Thiol-Reactive or Redox-Active: Revising a Repurposing\nScreen Led to a New Invalidation Pipeline and Identified a True Noncovalent\nInhibitor Against Papain-like Protease from SARS-CoV‑2

The SARS-CoV-2 papain-like\nprotease PLpro has multiple roles in\nthe viral replication cycle, related to both its polypeptide cleavage\nfunction and its ability to antagonize the host immune response. Targeting\nthe PLpro function is recognized as a promising mechanism to modulate\nviral replication, while supporting host immune responses. However,\nthe development of PLpro-specific inhibitors remains challenging.\nComprehensive investigations utilizing enzymatic, binding studies,\nand cellular assays revealed the previously reported inhibitors to\nact in an unspecific manner. At present, GRL-0617 and its derivatives\nremain the best-validated compounds with demonstrated antiviral activity\nin cells and in mouse models. In this study, we refer to the pitfalls\nof the redox sensitivity of PLpro. Using a screening-based approach\nto identify inhibitors of PLpro’s proteolytic activity, we\nmade extensive efforts to validate active compounds over a range of\nconditions and readouts, emphasizing the need for comprehensive orthogonal\ndata when profiling putative PLpro inhibitors. The remaining active\ncompound, CPI-169, was shown to be a noncovalent inhibitor capable\nof competing with GRL-0617 in NMR-based experiments, suggesting that\nit occupied a similar binding site and inhibited viral replication\nin Vero-E6 cells, opening new design opportunities for further development\nas antiviral agents.