K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.

Current address Biological Sciences Division Pacific Northwest National Laboratory 902 Battelle Blvd Richland WA 99353

Department of Biochemistry and Biophysics Texas A and M University 301 Old Main Drive College Station Texas 77843

Department of Chemistry Texas A and M University 301 Old Main Drive College Station Texas 77843

Department of Medicine Division of Infectious Diseases and Global Public Health University of California San Diego La Jolla CA 92037 USA

Department of Microbiology and Immunology University of Texas Medical Branch 3000 University Boulevard Galveston Texas 77755 1001

Institute for Antiviral Research Department of Animal Dairy and Veterinary Sciences 5600 Old Main Hill Utah State University Logan Utah 84322

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic 16610 Prague Czech Republic

Selva Therapeutics Department of Animal Dairy and Veterinary Sciences 5600 Old Main Hill Utah State University Logan Utah 84322

Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego La Jolla CA

ACS Chem Biol. 2021 Apr 16;16(4):642-650. doi: 10.1021/acschembio.0c00875. PubMed

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