key: cord-0958446-h5igxjji
authors: Abdullah Alharbi, Raed
title: Structure Insights of SARS-CoV-2 open state envelope protein and inhibiting through active phytochemical of ayurvedic medicinal plants from Withania somnifera
date: 2021-03-18
journal: Saudi J Biol Sci
DOI: 10.1016/j.sjbs.2021.03.036
sha: e1000718c84e1cd77799720947fec6672ee40b97
doc_id: 958446
cord_uid: h5igxjji

Coronaviruses have been causing pandemic situations across the globe for the past two decades and the focus is on identifying suitable novel targets for antivirals and vaccine development. SARS-CoV-2 encodes a small hydrophobic envelope (E) protein that mediates envelope formation, budding, replication, and release of progeny viruses from the host. Through this study, the SARS-CoV-2 E protein is studied for its open and closed state and focused in identifying antiviral herbs used in traditional medicine practices for COVID-19 infections. In this study using computational tools, we docked the shortlisted phytochemicals with the envelope protein of the SARS-CoV-2 virus and the results hint that these compounds interact with the pore-lining residues. The molecular level understanding of the open state is considered and the active inhibitors from the phytochemicals of Ayurvedic medicinal plants from Withania somnifera. We have thus identified a potential phytochemical compound that directly binds with the pore region of the E protein and thereby blocks its channel activity. Blocking the ion channel activity of E protein is directly related to the inhibition of virus replication. The study shows encouraging results on the usage of these phytochemicals in the treatment/management of SARS-CoV-2 infection.

The RNA virus is terrible in nature due to its tendency to cause sudden outbreak and among that, the coronavirus is highly cause for recent pandemic situations (Paital, 2020) .

Respiratory Syndrome) are considered high-lethality viruses that trigger infections of the common cold to fatal pneumonia (Hajjar et al., 2013) . In the follow-up, SARS-CoV-2 also joins the game, and the 2019 outbreak caused by the novel coronavirus (2019-nCoV) and disease referred to as COVID-19. In 2020, COVID-19 is the most recognized word because of its outbreak and has resulted in infections of up to 111 million people by February 2021 (Selvaraj et al., 2020c) . The genomic architecture of SARS-CoV-2 has RNA ~ 30 kB on translation, converts to four structural proteins, sixteen non-structural proteins and ten accessory proteins.

The structural proteins are Spike protein (S), Envelope protein (E), Membrane protein (M), Nucleocapsid protein (N) is playing the vital function of forming the virus protein interface to the external environment (Satarker and Nampoothiri, 2020) . The S protein fuses with ACE2 (Angiotensin-Converting Enzyme 2) in the first step with the receptor binding domain (RBD) and initiates the virus host recognition mechanism. Another structural protein, N and M respectively involved in forming the nucleocapsid proteins and provide shape to the viral envelope (Tang et al., 2020) . Here S interaction with M required for retaining the S in ERGIC/Golgi complex and also for integrating the new virions, M interactions with N plays stabilizing the capsid stability/assembly and M interactions with E playing the role in viral envelope formations and also in release of new virions. They are all active from entry to exit mechanism of viral pathogenesis inside the human host cell (Mukherjee et al., 2020) .

Among the structural proteins, E protein is integral membrane ion channel protein, but short in length comprises 76-109 amino acids. In this, 07 th to 12 th amino acid is hydrophilic, followed by long hydrophobic transmembrane domain (TMD) and a long hydrophilic carboxy terminus (Bianchi et al., 2020) . In E protein, the specialized structural phenomenon TMD contains, an amphipathic α-helix which oligomerize to form ion-conducting pore in membranes. In this ion channel, the ions are allowed to pass through the lipid channels in ERGIC/Golgi membranes, which results in activation of NLRP3 inflammasome and production of IL-1β (Farag et al., 2020) . E protein plays imperial role in the human cell disruption, immune evasion, pathogenesis, virion structure formation and exit (Tang et al., 2020) . This makes E protein as an attractive target for the drug design after the spike and main proteases (Nayarisseri et al., 2020) . For identifying the inhibitors, there are several methods to find suitable drug candidates and now a days, powerful computational screening is available (Shah et al., 2020) . But the intention of the work is to find the potential phytochemical compounds from natural herbs for treating SARS-CoV-2, by considering channel form of E protein. For tracking suitable inhibitors, the traditional medicinal plants are analysed and found that, compounds from Withania somnifera may have the ability to interact with E protein and inhibit the viral pathogenesis . For this, the desired compounds from Withania somnifera are docked with pentamer chain E protein of SARS-CoV-2 and the results are provided. From this work, the results show that Withania somnifera based inhibitors are having high potential and can effectively block the critical viroporin with high specificity and affinity.

Protein-Protein Interaction analysis

The viral E protein interactions with human protein for both SARS-CoV (Pfefferle et al., 2011) and SARS-CoV-2 (Gordon et al., 2020) is analysed with CoVex protein-protein interaction prediction server. Here, each viral protein will be displayed with interacting host proteins and for this study, the E proteins interactions with host proteins, as per literature is visualized and provided (Sadegh et al., 2020; Sivakamavalli et al., 2014) .

The E protein sequence from SARS-COV (Sequence information from PDB ID: 2MM4) and SARS-CoV-2 is visualized for sequence alignment. But the NMR solved structure for E protein from SARS-CoV-2 is not solved fully (Mandala et al., 2020; Yadav et al., 2014) , and so the whole sequence (Uniprot ID: P0DTC4) is taken and visualized for multiple sequence alignment using Praline sequence viewer available in https://www.ibi.vu.nl/programs/pralinewww/ (Dijkstra et al., 2019; Selvaraj et al., 2014) .

As stated above, the full length and open state of E protein has not yet solved and so, the sequence of E protein from SARS-CoV-2 (Uniprot ID: P0DTC4) is taken from uniport and searched for suitable templates using the blast server (https://blast.ncbi.nlm.nih.gov/) (Altschul et al., 1990; Fazil et al., 2012) . Suitable template for building the whole proteins is chosen for multiple template-based modelling and subjected to modeller 9v7 (Beema Shafreen et al., 2014; Choudhary et al., 2020) . Based on the templates, three models are generated and multiple chains are formed as homopentameric ion channel. The final protein structure is visualized for the validation server using SAVS server 3 and based on Ramachandran plot, the final protein is chosen for the study (Selvaraj et al., 2020a) .

The Modelled protein, along with SARS-CoV E protein (PDB ID: 2MM4) and SARS-CoV-2 transmembrane domain protein (PDB ID: 7K3G) from protein data bank (PDB) is taken for the study (Mandala et al., 2020) . 

As of now, the active site information of SARS-COV-2 E protein is lacking the active site information, and so the homopentameric modelled E protein is subjected to Active site analysis using sitemap . The regions predicted from sitemap is marked and those regions are provided as input for grids. The molecular interactions between the (Umesh et al., 2020) . Lower energy conformations are evaluated and chosen for the molecular visualization using the Schrödinger 2D interaction visualizer.

The apo and docked ligand complex with best docking profiles and interactions are subjected to molecular dynamics simulations through Desmond molecular dynamics package (Selvaraj et al., 2018) . The E protein is a membrane bound structure and thus, the protein-ligand complex is placed in the POPC membrane along with TIP3P water model (Loschwitz et al., 2020) . The distance between the complex to edge of the orthorhombic box is measured to have 10Å distance and minimized using steepest descent up to 2000-time steps. Temperature is changed to 310 K as per human body temperature, and pressure and pH is set to default. Pressure is maintained using the Berendsen thermostats and barostat method and NPT ensemble is performed for 12 ps followed by NVT ensemble for 24ps (Muralidharan et al., 2015) . The timestep is maintained with NVT, NPT and MD simulation using the RESPA integer and the whole complex is simulated for 30ns of MD simulations. The 30ns MD simulations are analysed for the results from the simulated trajectories using the VMD molecular dynamics visualization tool. The MD simulations are performed for understanding the molecular stability of ligands inside the ion channel and so the results of RMSD values are extracted and the values are plotted (Humphrey et al., 1996) .

The 

As mentioned, the E protein in SARS-CoV-2 is a membrane integrating ion channel protein that plays the imperial role in budding, the release of progeny viruses from the host cell, and in the activation of host inflammasome. In this, five chains of E protein join to form All the best four compounds are having the aromatic ring feature, which are able to bind with hydrophobic regions in the pore regions. While dissecting the interactions, all these four compounds are able to interact with D and E chains of the homo pentamer. All the compounds showing prominent interactions, and 2D interaction viewer showed in figure 6 shows, hydrophobic residues surrounded the new leads. On repeating analysis also shows the interactions stronger with the same residues Arg74 (D), Asn77 (D), Thr43 (E), Cys46 (E), Ile59 (E), and Val60 (E). This shows that the above-mentioned residues are having the tendency to interact with ligands and function for the active sites. Final best leads are perfectly found to binding inside the pore region and well interacted with in the pore-lining amino acids. This clearly indicate the inhibitor insights and also the potential active sites residues of Arg74 (D), Asn77 (D), Thr43 (E), Cys46 (E), Ile59 (E), and Val60 (E), which should be targeted for the inhibition of E protein in SARS-CoV-2.

It is clear that high hydrophobic nature of E protein and integrated with membrane layer and the E protein is placed inside the lipid bilayer membrane as shown in the figure 7. For that C terminal is reported to make it presence in the cytoplasm and high hydrophobic N terminal is reported as inserted within the Golgi membrane. So, the membrane is fixed with N-terminal region along with best compounds and simulated for 30 ns. The purpose of the MD simulations is to understand the ligand stability in the dynamic environment along with lipid membrane for providing lively environment and also to understand the dynamic reaction of lead molecules inside the large binding pocket. The figure 7 explains the RMSD values plotted with respect to deviations occurs for the apo and ligand bound complex in the membrane. The results show that, the apo and ligand complex are stable inside the membrane-based MD simulations throughout the 30 ns of the MD timescale. Ligands are bit moving due to the pore space, but the amino acids Arg74 (D), Asn77 (D), Thr43 (E), Cys46 (E), Ile59 (E), and Val60 (E) are strongly holding the ligands. For stability, both apo and ligand bound complex lies in between the RMSD values of ~ 2.2Å which shows the MD simulations suggest the compounds as best compounds in the dynamic environment.

As of now, there has been several drug targets from both SARS-CoV-2 and host is considered for the drug design and for that, the Spike protein, Main protease and few other proteins are considered with full focus. Along with the above-mentioned proteins, E protein role is imperial and from beginning of the cell entry to exit of cell, E protein plays the major role. The importance of E protein is compared with both SARS-CoV and SARS-CoV-2.

Protein-protein interactions based on literature says, that E protein has survival capacity more in SARS-COV-2 than the SARS-COV. This clearly, represent the E protein is an attractive target for drug discovery. Considering this, the work concentrated on describing the structural 

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The author RAA has consent to publish this manuscript in Saudi journal of Biological Science

Data will be available on request to corresponding or first author

Not Applicable Fig. 1 . Protein-protein interactions predicted by CoVeX prediction server (a) represents the protein-protein interactions between the SARS-CoV E protein with host protein and (b) represent the protein-protein interactions between the SARS-CoV-2 E protein with host proteins. 

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