Targeting SARS‐CoV‐2 with Chaga mushroom: An in silico study toward developing a natural antiviral compound

Abstract The novel coronavirus (SARS‐CoV‐2) has caused large‐scale global outbreaks and mainly mediates host cell entry through the interaction of its spike (S) protein with the human angiotensin‐converting enzyme‐2 (ACE‐2) receptor. As there is no effective treatment for SARS‐CoV‐2 to date, it is imperative to explore the efficacy of new compounds that possess potential antiviral activity. In this study, we assessed the potential binding interaction of the beneficial components of Chaga mushroom, a natural anti‐inflammatory and immune booster with that of the SARS‐CoV‐2 receptor‐binding domain (RBD) using molecular docking, MD simulation, and phylogenetic analysis. Beta glycan, betulinic acid, and galactomannan constituents of Chaga mushroom exhibited strong binding interaction (−7.4 to −8.6 kcal/mol) forming multivalent hydrogen and non‐polar bonds with the viral S1‐carboxy‐terminal domain of the RBD. Specifically, the best interacting sites for beta glycan comprised ASN‐440, SER 373, TRP‐436, ASN‐343, and ARG 509 with average binding energy of −8.4 kcal/mol. The best interacting sites of galactomannan included ASN‐437, SER 373, TRP‐436, ASN‐343, and ALA 344 with a mean binding energy of −7.4 kcal/mol; and the best interacting sites of betulinic acid were ASN‐437, SER 373, TRP‐436, PHE 342, ARG 509, and ALA 344 that strongly interacted with the S‐protein (ΔG = −8.1 kcal/mol). The docking results were also compared with an S‐protein binding analog, NAG and depicted similar binding affinities compared with that of the ligands (−8.67 kcal/mol). In addition, phylogenetic analysis using global isolates depicted that the current SARS‐CoV‐2 isolates possessed a furin cleavage site (NSPRRA) in the RBD, which was absent in the previous isolates that indicated increased efficacy of the present virus for enhanced infection through increased interaction with ACE‐2. The results showed that Chaga could be an effective natural antiviral that can supplement the current anti‐SARS‐CoV‐2 drugs.


| INTRODUC TI ON
The recent emergence of a novel severe acute respiratory syndrome corona virus (SARS-CoV-2) from China with more than 30 million confirmed cases and 900,000 deaths worldwide has brought a paradigm shift in the global epidemiology (Chen et al., 2020;WHO, 2020). SARS-CoV-2 exhibits a range symptoms including mild fever, sore throat, loss of taste and smell, respiratory distress, and fatal multi-organ failure (Machhi et al., 2020;Sharma et al., 2020;Zimmermann and Curtis, 2020). SARS-CoV-2 possesses a unique structural protein, the spike glycoprotein, or S-protein, which is responsible for aiding entry into the host cell via interaction of the receptor-binding domain (RBD) with the peptidase domain of the angiotensin-converting enzyme-2 (ACE-2) expressed on the host immune cells (Shang, Wan, Liu, et al., 2020;Wang et al., 2020;Yan et al., 2020). More detailed structural analysis has shown that the RBD comprises certain conservative amino acids, spanning from 326 to 580 amino acids that primarily aid in the S-protein recognition and binding to ACE-2 (Tai et al., 2020;Walls et al., 2020;Xia et al., 2020).
The coronavirus attachment to the host cell is initiated by the interaction of S-protein with the host receptor through membrane fusion and endocytosis (Shang, Wan, Luo, et al., 2020;Walls et al., 2020). The S-protein comprises S1 receptor-binding subunit possessing the S1-N-terminal domain (NTD) (15-300 aa) and S1-C-Terminal domain (CTD) (326-567 aa), and the S2 membrane fusion domain that is involved in aiding viral RNA entry into the host cell post S1-RBD-ACE-2 interaction. In particular, S1-CTD serves as the RBD for all human coronaviruses. Receptor binding induces structural changes in the S-protein that comprises S1 and S2 domains, which eventually splices the two domains with the help of protease. Post receptor binding, acid-dependent proteolytic cleavage of S-protein occurs mediated by protease enzymes, along with the fusion of S2 domain that assists in the release of viral RNA into the host cell. Then, the viral replisome complexes are assembled, followed by the synthesis of genomic and sub-genomic RNAs, which are produced through negative-strand intermediates, and from which the sub-genomic RNA integrates with the host RNA and undergoes nested transcription (Shang, Wan, Luo, et al., 2020). The spike, membrane, and envelope structural proteins are first translated through the host-viral mRNA machinery and are exported to the host endoplasmic reticulum (ER) that translocate along a secretory pathway into the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) (Shang, Wan, Luo, et al., 2020;Tai et al., 2020;Walls et al., 2020;Xia et al., 2020). Then, the viral nucleocapsids bud into the ERGIC membranes that comprise viral structural proteins, thereby producing mature virions ( Figure 1). Then, the virions are transported by the vesicles to the cell surface and are released by exocytosis.
The cornerstone for controlling the virus might be effective vaccines or drugs that are still under research pursuit, and due to which almost a third of the global population is affected by the virus.
Studies aimed at finding key drug compounds or vaccine candidates targeting the S-protein RBD-ACE-2 complex have been under pursuit for prompt treatment and research purpose. As there is no proper treatment for SARS-CoV-2 at present, it is highly crucial to search for direct therapeutics as well as natural therapeutics or immune boosters or virucidal products that can assist in containing the spread of the virus. Natural substances from herbs or mushrooms have been shown to possess potent antiviral properties that can be explored as therapeutics for SARS-CoV-2 (Shahidi and Camargo, 2021;Mohiuddin, 2021;Shahzad et al., 2020;Pan et al., 2013). In this regard, Chaga (Inonotus obliquus), a traditional edible mushroom, is well known for its therapeutic value. Chaga mushroom polysaccharides have been found in several studies to possess biologically active substances, in particular long chain homopolysaccharide beta-glucan, galactomannan, and the unique terpenoid betulinic acid (Gao et al., 2005;Kim, 2005;Chen & Wang, 2014;Glamočlija et al., 2015;Szychowski et al., 2020;Peng and Shahidi, 2020;Lu et al., 2021;Basal et al., 2021). The virucidal activity of crude Chaga extract was previously proved against feline coronavirus (FCoV) (Tian et al., 2017) and hepatitis virus (Pan et al., 2013), suggesting a promising potential application in developing antiviral regimens against the current novel pandemic due to SARS-CoV-2. We hence attempted to target the S-protein of SARS-CoV-2 with the unique components of Chaga mushroom for developing a potent natural therapeutic that can perform dual roles: aid in inhibiting the viral entry by interacting with the specific viral S-protein interacting sites involved in ACE-2-RBD interaction, and boosting the immunity and reducing inflammation in the hosts, thereby assisting in preventing the cytokine storm and sudden inflammatory spurt that is responsible for maximum fatalities.

| Spike protein amino acid sequence retrieval and phylogenetic analysis
Global SARS-CoV-2 S-protein amino acid sequences (n = 202) were retrieved from Uniprot and comprised a set of sequences from bat, pangolin, and human samples involved in previous and recent outbreaks. Multiple sequence alignment (MSA) was performed using Clustal W program embedded in Mega software ( Figure S1).
Maximum-likelihood method and generalized time-reversible model of amino acid substitution were utilized to perform the phylogenetic analysis in Mega 5 software (Tamura et al., 2011). Bootstrap replications of 1,000 values of the Nearest-Neighbor Interchange procedure were used to estimate the robustness of each node, along with estimating the genetic distance using the maximum likelihood method.

| Preparation of receptor protein
The cryo-electron microscopy structure of SARS-CoV-2 protein (PDB ID: 6VSB) was chosen for initial structure preparation as it F I G U R E 1 Depicts the mechanism of SARS-CoV-2 entry, replication, and maturation of the virion mediated through the attachment to the host cell F I G U R E 2 The compact structure of spike glycoprotein showing the receptor-binding domain of SARS corona virus (COVID-19). Right. Active site pockets (red) of S-glycoprotein from CastP server based on maximum surface area (33,706.38) and surface volume (63,378.53) available for ligand binding is explicitly demonstrated with its ligand interacting sites (Wrapp et al., 2020). First, all the water molecules and heteroatoms were removed in Autodock tool. Then, hydrogen atoms were added to the model based on an explicit all atom model. Finally, Kollmann charges were added for ensuing interaction with the ligands, and the model was energy minimized.

| Ligand preparation
The 3D conformers of Chaga mushroom components beta glycan (CID: 439262), galactomannan (CID 439336), and betulinic acid (CID: 64971) were retrieved in the 3D conformer state from Pubchem ( Figure 2) and were prepared for docking in the Ligand Preparation tool of Discovery Studio v20.1.0.19295 (Studio, 2008). In addition, anuronosyl-beta-D-glucopyranose; CID: 5288907) that has potent binding affinity for the S-protein of SARS-CoV-2 was also retrieved from Pubchem for comparison purposes and prepared as described above. The root was detected for each ligand, which were eventually saved in.pdbqt files similar to that of receptor protein.

| Molecular docking and post scoring analysis
SARS-CoV-2 cryo-electron microscopic structure (PDB ID: 6VSB) was employed for the docking analysis. Because SARS-CoV-2 is a homotrimer consisting of chain A, B, and C, chain A of the spike protein was used for the docking experiment. The active site of the target protein was predicted using the binding site module of Discovery studio (Studio, 2008) and CastP server (Tian et al., 2018) based on maximum volume (Figure 3). Blind docking with the complete spike F I G U R E 3 Above. The architecture of S-protein showing the different types of domains involved in the host virus interaction and virus entry. All three natural ligands interacted with the S1-CTD of the spike protein, indicating their potential to specifically bind to the RBD. Below: 3D cartoon model of chain A of spike protein of corona virus showing the S1 and S2 domains and the specific interaction region (S1 carboxy-terminal domain, CTD) of the chaga mushroom components protein was performed using Autodock tools to predict the overall receptor-ligand interactions. First, both the receptor spike protein and the ligands were processed in Autodock tools as described above and converted to.pdbqt format. Autodock Vina (Trott & Olson, 2010) docking tool was used to assess the binding ability of chaga mushroom components and viral S-protein. Blind docking was performed to know the probable binding sites on the spike protein.   Figure S2). This revealed that the recent SARS-CoV-2 virus possesses a furin cleavage site near the S1/S2 junction from 680 to 686 bp (NSPRRA), which was absent in the previous virus. Furin cleavage site can cause the excision of the two segments by the furin enzyme which eventually assists in gaining entry into the host through precise fusion and proteolysis (Walls et al., 2020). This was a key difference in the SARS outbreaks from previous and current outbreaks and indicated that the RBD is being selectively evolving. Besides, several residues in the S1-CTD domain that have been reported to be key residues for binding of SARS-CoV-2 to human ACE-2 such as ASN-343, TRP-436, ASN-437, ASN-440, TYR-442, LEU-472, ASN-473, TYR-475, ASN-479, TYR-484, THR-486, and THR-487 (Chen et al., 2020;Shang, Wan, Liu, et al., 2020) were found to be positively selected and conserved in our phylogenetic analysis. Therefore, drugs/small molecules targeting these regions could play key roles in modulating the viral entry into the host cells.

| Molecular docking analysis
Docking analysis revealed that Chaga mushroom components (beta glycan, galactomannan, and betulinic acid) bound to the S1-CTD residues of SARS-CoV-2 (PDB: 6VSB) involved in ACE-2-RBD interaction (Figures 4-7). Computational assessment using Autodock tools for S-protein and the three ligands beta glycan, galactomannan, and betulinic acid revealed that the highest interaction occurred at the could modulate its binding and thus can cause inhibition of virus entry into the host cell. Besides, the docking results were at par with the binding affinity of control molecule NAG with S-protein that has been reported to exhibit strong binding affinity for S-protein (Tai et al., 2020). Therefore, Chaga mushroom would also exhibit similar effects by various interactions and modulate the virus-host cell interaction.
The versatile benefits of Chaga mushroom could be attributed to its unique composition through which it can specifically targeting the TA B L E 1 Amino acid residues of 6VSB.pdb involved in interaction with the chaga mushroom components: beta glycan, galactomannan, betulinic acid, and control molecule NAG S1-RBD of SARS-CoV-2 (Ibrahim et al., 2021). Chaga mushroom is a natural compound and indicates no side effects when administered in proper dosages . It also enhances specific innate immunity system and assists in lowering the proinflammatory cytokines, such as IL-6, IL-10, TNFα, MCp, and many others. (Van et al., 2009;Mishra et al., 2012;Balandaykin & Zmitrovich, 2015). This attribute will greatly assist in reducing the case fatalities of SARS-CoV-2 due to a phenomenon called "cytokine storm" that results in an uncontrollable amplification and recruitment of inflammatory cytokines and immune cells to combat the infection, eventually resulting in organ damage and death (Pedersen & Ho, 2020). Therefore, Chaga mushroom can be a health-promoting booster in severe SARS-CoV-2 cases that exhibit excessive inflammation. Hence, delineating the role of Chaga mushroom components in SARS-CoV-2 interaction using and laboratory-based studies and clinical trials could reveal promising potential for the development of natural antiviral therapeutics.
Chaga mushroom shows promise in interacting with the viral spike protein and can be further explored in clinical settings that can bolster the current treatment regime for SARS-CoV-2. This will assist in developing natural anti-coronavirus therapeutics in future that can greatly supplement the use of current anti-SARS-CoV-2 drugs.

ACK N OWLED G EM ENTS
Nil.

CO N FLI C T S O F I NTE R E S T
The authors declare that they do not have any conflict of interest. Writing -original draft (equal); Writing -review and editing (equal).

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

F I G U R E 8
Mechanistic diagram depicting the overall interaction of Chaga mushroom components beta glycan, galactomannan, and betulinic acid with the spike protein (S-protein) of SARS-CoV-2. The three Chaga components specifically interacted with the S1-carboxyterminal domain of the SARS-CoV-2 with relatively high binding energies, which could potentially modulate the S-protein and implicate in inhibition of virus entry into the host cell

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available in article supplementary material.