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.