Peptidomimetic α,β-Unsaturated Ethyl Esters Are Irreversible Inactivators of Human Cathepsin L and Are Potent Inhibitors of SARS-CoV-2 in Cellular Models of COVID-19.

Human cathepsin L (hCatL), a lysosomal cysteine protease, facilitates endosomal entry for SARS-CoV-2, making it a critical therapeutic target for COVID-19. Here, we report irreversible tripeptidomimetic inactivators containing a 3-pyridyl alanine residue at the P1 position, modified with either electrophilic α,β-unsaturated ethyl ester or sulfone warheads. These inhibitors had Ki* values between 0.4 and 35 nM. The most potent inhibitors, VK-168 (Ki* = 0.58 nM) and VK-261 (Ki* = 0.38 nM), demonstrated robust anti-SARS-CoV-2 activity in Vero E6 (EC50 = 120-310 nM) and A549/ACE2 cells (EC50 = 78-120 nM). Additionally, VK-80 inhibited infection (EC50 < 156 nM) and showed partial efficacy in a murine COVID-19 model. Native mass spectrometry and dilution assays confirmed irreversible hCatL inactivation. Notably, these enoates acted as reversible inhibitors of the homologue cathepsin B (hCatB). Molecular modeling suggests that hCatL's active-site cysteine is better positioned for nucleophilic addition to the vinyl group than hCatB's.