20.500.12556/DiRROS-23338 Thiol-reactive or redox-active revising a repurposing screen led to a new invalidation pipeline and identified a true noncovalent inhibitor against papain-like protease from SARS-CoV-2 The SARS-CoV-2 papain-like protease PLpro has multiple roles in the viral replication cycle, related to both its polypeptide cleavage function and its ability to antagonize the host immune response. Targeting the PLpro function is recognized as a promising mechanism to modulate viral replication, while supporting host immune responses. However, the development of PLpro-specific inhibitors remains challenging. Comprehensive investigations utilizing enzymatic, binding studies, and cellular assays revealed the previously reported inhibitors to act in an unspecific manner. At present, GRL-0617 and its derivatives remain the best-validated compounds with demonstrated antiviral activity in cells and in mouse models. In this study, we refer to the pitfalls of the redox sensitivity of PLpro. Using a screening-based approach to identify inhibitors of PLpro’s proteolytic activity, we made extensive efforts to validate active compounds over a range of conditions and readouts, emphasizing the need for comprehensive orthogonal data when profiling putative PLpro inhibitors. The remaining active compound, CPI-169, was shown to be a noncovalent inhibitor capable of competing with GRL-0617 in NMR-based experiments, suggesting that it occupied a similar binding site and inhibited viral replication in Vero-E6 cells, opening new design opportunities for further development as antiviral agents. SARS-CoV-2 drug repurposing papain-like protease redox STD-NMR CPI-169 GRL-0617 biokemija SARS-CoV-2 ponovna uporaba zdravila true false true Angleški jezik Slovenski jezik © 2024 The Authors. Published by American Chemical Society Neznano 2025-08-20 08:36:08 2025-08-20 08:54:49 2025-10-24 03:54:08 0000-00-00 00:00:00 2025 0 0 Nasl. z nasl. zaslona; Opis vira z dne 10. 1. 2025; str. 66–77 iss. 1 Vol. 8 Jan. 10, 2025 0000-00-00 Zaloznikova Objavljeno apc 0000-00-00 0000-00-00 2025-01-10 577 2575-9108 10.1021/acsptsci.4c00325 213383683 https://pubs.acs.org/doi/epdf/10.1021/acsptsci.4c00325 1 f5ac3b95-7d8f-11f0-9bb7-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=34477 RAZ_Kuzikov_Maria_2025.pdf RAZ_Kuzikov_Maria_2025.pdf 1 CFFE6BC29B0ECC881928D89D786DC1EB 60374cb64ce37061260d6d0a95ff97b2951ab21c40f5542b1000378924613110 2191d50a-7d92-11f0-9bb7-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=34478 Kemijski inštitut 0 0 0