Peptidomimetic Analogues Act as Effective Inhibitors against SARS-CoV-2 by Blocking the Function of Cathepsin L

Cathepsin L (CatL) is a promising antiviral drug target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as an important protease for cleaving the SARS-CoV-2 spike protein and enhancing viral entry to cells. We identified a tripeptide aldehyde candidate, D1-1, which exhibited inhibitory effects against SARS-CoV-2 in Vero E6 cells. The protease screening analysis and protein pull-down assays demonstrated the direct binding of D1-1 to CatL. Guided by molecular docking, we synthesized 72 analogues. Upon analyzing the structure-activity relationships of these inhibitors, the D6 series was developed. Among them, D6-3 functioned as the most potent CatL inhibitor (IC₅₀ = 0.27 nM, EC₅₀ = 0.26 μM). D6-3 effectively blocked the CatL function and substantially hindered the entry of the SARS-CoV-2 pseudovirus to cells. Our work presented novel compounds for targeting and inhibiting CatL, offering valuable insights into the development of SARS-CoV-2 antivirals.