key: cord-320619-r466dc5t
authors: Chand Dakal, Tikam
title: SARS-CoV-2 Attachment to Host Cells is Possibly Mediated via RGD-Integrin Interaction in a Calcium-dependent Manner and Suggests Pulmonary EDTA Chelation Therapy as a Novel Treatment for COVID 19
date: 2020-11-05
journal: Immunobiology
DOI: 10.1016/j.imbio.2020.152021
sha: 
doc_id: 320619
cord_uid: r466dc5t

SARS-CoV-2 is a highly contagious virus that has caused serious health crisis world-wide resulting into a pandemic situation. As per the literature, the SARS-CoV-2 is known to exploit humanACE2 receptors (similar toprevious SARS-CoV-1) for gaining entry into the host cell for invasion, infection, multiplication and pathogenesis. However, considering the higher infectivity of SARS-CoV-2 along with the complex etiology and pathophysiological outcomes seen in COVID-19 patients, it seems that there may be an alternate receptor for SARS-CoV-2. I performed comparative protein sequence analysis, database based gene expression profiling, bioinformatics based molecular docking using authentic tools and techniques for unveiling the molecular basis of high infectivity of SARS-CoV-2 as compared to previous known coronaviruses. My study revealed that SARS-CoV-2 (previously known as 2019-nCoV) harbors a RGD motif in its receptor binding domain (RBD) and the motif is absent in all other previously known SARS-CoVs. The RGD motif is well known for its role in cell-attachment and cell-adhesion. My hypothesis is that the SARS-CoV-2 may be (via RGD) exploiting integrins, that have high expression in lungs and all other vital organs, for invading host cells. However, an experimental verification is required. The expression of ACE2, which is a known receptor for SARS-CoV-2, was found to be negligible in lungs. I assume that higher infectivity of SARS-CoV-2 could be due to this RGD-integrin mediated acquired cell-adhesive property. Gene expression profiling revealed that expression of integrins is significantly high in lung cells, in particular αvβ6, α5β1, αvβ8 and an ECM protein, ICAM1. The molecular docking experiment showed the RBD of spike protein binds with integrins precisely at RGD motif in a similar manner as a synthetic RGD peptide binds to integrins as found by other researchers. SARS-CoV-2 spike protein has a number of phosphorylation sites that can induce cAMP, PKC, Tyr signaling pathways. These pathways either activate calcium ion channels or get activated by calcium. In fact, integrins have calcium & metal binding sites that were predicted around and in vicinity of RGD-integrin docking site in our analysis which suggests that RGD-integrins interaction possibly occurs in calcium-dependent manner. The higher expression of integrins in lungs along with their previously known high binding affinity (∼K(D) = 4.0nM) for virus RGD motif could serve as a possible explanation for high infectivity of SARS-CoV-2. On the contrary, human ACE2 has lower expression in lungs and its high binding affinity (∼K(D)= 15nM) for spike RBD alone could not manifest significant virus-host attachment. This suggests that besides human ACE2, an additional or alternate receptor for SARS-CoV-2 is likely to exist. A highly relevant evidence never reported earlier which corroborate in favor of RGD-integrins mediated virus-host attachment is an unleashed cytokine storm which causes due to activation of TNF-α and IL-6 activation; and integrins role in their activation is also well established. Altogether, the current study has highlighted possible role of calcium and other divalent ions in RGD-integrins interaction for virus invasion into host cells and suggested that lowering divalent ion in lungs could avert virus-host cells attachment.

Ever since the recent emergence of novel coronavirus (SARS-CoV-2, earlier known as 2019-nCoV) in the Wuhan city of China and its subsequent transmission in other countries has resulted into serious heatth crisis as well as breakdown of socio-economic development. Scientific community from all over the world is industriously engaged in and committed to find a potent therapeutic solution for the treatment of COVID-19. As of 10 th April, 1,521,252 confirmed cases and 92,798 deaths were recorded as a cumulative data from different parts of the world (WHO Situation Report no. 81 available at who.int accessed on 12-04-2020). At the onset of the disease, the infected symptomatic patients experience hyperthermia, pharyngeal congestion, cough, and anosmia (in some cases); however, as disease progress more than fifty percent patients develop severe labored breathing, clinically referred to as dyspnoea or tachypnoea Qui et al., 2020; . Advance stages are characterized by severe pulmonary inflammation, fibrosis and obstructions of the bronchioles resulting in a pneumonia-like pathophysiological condition Qui et al., 2020; . In both symptomatic and asymptomatic COVID-19 patients, SARS-CoV-2 manages to cause significant damages to multiple organs before any patients could realize they are infected with SARS-CoV-2. This is because neither any neurological indications nor any signs of heart, kidney and liver failure are seen at the onset of disease in these patients (Qui et al., 2020).

As of today, there is no specific antiviral therapy available in any form, either vaccine or drug or others, to combat COVID-19 and its infection. The neutralizing antibodies that were previously tested and found successful against SARS-CoV-1 have displayed inappreciable cross-reactivity against SARS-CoV-2.For some antibodies (such as CR3022) that could bind to SARS-CoV-2, their neutralizing efficacy has not been tested and appreciated yet Yuan et al., 2020; Wrapp et al., 2020) . It has been asserted that the binding sites for these monoclonal antibodies on SARS-CoV-2 are vulnerable and it is only an assumption that antibodies that could bind strongly would possibly neutralize the SARS-CoV-2 also. Moreover, cross-reacting neutralizing antibodies are also doubted for their ability to confer prolonged protection against SARS-CoV-2. So far,we have no other choice than to use previously tested drugs or to solely oblige known tactics as precautionary measures against COVID-19. Recently, some researchers suggested the use of combination of remdesivir (a broad-range antiviral drug) and chloroquine for effective control of SARS-CoV-2 infection under in vitro condition (Devaux et al., 2020; . Similarly, hydroxychloroquine and azithromycin has also been referred to as a potent therapeutic weapon against the SARS-CoV-2 virus . Drug repurposing approach, which involved a screening of a thousand of molecules showed that HIV protease inhibitors, RNA-dependent RNA polymerase (RdRp) inhibitors and some other inhibitors and agonists such as methisazone (an inhibitor of protein synthesis), CGP42112A (an angiotensin AT2 receptor agonist) and ABT450 (an inhibitor of the non-structural protein 3-4A) could become promising treatment options for COVID-19 (Gordon et al., 2020; Li et al., 2020; Shah et al., 2020) . Lu (2020) suggested some treatment options for COVID-19 that include use of nucleoside analogues, neuraminidase inhibitors, lopinavir or ritonavir, remdesivir, 3TC/TDF/EFV monotherapy or combination therapy (DNA polymerase inhibitors), anti-inflammatory or immune-suppressive drugs, just to name a few. Besides this, some traditional Chinese medicine, for instance,ShuFengJieDu and Lianhuaqingwen capsules could also be useful (Lu, 2020) . Using virtual screening (Kandeel and Al-Nazawi, 2020), epigenetic dysregulation (Sawalha et al., 2020) , proteinprotein interaction mapping (Cava et al., 2020) , integrated network pharmacological approach , and similar such approaches, a number of other drugs have also been repurposed for COVID-19 treatment. However, most of the drugs are either in developmental stages or under clinical trials (https://clinicaltrials.gov/). In the midst of this pandemic situation, it is obvious that there exists no preventive therapy for highly contagious SARS-CoV-2 and this is strikingly alarming to all of us.

A number of studies demonstrated the key role of human ACE2 in virus attachment to host cells (Wan et al., 2020; Zhao et al., 2020) . The three-dimensional crystal structure of SARS-CoV-2 spike receptor binding domain complexed with its receptor, human ACE2 (Angiotensin converting enzyme 2), has been already solved .All other structural, functional and antigenicity related information related to SARS-CoV-2 are also available (Walls et al., 2020) . Based on biophysical data, it has been demonstrated that the human ACE2 binds to SARS-CoV-2 with greater affinity than SARS-CoV-1 (Wrapp et al., 2020) . However, owing to their low copy number (protein expression) (Chen et al., 2020), their high affinity (for SARS-CoV-2 than SARS-CoV-1) alone could not manifest reasonable virus-host cell attachment, at least in lung cells. When this manuscript study was under progress, Sigrist and coauthors (2020) showed that SARS-CoV-2 harbors a RGD motif and integrins (that display high affinity for RGD motifs) may be involved in facilitating virus entry into host cells. However, the study did not explain the full mechanistic state of affairs involved in RGD-integrins interaction and virus entry into the host cells.I also found RGD motif in the spike receptor binding domain of SARS-CoV-2 and studied mechanistic basis of RGD-integrin (and other ECM protein such as ICAM1) mediated virus invasion into host cells. This study is the first study to present striking evidence (substantiated by existing facts in literature) favoring the role of calcium and other divalent ions (magnesium, manganese etc.) in RGD-integrins mediated virus attachment with the host cells for and that lowering the concentration of calcium and other divalent ions in lungs could be a possible mechanism to avert SARs-CoV-2 infection and invasion. Herein, I did comparative protein sequence analysis, motif scanning, gene expression data analysis and bioinformatics based molecular docking using most trustworthy tools and techniques. The 

The coronavirus related nucleotide and protein sequences were retrieved from GenBank (https://www.ncbi.nlm.nih.gov/), UniProtKB (https://www.uniprot.org) and SARS Coronavirus 2 data hub of the NCBI (https://www.nih.gov/coronavirus).

The protein sequence of SARS-CoV-1 and SARS-CoV-2 coronaviruses were subjected to pair-wise 

The Pfam, Prosite and HAMAP profiles of the S protein from the coronavirus was ascertained using

MyHits Motif Scan (SIB, Switzerland) (https://myhits.isb-sib.ch/cgi-bin/motif_scan). The putative Nlinked glycosylation and other post-translation modification sites were predicted as Frequent Pattern in MyHits outputs (Pagni et al., 2007) .

The expression profile of ACE2 receptor protein and integrins in lungs was ascertained using Gene Expression Database at EBI (https://www.ebi.ac.uk/gxa/home) and the Human Protein Atlas 

In order to understand the role of divalent ions in RGD-integrins mediated virus-host cells attachment. A composite calcium and magnesium binding-site in integrins were predicted using IonCom (https://zhanglab.ccmb.med.umich.edu/IonCom/), which used an integrated approach based onab initio training and template-based transferals (Hu et al., 2016) and predicts calcium binding sites in a given protein by searching four or more oxygen atoms on protein surface arranged in a spherical manner. The input PDB file of integrins such as α5β1 (PDB ID: 3VI3) and αvβ6 (5FFG) were subjected for ion binding sites' prediction to specifically predict calcium and magnesium binds sites.

The PDB file of the spike receptor binding domain (PDB ID: 6LZG) and integrins such as α5β1 (PDB ID:

3VI3 and 3VI4) and αvβ6 (PDB ID: 5FFG) were retrieved from Protein Data Bank Protein-ligand binding modes were clustered according to their rank based on average FullFitnessin output data (Grosdidier et. al., 2007) .

Pairwise sequence alignment of SARS-CoV-1 and SARS-CoV-2 spike protein revealed that the Nterminus (especially upto 250 aa) of SARS-CoV-2 is highly divergent than that of the SARS-CoV-1

( Figure 1 ). The S1 Glycoprotein (from 268-304 aa in SARS-CoV-1 and from 282-317 aa in SARS-CoV-2) domain which plays an important role in recognition of host cell receptor were found divergent at 10 amino acid positions. The spike receptor binding domain is of 253 amino acid residues in SARS-CoV-1 and ranges from 317 to 569 aa; whereas, it is of 254 amino acid residues in SARS-CoV-2 and stretch from 330 to 583 aa. There were 49 mismatches and 1 insersion/deletion that render SARS-CoV-1 and SARS-CoV-2 approximately 80% sequence similarity. The S2 glycoprotein domain is a large 544 amino acid residues long region at the C-terminus of the spike protein which plays crucial role in virus fusion with host cells. This region has been predicted to be less divergent than S1 glycoprotein and RBD. There I also found two insertion sequences with the stretch HVSGTNGTKRFD 69-80 and RFQTLLALHRSYLTPGDSSSGWTAG 236-261 at the N-terminus region of spike protein and these were seen as discontinuous in the amino acid sequence as predicted by the pair-wise sequence alignment.

However, considering that these inserts are very short and appeared in the hypervariable region of viral spike protein, the most likely assumption for their origin is that they might have arisen naturally. Besides this, the receptor binding domain of the spike protein in SARS-CoV-2 has a stretch of sequence (TEIYQAGSTPCNGVEGF 470-486 ) showing mismatch with SARS-CoV-1.

The number and position of putative N-linked glycosylation sites, post-translation modification sites that can induce cAMP, CK2, Tyr, PKC signaling pathways and Myristyl sites were predicted to be varying in SARS-CoV-1 and SARS-CoV-2 (Table 1) . Although both coronaviruses have equal number of N-linked glycosylation sites but the position of sites differs (Table 1) 

The spike protein sequence of SARS-CoV-1 and SARS-CoV-2 were subjected to motif scanning and prediction using MyHits Motif Scan at SIB server. A number of motifs were predicted in the spike protein sequence such as RGD (from 403-405 aa in receptor binding domain of SARS-CoV-2) ( Table 2 ). The RGD motif (K403R substitution) was predicted in spike receptor binding domain of SARS-CoV-2 and the same was not predicted in the SARS-CoV-1 spike protein and other previous known coronaviruses.

This motif was originally found in fibronectin, which is an extracellular matrix protein and this motif plays a major role in cell adhesion and attachment. A number of proteins are known to interact with these RGD motifs. One such group of proteins are integrins that have strong affinity for RGD motif (~KD=4.0nM) and they employ this motif to efficiently mediate cell adhesion. Eight out of approximately twenty known integrins recognize and exploit RGD motifs for cell adhesion (Ruoslahti, 1996 (Table 2) .

Presence of RGD motif in the spike receptor binding domain of the SARS-CoV-2 and the fact that integrins display a strong affinity for proteins harboring RGD motifs (Liu, 2009) have been found to be affected by SARS-CoV-2 infection (Figure 2A-C) . These three integrins were used for molecular docking experiments. I also found that expression of an ECM protein ICAM1 is significantly higher (even higher than integrins) in lungs ( Figure 2D ) suggesting that SARS-CoV-2 spike protein might be using these integrins and/or ECM proteins such as ICAM1 (or others) for invading host cells. On the contrary, the ACE2 RNA is detected in very low quantity (almost unidentifiable) in lungs and detectable expression peaks were detected in other organs such as duodenum, small intestine, colon, gallbladder, kidney, testis, and heart muscles ( Figure 3 ). In congruence with this, Xu and coauthors (2020) found high expression of ACE2 in oral mucosa and Zhang and co-authors (2020) demonstrated digestive route as a potential mechanism of ACE2 mediated virus infection based on the single-cell transcriptomic analysis. On the contrary, Chen and coauthors (2020) observed relatively low levels of ACE2 mRNA expression in lungs which further supports my claims.

The RGD motif is the minimal indispensable requirement for integrins to bind with any viral protein which virus can use for attachment with host cells (Hussein et al., 2015) . Nagae Figure 4B ). ICAM1 is also known to bind integrins and this mechanism is exploited by some viruses (such as Rhinoviruses) to gain entry into respiratory system for pathogenesis (Bella et al., 1998) .

The RGD motifs are well known in the field of cell biology, cell therapy and tissue engineering because (Table 3 ).

In any disease or pathological condition where integrin expression would be high, I can expect patient to proteinurea and renal insufficiency are known to associate with EDTA chelation therapy and I suggest that a strict monitoring protocol, comprising routine cytokine profiling, blood examination, urine test and CT scanning etc., must be practiced while treating patients with pulmonary EDTA chelation therapy.

In combination with the EDTA/EGTAbased pulmonary chelation therapy, modulation of integrins expression using integrins inhibitors or anti-integrin antibodies could also serve as a mechanism to treat . The docking was done using HDOCK server (hdock.phys.hust.edu.cn/). Spike protein is shown in red and synthetic RGD peptide is shown in green. FIGURE 5. Pulmonary EDTA chelation therapy which can be clinically executed through a nebulizer or inhaler to allow sodium-EDTA to pass into the lungs. The sodium-EDTA will chelates Ca +2 ions and other divalent ions making them unavailable for RGD-integrin mediated virus attachment to the host cells. A novel strategy for safe, technically simple, quick, cost-effective and efficient treatment of COVID 19 patients. 

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I acknowledge Mohanlal Sukhadia University, Udaipur for the infrastructure facility.

The work has been conducted in absence of any funding.