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.