Multiple Sites on Sars-Cov-2 Spike Protein Are Susceptible to Proteolysis By Cathepsins B, K, L, S, and V

SARS-CoV-2 is the coronavirus responsible for the COVID-19 pandemic that has killed more than 215,000 Americans. COVID-19 is also a health disparity with a disproportionate amount of infections and deaths occurring among African-American, Hispanic, and indigenous populations. Proteases are central to the infection process of SARS-CoV-2. Cleavage of the spike protein on the virus’s capsid into S1/S2 subunits causes the conformational change that leads to membrane fusion and viral entry into the target cell. Since inhibition of one protease, even the dominant protease like TMPRSS2, may not be sufficient to block SARS-CoV-2 entry into cells, other proteases that may play an activating role and hydrolyze the spike protein must be identified. We identified amino acid sequences in all regions of spike protein, including the S1/S2 region critical for activation and viral entry, that are susceptible to cleavage by furin and cathepsins B, K, L, S, and V using PACMANS, a computational platform that identifies and ranks preferred sites of proteolytic cleavage on substrates, and verified with molecular docking analysis to determine if binding of these proteases can occur on the spike protein that were identified as possible cleavage sites. Together, this study highlights cathepsins B, K, L, S, and V for consideration in SARS-CoV-2 infection and presents methodologies by which other proteases can be screened to determine a role in viral entry or rationale for inhibition in patients with inflammatory cardiovascular or pulmonary disease.