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Natural products may interfere with SARS-CoV-2 attachment to the host cell.

Objectives: SARS-CoV-2 has been emerged in December 2019 in China, causing deadly (5% mortality) pandemic pneumonia, termed COVID-19. More than one host-cell receptor is reported to be recognized by the viral spike protein, among them is the cell-surface Heat Shock Protein A5 (HSPA5), also termed GRP78 or BiP. Upon viral infection, HSPA5 is upregulated, then translocating to the cell membrane where it is subjected to be recognized by the SARS-CoV-2 spike. In this study, some natural product compounds are tested against the HSPA5 substrate-binding domain β (SBDβ), which reported to be the recognition site for the SARS-CoV-2 spike.Methods: Molecular docking and molecular dynamics simulations are used to test some natural compounds binding to HSPA5 SBDβ.Results: The results show high to a moderate binding affinity for the phytoestrogens (Diadiazin, Genistein, Formontein, and Biochanin A), chlorogenic acid, linolenic acid, palmitic acid, caffeic acid, caffeic acid phenethyl ester, hydroxytyrosol, cis-p-Coumaric acid, cinnamaldehyde, thymoquinone, and some physiological hormones such as estrogens, progesterone, testosterone, and cholesterol to the HSPA5 SBDβ. Based on its binding affinities, the phytoestrogens and estrogens are the best in binding HSPA5, hence may interfere with SARS-CoV-2 attachment to the stressed cells.Conclusion: These compounds can be successful as anti-COVID-19 agents for people with a high risk of cell stress like elders, cancer patients, and front-line medical staff.


Introduction
The Chinese National Health Commission reports a novel human coronavirus (SARS-CoV-2) in December 2019 (Bogoch et al., 2020;Hui et al., 2020). It was after that declared as a pandemic two months later by the World Health Organization (WHO) (Bogoch et al., 2020;World Health Organization, 2020a, 2020b, 2020c. Pneumonia associated with SARS-CoV-2, termed COVID-19, is suspected to be due to the first animal to human transmission in a seafood market in Wuhan city in November 2019 (Hui et al., 2020;Parr, 2020 ). On 20 January 2020, Chinese authorities confirmed the human-to-human route for virus transmission (Hui et al., HSP5A is the master of the unfolded protein response (UPR) in the lumen of the endoplasmic reticulum (ER) (Ibrahim et al., 2019).HSPA5 is responsible for protein homeostasis in the lumen of the ER. Upon cell stress, such as under the condition of viral infection or in the case of cancer cells, HSPA5 is upregulated and translocated to the cytoplasm and cell membrane complexing with other proteins (Al-Hashimi et al., 2018;Chen & Xu, 2017;Misra et al., 2005;Nain et al., 2017;Pujhari et al., 2017). HSPA5 is reported to be cell-surface exposed and responsible for pathogen entry (such as the fungus Rhizopus oryzae and many viruses like Human Papillomavirus, Ebola virus, Zika virus, and human coronaviruses) (Elfiky, 2019;Elfiky, 2020a;Elfiky, 2020b;Ibrahim et al., 2019;Ibrahim et al., 2020;Pujhari et al., 2017).
Different natural products have plenty of active molecules that can block the recognition site of the cell-surface HSPA5 and compete for the viral spike recognition.
The four phytoestrogens daidzein, genistein, formononetin and biochanin A are found in Cicer arietinum and proved its estrogenic activity for binding human and murine estrogen receptors alpha and beta in silico and it's in vivo restoration of the bone thickness for ovariectomized mice in a previous study (Sayed & Elfiky, 2018).
It was reported that both palmitic and linoleic acids alone (250 lM) induce ER stress in H4IIE liver cells, while the cotreatment of the hepatic cells with palmitic acid (250 lM) and linoleic acid (125 lM) abolished apoptosis (Zhang et al., 2012). Linoleic acid (125 lM), but not palmitic acid (250 lM), is responsible for cytochrome C release from the mitochondria to the cytoplasm during apoptosis (Zhang et al., 2012). Additionally, the lipotoxicity of saturated fatty acids like palmitic acid is reversed by the treatment of unsaturated fatty acids, such as a-Linolenic acid in the renal proximal tubular cell line, NRK-52E and chlorogenic acid in rat hepatocytes (Katsoulieris et al., 2009;Zhang et al., 2018).
The pre-treatment of hydroxytyrosol, the bioactive component of olive leaf extract, was successful in ameliorating myocardial infarction-mediated apoptosis, which was induced by the administration of isoproterenol to H9c2 cells .
Grape skin polyphenols, including caffeic acid and p-Coumaric acid, protect retinal pigment epithelial cells from photooxidative damage in a previous study (Zhao et al., 2014). The administration of grape skin extract before exposing the ARPE-19 cells to blue light was successful in reducing apoptosis in a dose-dependent manner. At the same time, GRP78 knockdown inhibited this protective role of the extract (Zhao et al., 2014). The honeybee hive propolis bioactive component, caffeic acid phenethyl ester (CAPE), induce oxidized protein-mediated ER stress in an autophagy-dependent manner (Tomiyama et al., 2018). CAPE treated human SH-SY5Y neuroblastoma cells overexpress ER stress-related genes like HSPA5 and enhance the expression of the autophagy marker, LC3-II (Microtubuleassociated protein 1 A/1B-light chain 3-phosphatidylethanolamine conjugate) (Tanida et al., 2008;Tomiyama et al., 2018).
Cinnamaldehyde (found in cinnamon) reported reducing the ER stress in the rat obesity animal model (Neto et al., 2020). The anticancer, oxidative and antioxidative properties of cinnamaldehyde are responsible for its potential to be used against breast cancer, prostate cancer, colon cancer, leukemia, HCC and oral cancers (Hong et al., 2016).
Thymoquinone (found in Nigella sativa seeds) was reported to prevent ER stress and mitochondria-induced apoptosis in rat animal model for ischemia-reperfusion in the liver (Bouhlel et al., 2017). It reduced the expression of the ER stress determinants, including HSPA5 in rats, while it improved the mitochondrial function leading to liver cell protection against ischemia-reperfusion associated apoptosis (Bouhlel et al., 2017). Thymoquinone was used in free and encapsulated formulations to prevent de-myelination in different brain compartments of Wistar rats while it acts as an anti-inflammatory and remyelinating agent (Fahmy et al., 2019;Fahmy et al., 2014;Noor et al., 2015).
In this study, I tested the active components found in some natural products, known by its involvement in ER stress, against the host cell chaperone protein, HSPA5. Additionally, some physiological hormones and compounds are also tested against the chaperone protein (estrogens, hydrocortisone, cholesterol, progesterone, and Testosterone) aiming to find possible natural sources that can alleviate the rapid spread of the newly emerged coronavirus (SARS-CoV-2) and reduce its impact on patients who have a higher affinity to be infected such as cancer patients.
The only available solved structure in the Protein Data Bank (PDB) for the wild-type and full-length HSPA5 in the open configuration is 5E84 (Yang et al., 2015;Yang et al., 2017). The coordinates of HSPA5 were downloaded and prepared for the docking study (water molecules and ligands are removed while missing Hydrogen atoms are added). National Center for Biotechnology Information (NCBI) nucleotide database was used to retrieve the gene (NC_045512.2) from which spike protein was translated (Expassy translate tool). A model was built with the aid of Swiss Model portal, where SARS HCoV (PDB ID: 6NUR, chain A) was used as a template in a previous study by the author (Biasini et al., 2014;Ibrahim et al., 2020;NCBI. , 2020). Structure analysis and verification server (SAVES) of UCLA was used to validate the model (SAVES. , 2020). The validated model of the SARS-CoV-2 spike was energy-optimized using the computational chemistry workspace SCIGRESS in order for the spike structure to be ready for the molecular docking experiments. The minimization of the model was performed using classical mechanics (MM3 force field) after Hydrogen atoms addition (Lii & Allinger, 1989).

Molecular docking
Docking experiments (AutoDock Vina software) are performed using the HSPA5 solved structure (PDB ID: 5E84) after 50 ns of classical molecular dynamics simulation (performed using NAMD software) (Humphrey et al., 1996;Phillips et al., 2005;Trott & Olson, 2009). Four different conformations of HSPA5 representing the main four clusters (Chimera software) are used to test the ligands binding (Pettersen et al., 2004).
Thirteen different natural products-derived compounds are tested against the four different conformations of the host cell chaperone HSPA5 SBDb, including; daidzein, genistein, formononetin, biochanin A, palmitic acid, linolenic acid, chlorogenic acid, hydroxytyrosol, caffeic acid, caffeic acid phenethyl ester, p-Coumaric acid, cinnamaldehyde, and thymoquinone. Additionally, six different physiological compounds are also docked to the HSPA5 SBDb for comparison, including; estriol, estradiol, hydrocortisone, cholesterol, progesterone, and testosterone. All the dockings are done using flexible ligand into flexible active site protocol, where both the ligands and the active site residues (I426, T428, V429, V432, T434, F451, S452, V457, and I459) are treated as flexible during the search for a possible docking conformation using the vina scoring function of AutoDock Vina software (Trott & Olson, 2009;Yang et al., 2015). The grid boxes for the docking experiments were chosen to be of size 48 Â 46 Â 56 Å centered at (42.3, 54.9, À29.2) Å (with little differences between the different conformations of the HSPA5).
HADDOCK 2.4 web server is utilized to dock the spike model for SARS-CoV-2 against HSPA5 and the complex of HSPA5 with its docked ligands (van Dijk & Bonvin, 2006). The HADDOCK 2.4 easy interface was utilized in the study since there are no restraints to be defined (de Vries et al., 2010). Again the HSPA5 active site (I426, T428, V429, V432, T434, F451, S452, V457, and I459) is treated as flexible. In contrast, the C480-C488 region of the SARS-CoV-2 spike is treated as the active residues (binding site) in HADDOCK 2.4, as reported in a previous study by the author (Ibrahim et al., 2020).
After docking, the complexes are examined using the Protein-Ligand Interaction Profiler (PLIP) web server (Technical University of Dresden) (Salentin et al., 2015).
Results and discussion Figure 1 shows the 2 D structures of the natural product compounds (A) and physiological compounds (B) tested for its binding affinity to cell-surface chaperone HSPA5. The structure of HSPA5 (PDB ID: 5E84) is subjected to 50 ns of molecular dynamics simulation (MDS) to equilibrize its atoms in the presence of 0.154 M NaCl solution (TIP3P water model) at 310 K using the CHARMM 36 force field (Ibrahim A. Noorbatcha et al., 2010;Mark & Nilsson, 2001;Phillips et al., 2005;Rappe et al., 1992). Figure 2A shows  Figure 3A shows the average binding affinities of different natural compounds to the HSPA5 SBDb four different conformations with the error bars representing the standard deviations (SD). Pep42 (red column) is a cyclic peptide that recognizes explicitly cell-surface HSPA5 in vivo (Ibrahim et al., 2019;Kim et al., 2006). The average binding affinity of Pep42 is -6.73 ± 1.13 kcal/mol, which is used here as a reference to judge other compounds' binding affinities. Phytoestrogens (green columns) show excellent average binding energies to HSPA5 ranging from -6.98 ± 0.19 kcal/mol (biochanin A) up to -7.80 ± 0.91 kcal/mol (daidzein). Compared to Pep42, the phytoestrogens have at least the same binding affinity to HSPA5 SBDb. This means that a dietary supplement of phytoestrogens (found in Cicer arietinum) may contradict the binding of the SARS-CoV-2 spike to the cell-exposed HSPA5 preventing its recognition by the virus.

Docking results
For the saturated (palmitic) and unsaturated fatty acids (linoleic and chlorogenic acids) (yellow columns) the same conclusion can be drawn, with a better average binding affinity to HSPA5 for chlorogenic (À7.10 ± 0.96 kcal/mol) acid compared to other fatty acids (À6.05 ± 0.51 and À5.50 ± 0.46 kcal/mol for linoleic and chlorogenic acid, respectively). This pattern of HSPA5 binding affinities is in good agreement with the previous reports of the antagonistic effect of unsaturated fatty acids, chlorogenic and linoleic acids, against the saturated, palmitic, fatty acid which induces ER stress (Katsoulieris et al., 2009;Zhang et al., 2018;Zhang et al., 2012). Palmitic, linoleic, and chlorogenic acids may be used to counteract the SARS-CoV-2 recognition of the host cell-surface HSPA5 and hence may reduce the viral attachment. Additionally, the saturated fatty acid, palmitic acid, may be used to target stressed HSPA5-exposed cells (viral infected or cancer cell) and induce ER stress leading to cell apoptosis.
The bioactive component of olive leaf extract, hydroxytyrosol, (bink column) shows moderate average binding affinity (À5.20 ± 0.35 kcal/mol) to HSPA5 SBDb. Hydroxytyrosol succeeded in a previous study as a prophylactic agent against myocardial infarction-mediated apoptosis . For the caffeic and p-Coumaric acids (light blue columns), that are found in grape skin, the average binding affinities to HSPA5 SBDb are À6.3 ± 0.60 and À5.63 ± 0.57 kcal/mol, respectively. These values are slightly less than Pep42 (À6.73 ± 1.13 kcal/mol), but the differences are not significant. Caffeic and p-Coumaric acids may bind to cell-surface HSPA5 competing for its recognition by viral spike protein and contradict the attachment. The same effect can be concluded from the caffeic acid phenethyl ester (CAPE) (dark blue column) that can be found in honeybee hive propolis (average binding affinity to HSPA5 SBDb is -7.13 ± 0.95 kcal/mol). This  . The average binding affinity (in kcal/mol) calculated using AutoDock Vina software for the docking of the natural products bioactive compounds (A) and physiological compounds (B) into the four different conformations of the HSPA5 SBDb. The cyclic peptide Pep42 (red column) is used as a reference due to its specificity in binding HSPA5 in vivo. Estrogens and phytoestrogen are among the best binders to HSPA5 SBDb. One docking trial is selected here to represent one conformation of the HSPA5 during 50 ns MDS. Bold residues are interacting through p-Stacking, while underlined residues are forming salt bridges. One docking trial is selected here to represent one conformation of the HSPA5 during 50 ns MDS. Bold residues are interacting through p-Stacking. average binding energy value is better than the highly selective cyclic peptide, Pep42, which indicates the potential of CAPE as an HSPA5 SBDb binder. Additionally, CAPE was reported to induce ER stress in an autophagy-dependent manner in human SH-SY5Y neuroblastoma (Tanida et al., 2008;Tomiyama et al., 2018). Cinnamaldehyde (cyan column) and thymoquinone (violet column) show À6.25 ± 1.10 and À5.520 ± 0.12 kcal/mol average binding energies to HSPA5 SBDb. These binding energies are comparable to the Pep42 cyclic peptide (À6.73 ± 1.13 kcal/mol) and hence the active components of cinnamon and the seeds of Nigella sativa may tightly bind to cell-surface HSPA5 and could be successful in contradicting SARS-CoV-2 spike recognition and attachment. Not only the natural compounds can bind HSPA5 SBDb with high affinity, but also other physiological molecules. Figure 3B shows the average binding affinities for the binding of estrogens (estriol and estradiol), cholesterol, progesterone, testosterone, and hydrocortisone (cortisol) to HSPA5 SBDb. As implicated from the binding energy values, all the physiological compounds can tightly bond the HSPA5 SBDb with values ranges from -7.20 ± 0.58 kcal/mol (Hydrocortisone) up to -8.40 ± 0.98 kcal/mol (estradiol). These values are lower (better) than that of the Pep42 cyclic peptide, which reported to target cell-surface HSPA5 (GRP78) in vivo selectively. It is important here to point out that HSPA5 SBDb may act as a receptor for such hormones. The binding energies indicate that cell-surface HSPA5 may be critical for these hormones recognition and hence internalization which not only may downregulate the concentration of cell-surface HSPA5, and its associated chemotherapeutic resistance, but also may play an essential role in hormone internalization for cell-signaling (Niu et al., 2015;Zhang et al., 2015). As a consequence, these hormones may also be used as protective molecules during chemotherapy to revert the chemoresistance of the HSPA5 presenting cancer cells.
Tables 1 and 2 summarize the interactions established between the small molecules and the HSPA5. Two types of interactions are dominant, the H-bonding and the hydrophobic interactions. Additionally, p-stacking (residues in bold in the tables) is reported between the residue F451 and the estrogens (estriol and estradiol), phytoestrogens (daidzein, genistein, and formononetin), caffeic acid phenethyl ester (CAPE), and cis-p-Coumaric acid. Also, salt bridges (underlined residues in Table 1) are formed between the residue K460 and both linolenic acid and cis-p-Coumaric acid. Hydrophobic interactions are more dominant compared to the H-bonding, as can be seen from almost all the natural and physiological compounds. This is in good agreement with previous reports defining the function of HSPA5 SBDb in the lumen of ER as to recognize unfolded proteins in the lumen of the ER mediating its degradation or refolding using cellular machinery (Ibrahim et al., 2019;Pfaffenbach & Lee, 2011;Roller & Maddalo, 2013). On the other hand, hydrocortisone and chlorogenic acid have more H-bonds than hydrophobic interactions (5:3 and 7:5 for hydrocortisone and chlorogenic acid, respectively). Additionally, caffeic acid forms four H-bonds and four hydrophobic contacts with the HSPA5 SDBb. Figure 4 shows the interaction analysis made by the PLIP web server for the docked structures of HSPA5 to estrogen (estradiol) (A), phytoestrogens (daidzein) (B), and biochanin A (C) as an example. The HSPA5 is shown in colored surface representations with its domains labeled. The ligands are represented in yellow sticks, where it appears how it fit in the binding site groove of the SBDb. Enlarged views of the binding sites show how the interactions established upon docking. Residues in the binding site of HSPA5 SBDb are represented in blue sticks and labeled with its one-letter code. In Figure 4, the hydrophobic interactions are described in dashed-gray lines, while H-bonds and p-stacking are depicted in solid blue lines and dashed-green lines, respectively. Docking scores are listed to reflect a binding affinity for each complex. Noticeably, the interacting residues are mainly hydrophobic, while hydrophobic interactions are dominant all the docking complexes. For estradiol, only one H-bond is formed through T458, while none is reported in daidzein. On the other hand, the biochanin A-HSPA5 complex show 5 Hbonds. Estradiol and daidzein but not biochanin A form p-stacking with residue F451 of HSPA5.
I performed molecular docking experiments using the four different conformations of HSPA5 after the 50 ns MDS using HADDOCK 2.4. The average docking score is À66.3 ± 5.7 indicating high binding affinity between the interacting proteins. Additionally, I tried to dock the small molecules-HSPA5 complexes to the SARS-CoV-2 spike protein model, but the spike doesn't fit the HSPA5 binding site; this may be due to the presence of the small molecules in the SBDb of HSPA5. The small molecules prevent the spike from binding to HSPA5 SBDb in silico. The results support the effectiveness of natural products and physiological hormones to block HSPA5 SDBb, preventing SARS-CoV-2 spike recognition (see the graphical abstract). The small molecules tested in this study may be used as prophylactic agents for highrisk personals like elders, medical staff in the front-line, or cancer patients.

Conclusion
The newly emerged human coronavirus pandemic is the health crisis we encounter in the 21 century, leaving more than 100000 deaths and 1.6 million reported cases. Natural products are known historically for its pharmaceutical properties. In this study, we tried to illuminate the route that some natural product active compounds may utilize though the human cell-surface receptor HSPA5 and its impact on SARS-CoV-2 attachment. These natural compounds or hormones may be used to reduce the risk of COVID-19 for highrisk people like elders and cancer patients or the front-line medical staff.

Competing interest
The author declares that there is no competing interest in this work.

Data availability
The docking structures are available upon request from the corresponding author.