Regulation of the binding of the SARS-Cov2 spike protein receptor-binding domain (RBD) to its cognate receptor by heparan sulfate analogs

Abstract

The novel SARS-CoV-2 pandemic began in the Chinese city of Wuhan at the end of the year 2019 with more than 27 million individuals infected worldwide. Due to the severe nature of the symptoms that are most often associated with infection from this novel virus, it is imperative to understand the underlying pathogenesis of the disease, the infectious pattern and rates of transmission of the disease in order to screen for candidate drug targets that might prove efficacious in halting the spread of this disease. The clinical manifestation of the disease in infected individuals is high fever above 39C, acute respiratory disease syndrome (ARDS), severe inflammation and the formation of microthrombi in lung tissues and other major organs. Several studies have demonstrated that infection of alveolar cells by the SARS-COV-2 virus causes inflammation to occur through the recruitment of macrophages that secrete chemokines and cytokines resulting in a cytokine storm. The coagulation effect occurs as a result of the inflammatory exudates that activate the secretion of coagulation factors that enter the alveolar space and in turn cause the formation of the hyaline membrane which is responsible for the formation of microthrombi. To this effect, it has been hypothesized that the treatment of patients with anticoagulants such as heparin sulphate might be effective in preventing the formation of microthrombi. As such, this study aimed to determine the inhibitory potential of heparin sulphate and its mimetics ZU545, tafoxiparin and OKC4 in inhibiting the binding of the receptor-binding domain (RBD) of the spike protein to the ACE-2 receptor expressed on the surface of the Vero cells. To this effect, the RBD was expressed from transformed E. coli using IPTG and purified using gel filtration chromatography and the purified protein was inoculated to Vero cells that were subsequently treated with 1, 3, 10, 30 and 100g/ml of heparin sulphate, tafoxiparin, ZU545 and OKC4. It was observed that the highest concentrations of heparin sulphate and its mimetics (100g/ml) was most effective in inhibiting the binding of the RBD of the spike protein to the ACE-2 receptor expressed on the surface of the Vero cells. A comparison of the inhibitory potential of heparin sulphate and its mimetics at 100g/ml concentration demonstrated that tafoxiparin was most effective in inhibiting the binding of the RBD of the spike protein of the SARS-COV-2 virus. Lastly, the effect of freezing on the binding affinity of the RBD of the spike protein of the SARS-COV-2 virus. It was observed that binding affinity increased upon freezing of the RBD of the spike protein with 10% glycerol compared to only freezing the RBD of the spike protein in the absence of glycerol. In conclusion, heparin sulphate and its mimetics were effective in inhibiting the binding of the RBD domain of the spike protein of the SARS-COV-2 virus. The implication of these results in the fight against the novel SARS-COV-2 virus raises the possibility of a novel treatment strategy that might be effective in preventing the formation of blood clots which is the prevalent cause of mortality of SARS-COV-2 infected patients.