Dietary factors and SARS-CoV-2 contagion: in silico studies on modulation of viral and host proteins by spice actives

Abstract The SARS-CoV-2 contagion has had a huge impact on world population. It has been observed that despite massive spread of the contagion in India particularly during the second wave, the overall case fatality rates remain low. This prompted us to look into dietary factors that can possibly modulate the viral impact and/or host response. In silico studies were carried out on forty-two commonly used spices and their 637 known active compounds with an aim of identifying such compounds that may have propensity to reduce viral impact or boost host immune response. We chose to study SARS-Cov-2 helicase on account of its functional importance in maintaining viral load within the host, and the human tank binding protein (TBK1) for its important role in host immunity. We carried out in silico virtual screening, docking studies with 637 phytochemical against these two proteins, using in silico methods. Upon assessing the strength of the ligand-target interactions and post simulation binding energy profile, our study identifies procyanidin-B4 from bay leaf, fenugreekine from fenugreek seed and gallotannin from pomegranate seed as active interactors that docked to viral helicase. Similarly, we identified eruboside B from garlic, gallotannin from pomegranate seed, as strong interacting partners to human TBK1. Our studies thus present dietary spice constituents as potential protagonists for further experimentation to understand how spices in the diet might help the hosts in countering the viral assault and mount a robust protective response against COVID and other infections. Communicated by Ramaswamy H. Sarma


Introduction
The severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2, SCV) is the seventh Coronavirus to infect the human race after HCoV-NL63, HCoV-229E, HCoV-OC43, HKU1, SARS-CoV, and MERS-CoV (Andersen et al., 2020). Over the past two decades, SARS-CoV hit humans in 2002 infecting 8,000 people with a case fatality rate of $10%, followed by the MERS-CoV contagion in 2012 which was contracted by 2,500 people with a case fatality rate of 36%. SARS-CoV and MERS-CoV caused severe respiratory syndrome and exhibited higher case fatality rates, but their spread was contained thus minimizing the effect on human life (Gordon et al., 2020). The newly evolved sub-type SARS-CoV-2 (SCV), reported to have originated in December 2019, spread to 223 countries, infecting 169 million and proving fatal for over 3.5 million people worldwide (WHO, 2021). SCV is a large, single-stranded, positive-sense RNA virus. The cleavage of polyproteins by viral proteinases results in the generation of a total of 16 non-structural proteins (Nsp's) (Adedeji et al., 2012;Keum & Jeong, 2012;Seth et al., 2006). An intriguing factor has been the varying percentage of death rates between various countries and populations. Specifically, India, the second-most populous country of the world, has seen lower case fatality rates. As of 30th May 2021, according to WHO report, India has reported 27,894,800 cases and 325,972 deaths (WHO, 2021), with the case fatality rates being 1.16% against the average of 2.26% for the rest of the world. Amongst the reasons for how the Indian population has been mounting such protective response, various possibilities including higher basal immunity, immunization profiles including BCG vaccine, and its resulting immune memory have become subjects of discussion and research. We endeavoured to look at the aspect of 'what the Indian population eat' towards aiming to identify possible routine dietary ingredients that may have the potential of either limiting the viral impact or via activating the host immune system towards mounting a robust response. Amongst various dietary ingredients, ones that are consumed routinely are spices and condiments that form an ingredient of every meal an Indian consumes. Constituent flavonoids, terpenoids, and other metabolites from spices are known to protect by antioxidant, anti-inflammatory, antiviral and anti-microbial activities. We carried out in silico studies using 637 active constituents within 42 commonly used spices and condiments with a two-fold aim of (a) identifying such constituents that can be predicted to exhibit a propensity of checking the SCV replication or viral load (via studying SCV protein Nsp13), and (b) identifying the constituents that can boost the host immune system towards mounting an effective response (via studying host immune response protein TBK1), until the viral load is cleared.

Materials and methods
1. Mining of literature towards the identification of target proteins from SCV and its human host and identification of commonly used spices in India: The selection of proteins for the study was carried out in a hypothesis-driven manner of studying a protein that plays a critical role in viral replication and proliferation thus being important for maintaining viral load within the host. Similarly, literature was mined for identifying host (human) protein, related to the immune pathways that are activated in the case of SARS-CoV-2 infections. The spices were selected from the Food Safety and Standards, Regulations document based on the common usage across various parts of India overlapping these spices with Spice Board of India's cultivation data. 2. Ligand preparation: 3D structures of metabolites from 42 spices were procured using ChemSpider (https://www. chemspider.com/), and PubChem (https://pubchem.ncbi. nlm.nih.gov/) databases. Along with these structures, known inhibitors for each protein were also added to the list for screening. The downloaded structures were further processed using the LigPrep module of the Schr€ odinger suite (Schr€ odinger LLC, 2020) in the Maestro builder panel to add appropriate number of hydrogen atoms to satisfy the valency requirements of non-hydrogen atoms, ensure proper ionic states and 3D geometry of the compounds at a pH 7.0. The structures were optimized by energy minimization using the all-atom forcefield OPLS3e. 3. Preparation of protein structures and grid generation: Viral protein Nsp13 (Helicase) and host human TBK1 (Tank binding protein) protein structures were downloaded from PDB (Protein Data Bank, 2019). Hydrogen atoms, missing side-chain atoms and non-standard residues were fixed in these structures by using the protein preparation wizard (Schr€ odinger LLC, 2020). Potential binding sites were identified in these proteins using the SiteMap module (Schr€ odinger LLC, 2020). Sites having scores greater than one were used for downstream virtual screening and docking studies. XYZ-Coordinates for the selected sites were given as input, for making a grid using the 'Glide's Receptor Grid Generation' module. A cubic box was then generated around the active site of the target molecules. The grid box dimension was then adjusted to completely encompass the active site region of the target protein molecules. 4. Virtual screening and docking: The optimized structures of the ligand set were then docked using both SP and XP scoring functions using Glide (Schr€ odinger LLC, 2014). This Virtual screening using the natural compounds against the chosen target proteins (Host: TBK1; Virus: Nsp13) was then ranked based on the glide score. First, the ligands were subjected to standard precision (Friesner et al., 2004) docking. Resulting 32 hit ligands were further subjected to extra precision docking. OPLS3e force field with a pH ¼ 7 was used in all these studies. GlideScore is an empirical scoring function that approximates the ligand binding free energy. It is composed of many terms, including force field (electrostatic, van der Waals), GScore is the short form for GlideScore, used for comparing poses of different ligands which uses the equation below. vdW is Scaled van der Waals energy, Coul is Scaled Coulomb energy, Site is Polar interactions in the active site, CvdW is Non-bonded ligand/receptor interactions (Coul þ vdW), not included in GlideScore (though Coul and vdW are included individually, with separate weights), Intern is Internal energy of ligand, not included in GlideScore, Emodel is combination of GScore, CvdW, and Internal energy. These contributions and the terms rewarding or penalizing inter and intra molecular interactions known to influence ligand binding constitute the GScore. (Friesner et al., 2006). The receptor-ligand complexes were minimized after solvating with SPC water molecules in a box whose dimensions covered 10Å from the surface of the molecule in all directions. The solvated systems were energy minimized using steepest descent strategy until a gradient threshold of 1.0 kcal/mol/Å was approached with a minimum step size of 10. Periodic boundary conditions were used along with a 9.0 Å cut-off for electrostatic interactions. The MD simulation run consists of eight stages, including the pre-relaxation phase of the protein-ligand complexes. Unrestrained production runs were conducted at a temperature of 300 K and 1.01325 bar pressure, for 100ns. Constant pressure and temperature were maintained using Berendsen coupling method (Berendsen et al., 1981). No additional restraints were given for the hydrogen atoms of the protein or the ligand molecules. Coordinates and energy parameters were saved at every 25ps. The resultant 4000 frame trajectories of the simulated systems were analysed and visualized using the inbuilt tool 'simulation interaction diagrams' of the Maestro/Desmond module of the Schrodinger Suite. For analysing the hydrogen bonding interactions from the simulated trajectories, the following geometric criteria for a protein-water or water-ligand H-bond are used: a distance of 2.8 Å between the donor and acceptor atoms (D-HÁÁÁA); a donor angle of !110 between the donor-hydrogen-acceptor atoms (D-HÁÁÁA); and an acceptor angle of !90 between the hydrogen-acceptor-bonded atom atoms (HÁÁÁA-X), while for hydrogen bond is distance of 2.5 Å between the donor and acceptor atoms (D-HÁÁÁA); a donor angle of !120 between the donorhydrogen-acceptor atoms (D-HÁÁÁA); and an acceptor angle of ! 90 between the hydrogen-acceptor-bonded atom atoms (HÁÁÁA-X).Binding energy calculations were performed using MM-GBSA using the Prime (Schr€ odinger LLC, 2014) module of Schrodinger software on the snapshots taken at every nanosecond, where the dynamic shift in energy was measured between the unbound and bound state of the complex. For the run, simulation report was generated. The binding energy was calculated according to the equation:

1.
Identification of target proteins from SARS-CoV-2 and its human host and identification of commonly used spices in India: In context with viral machinery, the replicase/ transcriptase complex was chosen as a target in the virus system (human-SARS CoV2 interactomereference). Further understanding of helicase Nsp13 gave enthralling reasons for its vital existence in the virus. As its functionality varies from contributing to the cytoskeleton, signalling, vesicle trafficking to epigenetic, and gene expression regulation (Fung & Liu, 2014;Gordon et al., 2020). Similarly, human host proteins interacting with Nsp13 were procured from BioGrid (https://thebiogrid. org/), amongst the list TBK1; a Serine/Threonine kinase that plays an essential role in regulating inflammatory responses to foreign agents. To lead the idea of immunity and immune response to host-virus interaction TBK1was identified as target host protein. Especially, when virus hijacks host the ER system for cargo facility and amplification that in return creates stress response which activates the defence (https://thebiogrid.org/). Depending on age, gender, and other individual attributes this immune response can vary from being insensitive to moderately responsive by stimulating a defence against the cytokine storm. The Food safety and standards, regulations document was used as a primary criterion to select spices. By overlapping these spices with Spice Board of India's cultivation data (http://www.indianspices.com); forty-two most commonly used/cultivated spices that are rooted in India's geography were identified and selected for further studies (Table 1) . In addition, adamantane derivatives that have been used clinically for many years as antiviral treatments and as muscle relaxants were also included in the ligands to be used in the screening. In a different study, a demonstrated class of pyridoxal-conjugated trioxaadamantanes, known as the bananins, was shown to inhibit both the ATPase and helicase activities of the SARS Coronavirus helicase (Tanner et al., 2005). A representative of that class, bananins was taken to be a potent inhibitor for Nsp13 as a positive control. Whereas for TBK1 a known inhibitor from Gordon et al. (2020). ZINC95559591 was taken as screening/control. 3. Protein preparation and grid generation: Structure of Nsp13 (PDB code 6JYT) and human TBK1 (PDB code 4IM0) were downloaded from the PDB (www.rcsb.org). These structures were fixed for downstream computational studies using the Protein Preparation Wizard of the Schrodinger suite (Schr€ odinger LLC, 2020). The structures were energy minimized to remove short contacts and to ensure proper stereochemistry. Potential binding sites in the proteins were identified using the SiteMap module (Schr€ odinger LLC, 2020). Sites having scores more than 1 were taken for grid calculations as enlisted in Table 2. Five sites were identified in each of the proteins TBK1and Nsp13 and were ranked according to their score. Analysis of the 3D structure revealed that these sites are populated with residues that can potentially make electrostatic, hydrogen bonds as well as hydrophobic interactions.  Figure 3) and cosmosiin (Glide score: À11.7) from cumin, cyanin (Glide score: À11.8) from ginger, as selective interactors ( Figure S3). Whereas gallotannin (Glide score: À11.1 and À11.5) (See Supplementary file 2, Figure S2 and S3) from pomegranate seed was able to bind at both the sites. The results of these docking studies with Glide/docking score along with interacting residues are listed in Table 3. 5. MD Simulation and MM-GBSA analysis: Unrestrained molecular dynamic simulations studies were undertaken using the Desmond module of the Schrodinger Suite and analysed using the in-built modules of maestro/ Desmond. The RMSD plots (Figure 4) show that the protein-ligand complexes were stable during most of the 100 ns simulation time. At a few snapshots, the ligands have changed their pose but stayed within the binding site (protein-ligand contact map is provided in Supplementary file 3), which can be observed from the RMSD plot where the RMSD trajectories of the protein and its respective ligand overlap. MM-GBSA analysis shows that the binding is highly stable for 4 out of 5 ligands that we have studied with high binding energy values. Table 4 lists total binding energy along with the components of the energy terms from MM-GBSA analysis of the systems under study. MM-GBSA analysis, (Table 4) showed that for helicase Nsp13, eruboside B (À31 kcal/mol) has weak binding while gallotannin (À73.5 kcal/mol) is the strongest. Similarly, for TBK1 eruboside B (À120.7 kcal/mol) and gallotannin (À120.6 kcal/ mol) have strong binding with the receptor respectively. MM-GBSA analyses also show that eruboside B and gallotannin have strong binding affinity towards TBK1, while gallotannin has the highest affinity for Nsp13 followed by fenugreekine and procyanidin B4 (Figure 4). Eruboside B seems to be a potential candidate to inhibit both the viral helicase and the human TBK1 protein, though its interaction with the human TBK1 is stronger than that with the viral nsp13.
These can be comprehended by the schematic representation ( Figure 5). Here it can be seen that there were hydrogen bonds that were present in the maximum time of the simulation run, but also water bridges were formed. Here, only those residues were taken into account which stayed in interaction for more than 50% of the run. It can be observed that water bridges contributed by polar amino acid residues like (HIS, ASN, THR) contributed to the interactions for longer durations. Apart from these interactions there were Pi-Cationic and ionic contacts were also seen.

Discussion
In the current study, we have undertaken studies on Nsp13 as well as TBK1 protein using the dietary metabolites from spices to decipher if any of the spice constituents can act as stimulants for immunity towards providing rescue and protection from viral pathology. Structural features of TBK1 contain a predicted Ubiquitin-like domain (ULD) that is located between the N-terminal kinase domain (KD) and the C-terminal scaffolding/dimerization domain (SDD), a domain arrangement that appears to be shared among the IKK family of kinases ( Figure 2) (Shi et al., 2018). TBK1 triggered immune signalling cascade directs host's defence towards viral invasion. Here we present two sets of potential candidates, based on docking studies, that can function either in boosting immune response or providing protection in case of SARS-CoV-2 infection. After docking studies in Set-A metabolites (Site ID: Site-B), eruboside B and inulin from garlic, gallotannin from pomegranate seed, and hesperidin from hyssop, capsicum, and chili, were identified. Inulin is a non-digestible carbohydrate also used as a prebiotic which may have an impact on the immune system, especially in the area of the gut-associated lymphoid tissue [GALT]. Recent studies show that it modulates the immune system by activating immune cells in Peyer's patches including IL-10 production and natural killer [NK] cell cytotoxicity (Watzl et al., 2005). Hesperidin is a flavanone glycoside comprised of the flavanone hesperetin and the disaccharide rutinose. Various studies have reported it to defend airway hyper-responsiveness, inhibiting inflammatory cell infiltration, and mucus hyper-secretion. Also, it enhances mucosal and humoral immunity by increasing intestinal intraepithelial lymphocyte numbers and lymphoid organ indices. It is a strong chain-breaking antioxidant that provides potent cellular antioxidant defence against the damaging effects (Xiao et al., 2018). The second set of hit metabolites interacting with TBK1 are listed within Set-B [Site ID: Site-C], in order as the first hit being luteolin-7-glucoside from cumin; a glycosyloxyflavone that is luteolin substituted by a beta-D-glucopyranosyl moiety at position 7 via a glycosidic linkage. It plays a role in the modulation of Reactive Oxygen Species [ROS] and immune response involving NF-jB pathways (Jin et al., 2011;Maatouk et al., 2017). We also identified a metabolitecyanin, from ginger that boosts host immunity and creates a defense response to allergies and inflammation (Mashhadi et al., 2013). The third metabolite is Cosmosiin also known as apigetrin from cumin which participates in rescuing inflammatory response in the host that has lost its balance (Liu et al., 2017). With the results, it could be stated that flavonoids, terpenoids, and saponins are the major class of plant metabolites participating in interactions with the host and viral proteins. The experimentally known inhibitors used as controls for virtual screening did not dock in the initial standard precision mode for both the proteins. Thus, we are anticipating significantly higher activity than the control molecules used. It has been established that the Nsp13 has a putative binding site comprising of TYR-277, ARG-507 and LYS-508 wherein the inhibitor SSYA10-001 binds. Interestingly this (A O Adedeji et al., 2014) overlaps within our chosen dock site from SiteMap results. The hit metabolites were interacting in the same docked region if not directly to the same residues as before mentioned.  MD simulation and the MM-GBSA analysis show that the results of docking studies are not just pertaining to a specific snapshot of the interaction profile, but there is a strong energy dynamics involved in the active site that in return contributes to the stability of the protein-ligand complexes. Starting with the Nsp13 (helicase) complex, it is quite evident with the results that eruboside B is the weakest of all though it is strong enough to be considered as a potential candidate with a binding energy À31 kcal/mol; while gallotannin, fenugreekine and procyanidin B4 are strongly bound proving to be better ligands from the list. While in case of TBK1 complex eruboside B and gallotannin were seen to be positioned with strong interactions in the binding pocket as per MM-GBSA energy profile. From the root mean square deviation (RMSD) plots; it can be comprehended that the ligands that are bound to Nsp13 or TBK1 stayed in the binding pocket when the system is subjected to 100 ns simulation. The binding energy as reported by MM-GBSA run for Nsp13 from À31.0 kcal/mol to À73.5 kcal/mol. Eruboside B interaction with Nsp13 is primarily of Vander Waals interactions (VDW) in nature. Whereas for fenugreekine, gallotannin and procyanidin B4 the dominating donor was electrostatic/columbic interactions followed by VDW. The RMSD plots indicate that, in these systems too, though there are some frames along the trajectory where the binding orientations have changed, the drugs mostly remained in the tightly bound state. TBK1 where the average NBE of each ligand was around $ À121 k/cal/mol. These results can be further supported with the help of simulation interaction diagram, there it can be seen that hydrogen bonds and water bridges comprise majority of the interactions in the run along with few ionic and pi-cationic interactions. As it is known, that when a ligand is strongly bound to the receptor it can either induce physicochemical alterations or prevent the natural substrate from interacting with the receptor, thus affecting the downstream biological processes. Here, it can be understood in terms of two distinct modes of action hypothetically. The first one is with helicase protein, the bound ligand can alter the function viral protein, next is TBK1; a host protein that is responsible for immune response. Thus, it can contribute to the host defense system upon infection or as a preventive measure for the same.
Although this study opens up a fresh perspective, in silico results are subject to laboratory validation. Furthermore, this study performed is just a primary staging of wonders Indian spices sustain. As they are being part of the population's daily diet, they can be employed for the wellness of the society as a nutraceutical supplement. Especially with the growing development in the fields of using medicinal properties of hydrolyzing tannins, gallotannins can be further studied as a mutual target. While eruboside B for host protein and fenugreekine, procyanidin B4 for viral protein can be further validated at individual experimental studies to validate the precise mechanism involved and what are the upshots in terms of immunity and health of an organism. We propose potential nourishment in dietary spice metabolites such as   (Pyridines and derivatives like fenugreekine); saponins (eruboside B) and flavonoids (procyanidin B4) can be theoretically used to manage the outbreak of COVID-19, or any similar RNA virus or the new strains of SARS-CoV-2. We anticipate that the comprehensions gained in the current study may support the valued contribution for employing the spice components as a part of a fortified Nutraceuticals herbal immune boosting supplement, towards the production of natural origin supplements in fight against COVID-19. Such a development might add up to the ease in the restriction of infection and for support of the susceptible host health. Henceforth, this study proves to be a precious addition in the fight toward the viral infection front where an individual standing globally can have access to the kitchen spices and sustain their response to the viral concurrence.

Conclusion
For understanding the relationship and modulated dynamics of Nsp13 and TBK1 upon interaction with metabolites generated by the study, we present dietary spice actives as protagonists for further experimentation to understand how spices can potentially stimulate host's immune system and at the same time if these are also modulating the pathogenicity, or can these compounds be potential leads to inhibit viral replication and its consequent propagation, thus modulating effect of the virus on exposed human population. A combination of these spice actives could have translational potential as immunity-boosting health supplement.