., 2015).NSP3 is one of the important Non-structural proteins it plays a major role in host immune response.By inhibiting this NSP3 protein helps to preserve humoral immune response (Fireman et al., 2008, Lie et al., 2018,).ORF3a is one of the potential targets in treating nCov19 infection.ORF3a plays important role in viral pathogenicity (Gordon et al., 2020).
NSP13 is one of the important targets in controlling nCov19 infection.Studies showed that this hydrolase protein NSP13 plays a major role in this viral replication in the host cell (Mirza&Froeyen 2020).NSP10-NSP16 Protein complex possesses 2' -O-MTase activity which helps in host viral replication.So it may be one of the potential targets in nCov19 infection.Papain enzyme known for its medicinal value in this present study papain enzyme docked against selected target proteins and study the e cacy of papain enzyme selected target proteins (LanLonde et al., 1998).
This present study investigates the selected target proteins that will be docked against selected ligand molecules that are using traditionally to boost the immune system and also to treat the common cold and mild infections.
Selected ligands must be tested for their drug-likeness property.It is important to analyze the selected ligands should obey Lipinski's rule of ve.It helps to analyze the stability of the compound and ensures the ability to exhibit the proposed biological activity in our system.Based on Lipinski's rule the selected compounds ensure whether the selected ligand molecules will be orally active or not (Lipinski et al., 2001

Ligand Structures
The ligand selection is based on herbs that are used to treat the common cold.In this present study, these ligands were selected to study the e ciency of selected target proteins.There were no studies performed before with these target proteins against selected target proteins.Ligand structures were downloaded from the PubChem database.(

Molecular Docking
The protein structure was downloaded and prepared using autodock software, where Kollman charges and polar hydrogen were added and converted the structure format to .pdbqtle format.The ligand was prepared by detecting the rotatable bonds and set the aromatic criterion, and the ligand structure format converted to .pdbqtformat.The Grid parameter le should get prepared for further docking study.Grid generated by selecting grid size, Crystal structure of SARS COV-2 helicase NSP13 (PDB ID: 6ZSL)-90x90x90, Crystal structure of SARS COV-2 ADP-Ribose phosphatase NSP3 Proteins (PDB ID: 6w6y)-80x80x80, Crystal structure of SARS COV-2 ORF3a Protein (PDB ID: 6xdc) -90x90x90, RNA dependent RNA Polymerase-70x70x70, Crystal structure of SARS COV-2 ORF7A encoded accessory protein (PDB ID: 6w37)-60x70x60, Crystal structure of NSP10-NSP16 Complex-80x80x80, Crystal Structure of the SARS COV-2 Papain-like protease (PDB ID: 6wx4)80x70x80 before performing docking study.Grid size helps the ligand to identify its binding pocket.Docking parameter le should get prepared for docking calculations by choosing macromolecule, as well as ligand le and nally docking parameter le written using the Lamrickan algorithm.

Molecular Dynamics Simulations
In our study, we performed molecular dynamics simulations of protein-ligand, protein-protein complexes using DESMOND and GROMACS 4.0.6 software package.OPLSAA and charmm force elds were used for preparing the complexes and a 300 K constant temperature was maintained for the simulations.The molecular simulation time of protein-ligand, protein-protein complexes was set at 50ns and 30ns.xmgrace software was used to plot the RMSD graphs.

ADME/T Prediction
The drug-likeness property of selected ligands was analyzed so that it ensures oral bioavailability in the human system.Based on the results all the ligands were obeying the rule of ve.Results ensured that the selected ligands are exhibits the intended property in the human system Papain-like protease protein is one of the target proteins in this docking study.Ligands were selected based rationale based on their previous experimental studies for different diseases.Inhibitor e ciency of the selected ligands based on the binding a nity and hydrogen bonds formed in the active site pocket.Based on the Score Nimbin>Piperine> 14-deoxy-11,12didehydroandrographolide(AP3)>andrographolide(AP1)> Quercetin> Kaempferol> Curcumin> Vasicine>Thymoquinone>Cinnamaldehyde>Ascorbicacid>thymol> Eugenol>Gingerol>Diallaylsul de.Based on the hydrogen bond interactions we found that selected ligands were docked in the S1/S2 binding pockets which are said to be the active site pocket of papain-like protease (Rut et al., 2020).HIS73, ARG 82, ILE123, GLU124, LEU125, PHE127, ASP156, ASP164, ARG166, GLN174, HIS175, LEU178, LYS200, VAL202, GLU203, TYR273, THR301[Table 3].Piperine is a potential ligand structure base on its clinically proven therapeutic activity.It showed better binding a nity but it made no hydrogen bond with active site residues (Figure 2a).
Crystal structure of SARS COV-2 ORF7A encoded accessory protein NSP3 Protein was docked against selected ligands and it was docked well in the active site of the NSP3 protein.PHE6, GLY8, TYR9, LYS11, LYS19, GLU120, TYR152, TYR161(Figure 2d) are the interacting residues found in top-ranked docking poses.Nimbin was the top-ranked docking pose with greater binding a nity compared with other ligand structures (Table 3).
Crystal structure of SARS COV-2 helicase NSP13 NSP13 protein was docked against selected ligand structures.Based on the docking score and interacting amino acids con rmed that the ligands are bound well in the active site of the proteins.From all the results Nimbin structure binding a nity better than other ligand structures (Table 3).It may have the inhibitory effect of the NSP13 protein thereby controlling the nCoV19 infection.LYS146, VAL181, LYS192, GLN194, THR214, SER229, MET 233, SER236, ALA237, VAL340, CYS342, ASP344, THR351, LEU363, ARG390(Figure 3e).

Protein-Protein Interaction Study
Papain has interacted with target proteins.Papain enzyme ASN64, TYR61, ARG59, TYR67, GLN112, SER205 interacted with amino acid residues of nCov19papin like protease enzyme TYR137, ARG138, TYR71, LEU16, ASN15.Interacting residues were found in the active site of papain-like protease protein (Figure 3a).In Papain enzyme and RNA dependent, RNA polymerase docking following papain enzyme amino acid residues which are as follows GLN112 and TYR116 have interacted with GLN444 and ALA 443 of RNA dependent RNA polymerase enzyme (Figure 3b).
Papain enzyme ASN117, ARG191, ILE1 made hydrogen bonds with the following amino acid residues from the nCov19 ORF7A which are as follows TYR5, GLU1, PRO9, and HIS58.Interacting residues were in the active sites of the ORF7A protein (Figure 3c).
Papain enzyme SER216, THR223, SER58, GLN116 made hydrogen bonds with the following amino acid residues from the nCoV ORF3A which are as follows THR223, SER216, GLN116, SER58.Interacting residues were in the active sites of ORF3A protein (Figure 3d) Papain enzyme TYR116, ASN155, TYR59 made hydrogen bonds with the following amino acid residues from the nCoV NSP3 protein which are as follows PHE475, TYR476, GLU591 were in the active sites of NSP3 protein (Figure 3e).
Papain enzyme TYR61, ARG59 made hydrogen bonds with amino acid residues from the nCoV NSP3 protein which are as follows VAL49, GLY 48.Interacting residues were found in the active site of the target protein (Figure 3g).

Molecular dynamics simulations interpretation of docked complexes
In the molecular dynamics simulations, we used the RMSD of the backbone to examine whether the system reaches its stability and equilibrium.The prepared complexes were immersed in a periodic water box, ions added to the box and the equilibration was performed at 200 k constantly.The nal molecular dynamics was performed for 30ns for all the protein-protein and 50ns for protein-ligand complexes.
Figure 4 shows that the RMSD plot for 6w37-9pap complexes (red-protein, black -protein with ligand), its shows the stability of the proteinprotein complex.
The RMSD of the 6w37-piperine complex shows stable conformation after 25ns from starting point of the stimulation and it represents the steady-state up to 50ns (Figure 5 (a)).The RMSF of the protein and ligand plot shows the local characteristic changes and positions (Figure 5(b),(c)).Protein secondary structures like alpha-helix and beta-strands were interpreting throughout the simulations, alpha helices highlighted with red color and blue color indicates the beta-strands, and SSE plots show the residue index throughout the simulation (Figure 5 (d)).From the SSE plots, the residue index shows beta-strands completely.The protein-ligand contacts (Figure 5 (e)) show the bond interaction with the residues, hydrophobic interactions found with HIS4, VAL14, LEU15, LEU16, PHE48, TYR60, LEU62 residues.The hydrogen bond interactions were found with GLN6, LYS17, GLN61 residues, and only one water bridge was found with LYS17 and no ionic interactions were found between the protein-ligand.

Conclusion
This present study deals with the computational docking study using small molecules and an enzyme to control the replication of the virus in the host cell.Selected small molecules were studied for their drug-likeness property because they are herbal-based compounds.Based on this study Nimbin, Piperine and Quercetin are the lead molecules that were bound well in the active site of target proteins (Papain-like protease, RNA dependent RNA Polymerase, ORF7A protein, NSP3, NSP13, NSP10-NSP16, ORF3A).Based on the Scores and interactions ensure the binding a nity and protein-ligand stability.It ensures that the lead molecules were strong enough to inhibit the target proteins.Papain enzyme was also docked against the selected target proteins using the Protein-Protein interaction server.Molecular dynamics studies were performed for the proteins NSP13, 6w37 with piperine ligand results were ensured that the ligand can inhibit the target proteins.In the same way, proteinprotein dynamics studies were also performed and ensured that the papain enzyme structure also can control the target protein ORF7A.This present study was performed to choose multiple targets to control the infection with studied ligand structures.

Table 3
Molecular docking score and interacting residues for selected target proteins