Discovery of Novel Druggable Binding Pockets on SARS-CoV-2 PLpro via Fragment Screening and Insights for Structure-Based Inhibitor Design.

Coronaviruses are a large family of viruses capable of causing severe respiratory diseases, with recent outbreaks posing major threats to global public health. The SARS-CoV-2 papain-like protease (PLpro) is an essential viral enzyme involved in viral replication and host immune evasion, making it an attractive target for antiviral drug development. However, no PLpro-targeted drugs have yet been approved. Here, we employed a fragment-based drug discovery (FBDD) strategy combined with crystallographic screening and identified 23 fragment compounds with well-defined electron density. These fragments bind to seven distinct sites on PLpro and reveal multiple compound scaffolds and potential binding pockets through systematic structural analysis. Notably, Site 2 represents a newly identified pocket adjacent to the canonical substrate-binding site (Site 1) and shows strong potential for fragment linking to enable inhibitor optimization. Among them, Frag368 exhibited good enzyme inhibitory activity. Furthermore, based on structural similarities among the fragments, the FDA-approved drug 5-fluorouracil was identified as a ligand for Site 2. Biochemical assays confirmed that 5-fluorouracil exhibits inhibitory activity against PLpro. Collectively, these findings provide a robust structural framework for the rational design of PLpro inhibitors and support the development of novel antiviral therapeutics.