A Second Opportunity for the Peptide-Based Analogues with γ-Lactam at the P1 Position: Human Cathepsin S Inhibition

<b>Background/Objectives</b>: SARS-CoV-2 pandemic led to the identification of peptide-based main protease (M^pro) inhibitors. The overwhelming majority of them carry an electrophilic warhead and a γ-lactam at the P1 position. During the selectivity assessment of an <i>in-house</i> Michael acceptors targeting SARS-CoV-2 M^pro, we unexpectedly observed a significant inhibition of human cathepsin S (hCatS). <b>Methods</b>: The biological investigation of three compounds (i.e., <b>SPR38</b>, <b>SPR39</b>, and <b>SPR41</b>) against hCatS was performed. The binding mode of SPRs was investigated by docking and molecular dynamics simulations. <b>Results</b>: Biological investigation has corroborated that hCatS is sensitive to peptide-based analogues harbouring γ-lactam at the P1 position and a vinyl methyl ketone warhead. In silico studies revealed that despite being solvent exposed, the γ-lactam at P1 might be involved in water-mediated H-bonds that could be optimized to gain inhibition potency and selectivity. <b>Conclusions</b>: The molecules repurposing of peptide-based SARS-CoV-2 M^pro inhibitors carrying the γ-lactam at the P1 site could pave the way for the identification of novel potent and selective hCatS ligands.