key: cord-0705449-szg6yijl
authors: Bhowmik, Arijit; Biswas, Souradeep; Hajra, Subhadip; Saha, Prosenjit
title: In silico validation of potent phytochemical Orientin as inhibitor of SARS-CoV-2 spike and host cell receptor GRP78 binding
date: 2021-01-11
journal: Heliyon
DOI: 10.1016/j.heliyon.2021.e05923
sha: 7cbc74d41a04f461b68d89637f5e322a71c4a060
doc_id: 705449
cord_uid: szg6yijl

The present wellbeing worry to the whole world is the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also called COVID-19. This global health crisis first appeared in Wuhan, China around December 2019 and due to its extremely contagious nature it had spread to almost 187 countries. Still now no effective method of treatment or vaccine is developed for controlling the disease. Therefore, the sole obliging strategy is to take precautionary measures by repurposing drugs from the pre-existing library of therapeutically potent molecules. In this situation of pandemic this repurposing technique may save the labour-intensive and tiresome process of new drug development. Orientin is a natural flavonoid with several beneficial effects. This phytochemical can be isolated from different plants like tulsi or holy basil, black bamboo, passion flowers etc. and it’s antiviral, anti-inflammation, vasodilatation and cardioprotective, radioprotective, neuroprotective, anticarcinogeneic and antinociceptive effects are already established. In this research, it is intriguing to find out whether this molecule can interfere the interaction of SARS-CoV-2 spike glycoprotein and their host receptor GRP78. Our in silico docking and molecular dynamics simulation results indicate the binding of Orientin in the overlapping residues of GRP78 binding region of SARS-CoV-2 spike model and SARS-CoV-2 spike model binding region of GRP78 substrate-binding domain. Therefore, the results included in this research work provide a strong possibility of using Orientin as a promising precautionary or therapeutic measure for COVID-19.

binding domain. Therefore, the results included in this research work provide a strong possibility of using Orientin as a promising precautionary or therapeutic measure for COVID -19. Keywords: Orientin, SARS-CoV-2 spike, GRP78, In silico docking, MD Simulation, Phytochemical

The latest emergence of pandemic threat of novel corona virus disease (COVID-19) by the pathogenic Severe Acute Respiratory Syndrome Coronavirus 2 is responsible for global risk of public health [1] . More than 13 million cases and around 285 thousand deaths have been recorded worldwide upto third week of July, 2020, which triggers the urgent need of active antiviral agent identification. The major symptoms of this disease include shortness of breath, fatigue, fever, muscle aches, dry cough and sometimes lead to pneumonia [1, 2] . Patients with past medical history of other maladies like cancer, heart diseases, diabetes, asthma etc. along with elderly individuals and children below the age of 6 are in grave risk due to their compromised or weaker immune system. With the changing epicentres from Wuhan, China to countries like Italy, Spain and the USA this disease seems to increase its mortality rate [3] [4] [5] . A timely identification of causal agent called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) opened up new paths of antiviral research for COVID-19 treatment. SARS viruses have vivid plethora of host animals like birds, pangolins, domestic animals like dogs etc, whereas SARS-CoV-2 seems to be more pathogenic to human [6, 7] . This is a virus with single stranded RNA genome of around 30 Kbs with low number of encoded proteins involved in structural and non-structural features of this member of genus Betacoronavirus [8] [9] [10] .

The most prominent structural proteins are spike glycoprotein (S), envelope protein (E), membrane protein (M) and the nucleocapsid protein (N). On the other hand, proteases (nsp3 and nsp5) and

RdRp (nsp 12) are the major non-structural proteins of this type of virus [8] . Out of these different types of protein spike protein is vital for viral attachment and entry to the host cells as they work as recognition factor. The earlier known SARS spike proteins have around 75%-81% sequence similarity with SARS-coronavirus 2 [11] . Though these RNA viruses have high mutational frequency, but very fewer differences in the spike proteins have been observed in emergent SARS-coronavirus 2 variants [12] . At the time of infection to the host cell the spike proteins are mostly in open state as the closed state of the same is less vulnerable to the antibodies [12] . The receptor binding domain of spike which faces outside part of virion shows distinctive "corona", or crown-like appearance [13] . The ectodomain of SARS-CoV-2 spike proteins comprise of two major domains: a N-terminal domain which is responsible for receptor binding and a C-terminal domain responsible for fusion with the host cells [14] . Binding affinity can be observed in some of these regions with host receptor proteins of SARS-CoV-2 like GRP78 [13] .

The Glucose Regulating Protein 78 (GRP78) is a master chaperon protein which also known as Binding immunoglobulin protein (BiP) [15, 16] . It generally acts when unfolded or misfolded proteins accumulate [17, 18] . It has prominent role in cell death and differentiation as it causes the inactivation of enzymes involved in mentioned phenomena by binding to the lumen of the endoplasmic reticulum (ER) [19, 20] . However, cell stress can also increase the chance of translocation of GRP78 from ER to cell membrane [20] . This makes GRP78 more susceptible to virus recognition and entry. The key viral recognition region of GRP78 is the substrate-binding domain (SBD) [19] . It is intriguing to find out molecular blocker of this substrate binding domain of GRP78 as well as the blocker for binding regions of spike proteins of SARS-CoV-2. Effective phytochemicals from natural resources may provide promising results as molecular blocker for GRP78 and spike proteins with no or less side effects. Orientin is a natural flavonoid can be found in many plants like

Tulsi or Holy basil (Ocimum sanctum), Pheasant's eye (Adonis vernalis), Wilco (Anadenanthera colubrine), Cohoba (Anadenanthera peregrina), Black bamboo (Phyllostachys nigra), passion flowers (Passiflora species), Golden Queen (Trollius species), Bellyache Bush (Jatropha gossypifolia ) etc [21] . Most of these plants are known for their medicinal values. Tulsi or Holy basil is a commonly used medicinal plant in ancient India. In ayurvedic literature it is mentioned as an effective remedy for many diseases like cough, common cold, malarial fever etc [22] . From the leaves of Holy basil Nair et al. and Uma Devi et al. successfully isolated orientin which has a molecular formula of C 21 H 20 O 11 and a molecular weight of 448.3769 g/mol [21, 23, 24] . From different studies it is evident that orientin has antiviral, antioxidant, antiaging, anti-inflammation, vasodilatation and cardioprotective, radioprotective, neuroprotective, antiadipogenesis, anticarcinogeneic and antinociceptive effects which makes it promising therapeutic molecule [21] . Orientin is proved to possess antiviral activity against Para 3 virus which was demonstrated by Qiufeng et al. [25] and Li et al. [26] . Besides that, it also shows efficacy against Herpes Simplex Virus Type 2 (HSV-2) [27] . Therefore, it is fascinating to find out its antiviral effect against SARS-CoV-2.

In this study, we try to find out the in silico binding of orientin either with spike protein of SARS-CoV-2 or with GRP78. Here it is hypothesized that orientin has a scope to block the binding of spike protein and GRP78, thus it may restrict the recognition and entry of SARS-CoV-2 in GRP78 expressing host cells. Therefore, this report suggests that natural phytochemical orientin is a potential candidate for development of anti-SARS-CoV-2 drug.

Autodock Vina is used for protein-ligand docking and HADDOCK (ver. 4.2) is used for protein-protein interaction [28, 29] .

SARS-CoV-2 spike ectodomain structure (open state) is obtained from RCSB PDB (PDB id-6VYB) and also a new spike protein model is generated from SARS-CoV-2 sequence which is downloaded from UniProt (UniProtKB-P59594) and modelled by Phyre2 web server [30] . Superposition and pairwise sequence alignment of SARS-CoV-2 spike ectodomain structure (open state) and NEW SARS-CoV-2 spike model is also done by Phyre2 webserver [30] . GRP78 SBD domain (PBD id. 5e85) is downloaded from RCSB PDB (Protein Data Bank).

Orientin and various other plant derived compound like caffeic acid, isobavachalcone, lycorine, ellagic acid, galangin, an inhibitor of GRP78 i.e verrucosidin and clinically used anti-COVID19 drug chloroquine are screened against SARS-CoV-2model and GRP78 SBD domain. 3D conformations of orientin, caffeic acid, isobavachalcone, lycorine, ellagic acid, galangin, verrucosidin and chloroquine are collected from PubChem database. Optimization of the 3d structure is done in Autodock tools (ver. 1.5.7) which utilizes Iterated Local Search global optimizer that based on the Broyden-Fletcher-Goldfarb-Shanno (BFGS) method [31, 32] . Ligand preparation is done in Autodock tools under ligandprep function by removing water and adding hydrogen atom [33, 34] . Energy minimalization is done by applying Gasteiger charges and AMBER force field with RMSD cut of 0.02 Å to obtain the most stable conformer [35] .

In In silico docking is performed to identify interacting residues responsible for ligand (orientin and other compounds) docking within the GRP78 SBD domain and as well as in the NEW SARS-CoV-2 spike model [36] .

Docking between NEW SARS-CoV-2 spike model and GRP78 SBD domain is done by HADDOCK (ver. 4 .2) webserver with easy interface and without any restrains [29, 37] . In case of SARS COV-2 model spike protein N460, N487, D420, L455, K417, Y421, Y473, Y505, E484, N481, P479, E406, T478 are selected as active residues and for GRP-78 SBD domain Q492, K447, S452, Q449, E427, I450, I450, S448, G430, T428, V429, S452, T458, V457, V490 are selected as active residues as mention in previous report [13, 38] . Other than these residues all other surrounding amino acids are considered as passive residues. These active sides are directly involved in the interaction between two proteins where the passive residues are showing indirect interactions [37] . After docking the best docked clusters are ranked according to their HADDOCK score and all other parameters are represented with in separate statistical graphs.

The ranking of the clusters is based on the average score of the top 4 members of each cluster. The score is calculated as:

HADDOCK score = 1.0 × Evdw + 0.2 × Eelec + 1.0 × Edesol + 0.1 × Eair Where Evdw= intermolecular van der Waals energy, Eelec= intermolecular electrostatic energy, Edesol= empirical desolvation energy, Eair= Restrain energy.

Bindings of orientin, caffeic acid, isobavachalcone, lycorine, ellagic acid, galangin and verrucosidin with the new SARS-CoV-2 spike model and GRP78 SBD domain are executed by Autodock vina [28] .

Each protein is prepared for docking by Autodock tools software where Gasteiger charges and hydrogen atoms is added and also make sure that water molecules are removed [39] . AMBER force field are applied for optimization of each structure [35] . For molecular docking grid boxes are also generated by the Autodock tools software, grid cubic boxes are generated using 0.492 Å spacing and with a dimension of 60 Å× 60 Å× 60 Å around each of the target protein. Where ii) [40] Where N= no of heavy atom in structure a, min= minimum is over all atoms in structure b with the same element type as atom i in structure a.

Protein-Ligand Interaction Profiler (PLIP) web server of Technical University, Dresden is used to analyse the bond formation in docking complexes of NEW SARS-CoV-2 spike model-Orientin and GRP78 SBD domain-Orientin and two main interactions viz. hydrophobic interactions and hydrogen bonds are established [41] . Bond analyzation for Protein-protein docking complexes is performed in PIC (protein interaction calculator) and binding energy is predicted by PRODIGY for best model complexes of NEW SARS-CoV-2 spike model with GRP78 SBD domain and the residues in the interactions are tabulated [42] [43] [44] .

The stabilities of the docked complexes are probed by finite temperature classical molecular dynamics [34] . Calculations are done in the simple point charge (SPC) water model using OPLS_2005 all atom force field (Optimized potentials for liquid simulation) as implemented in Desmond [45, 46] .

The ensemble type of the simulation is NPT with constant number of molecules (N), a defined temperature T (298K) and pressure P (1 bar). Nose-Hoover thermostat and Martyna-Tobias-Klein barostat are used to maintain the temperature and pressure of the system, respectively [45] . Protein given as per the PDB structures 5e85 and 6vyb, respectively. Along with orientin and Verrucosidin 3D structure of chloroquine is also collected from PubChem database for MD simulation study.

New SARS-CoV-2 spike model generation and sequence alignment with SARS-CoV-2 spike ectodomain structure A potential 3D model of SARS-CoV-2 spike protein based on alignment to known protein structures is generated by Phyre2 (Protein Homology/analogY Recognition Engine V 2.0). This method can generate accurate protein models of about 70% of the domain similarity with a known structure where the core of the protein shows 2-4Å root mean square deviation from the native. Remote homology detection techniques such as profile matching and hidden Markov model (HMM) matching is used to detect and align protein sequences. This efficient technique is able to generate reliable models of proteins even if they have considerable divergence over evolutionary time. The newly generated SARS-CoV-2 spike model sequence is close to the SARS-CoV-2 spike ectodomain structure (open state) (PDB: 6VYB) with 81% sequence identity. Figure 1A shows colored cartoon of the known structure of SARS-CoV-2 spike ectodomain (PDB: 6VYB) which serves the roll of template for the new model generation. Figure 1B represents newly generated 3D model of SARS-CoV-2 spike (colored cartoon). The Root Mean Square Deviation (RMSD) between the two structures is 2.513 A˚.

The template modelling score or TM-score is 0.75 which indicates significant similarity between the structures of SARS-CoV-2 spike ectodomain and newly generated SARS-CoV-2 spike model. Figure   1C shows the superposition of SARS-CoV-2 spike model (blue cartoon) and SARS spike structure (PDB 

CoV-2 spike model HADDOCK software is used to perform GRP78-SARS-CoV-2 spike model docking. Figure 3A The various structural analyses are made with respect to the best HADDOCK model. Therefore, for the best-docked complex values of van der Waals energy is -71.5 +/-9.2, electrostatic energy is -90.0 +/-14.2, desolvation energy is -24.5 +/-2.0, restraints violation energy is 31.7 +/-29.0. In the graphs of figure 3B, best cluster of docked models is defined by lowest HADDOCK score in respect to lowest interface RMSD (i-RMSD) value, lowest HADDOCK score in respect to highest value of fraction of common contacts, lowest van der Waals score in respect to lowest interface RMSD, highest electrostatics energy in respect to lowest interface RMSD, lowest desolvation energy in respect to lowest interface RMSD and lowest restraints energy in respect to lowest interface RMSD. Therefore, cluster 4 is considered as best cluster as per the mentioned parameters and best binding model of cluster 4 is represented in figure 3A . Hence, it is evident from the best docked model that predicted binding residues of SARS-CoV-2 spike model lies between the sequences of 427-609 amino acids, whereas 427-652 amino acid sequence is the region for the binding of GRP78 SBD.

The water-soluble flavonoid orientin is a C-glycoside which has the IUPAC name of 2-(3,4dihydroxyphenyl)-5,7-dihydroxy-8-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2yl]chromen-4-one [21] . Figure 4A represents the chemical structure of orientin which consists of mostly phenol groups with two ether groups and one ketone group. After determination of residues responsible for interaction between SARS-CoV-2 spike model and GRP78, it is intriguing to find out Docking with orientin and other known phytochemicals against COVID-19 i.e caffeic acid, isobavachalcone, lycorine, ellagic acid, galangin and inhibitor of GRP78 verrucosidin suggest that orientin is comparable or better than the known anti-SARS-COV-2 phytochemicals in terms of in silico binding with GRP78 SBD and SARS-COV-2 spike model (Table 3 ) [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] . The in silico docking results suggest the binding of orientin with SARS-COV-2 spike model's receptor binding domain and also with GRP78 SBD with better affinity than the mentioned phytochemicals. Other than this, compared to the GRP78 inhibitor verrucosidin higher binding affinity of orientin is observed in case of docking with both GRP78 SBD and SARS-COV-2 spike model. Binding affinity of SARS-COV-2 spike model with caffeic acid, isobavachalcone, lycorine, ellagic acid, galangin and verrucosidin are -5.1

Kcal/mol, -6.0 Kcal/mol, -5.7 Kcal/mol, , -6.0 Kcal/mol, -5.8 Kcal/mol, -6.1 Kcal/mol respectively, lower than the binding affinity of orientin with SARS-COV-2 spike model (-6.2 Kcal/mol) ( Table 3) . On the other hand, binding affinity of caffeic acid, isobavachalcone, lycorine, ellagic acid, galangin and verrucosidin with GRP78 SBD domain are -5.0 Kcal/mol, 6.6 Kcal/mol, 7.2 Kcal/mol, -6.4 Kcal/mol, -6.1 Kcal/mol, -5.9 Kcal/mol respectively, comparable or lower than the binding affinity of orientin with GRP78 SBD domain (-7.2 Kcal/mol) ( Table 3) . Binding results also suggest that orientin can bind more efficiently with GRP78 than its natural inhibitor verrucosidin which in turn advocate the potential of orientin as an efficient binding molecule of GRP78 SBD. Therefore, all these data indicate that orientin could be used to inhibit SARS-CoV-2 spike binding to GRP78, which in turn may reduce the occurrence of COVID-19.

SARS-CoV-2 spike model is built in silico from pre-existing COVID-19 spike (pdb id.-6vyb). The spike model is prepared first because of incomplete sequence and breakage within the chain of SARS-CoV-2 spike protein reported in protein data bank. This spike protein covers the whole surface of corona virus and it is the most important primary target for phytochemicals. These spikes composed of three identical chains and appear as a distinctive crown-like structure under electron microscope.

Natural flavonoid orientin is found to bind in silico to the domain of SARS-CoV-2 spike model which is responsible for receptor binding. It is predicted that Orientin may bind to the overlapping amino acid residues of SARS-CoV-2 spike essential for SARS-CoV-2 spike -host cell receptor GRP78 binding. On the other hand, it is also observed from the data of in silico docking and MD simulation that orientin may also bind to the substrate binding domain of GRP78 which is essential for binding with SARS-CoV-2 spike. Overlapping binding residues of GRP78 -orientin and SARS-CoV-2 spike model -GRP78

suggests that orientin may cause inhibition of SARS-CoV-2 spike -GRP78 binding. As it is predicted that inhibition of the interaction between the COVID-19 spike protein and the host cell receptor GRP78 would possibly reduce the rate of viral infection, orientin could be an effective phytochemical to do the job. Therefore, the present in silico outlook suggests the possibility of using orientin as an inhibitor of SARS-CoV-2 spike protein-GRP78 binding which may pave the route for drug designers to develop suitable precautionary or therapeutic modality against COVID-19. Table 1 represents the hydrogen bonds generated between the new SARS-CoV-2 spike model and host cell-surface GRP78 SBD during HADDOCK based docking. In Table 2 hydrophobic interactions between residues of new SARS-CoV-2 spike model and host cellsurface GRP78 SBD are represented. 

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We are thankful to Dr. Uttam Pal (S.N. Bose National Centre for Basic Sciences) for his technical support in MD simulation. We are also thankful to Dr. Jayanta Chakraborty, Director, Chittaranjan National Cancer Institute (CNCI) and Ministry of Health and Family Welfare for providing the scope of research.