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

Abstract

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 = 4.0 nM) 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 = 15 nM) 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.

Fig. 1

Pair-wise sequence alignment of SARS-CoV-1 and SARS-CoV-2 spike protein RBD sequence using Clustal Omega.

Fig. 2

Gene expression profile of RGD binding integrins αvβ6 (A), α5β1 (B), α8β1 (C) and human ICAM1 (D) in lungs as obtained and plotted at the Human Protein Atlas. (https://www.proteinatlas.org/). The bar shows expression level of respective protein in different human tissues and organs. The expression peaks that correspond to lungs have been shown using red arrow to ease visualization and verification of data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Gene expression profile of ACE2 in lungs as obtained and plotted at Gene Expression Database at EBI (https://www.ebi.ac.uk/gxa/home) (A) and the Human Protein Atlas (https://www.proteinatlas.org/) (B). The expression peak that corresponds to lungs have been shown using red arrow to ease visualization and verification of data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Molecular docking of viral spike RBD domain onto the structure of α5β1 (PDB ID: 3VI3) and αvβ6 (PDB ID: 5FFG) showing interaction via the RGD motif present in the spike RBD domain (A). The molecular docking of viral spike RBD domain onto the structure of α5β1 (PDB ID: 3VI3) showed that the spike RBD is interacting with the α5β1 through its RGD motif in the same way (B, left panel) as synthetic RGD peptide binds and interact to α5β1 (PDB ID: 3VI4) (B, right panel) as already known in literature (Nagae et al., 2012). 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. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 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.