Natural biflavones are potent inhibitors against SARS-CoV-2 papain-like protease

Papain-like protease (PL^pro) is a key enzyme encoded by SARS-CoV-2 that is essential for viral replication and immune evasion. Significant suppression of viral spread and promotion of antiviral immunity can be achieved by inhibition of PL^pro, revealing an inspiring strategy for COVID-19 treatment. This study aimed to discover PL^pro inhibitors by investigating the national compound library of traditional Chinese medicines (NCLTCMs), a phytochemical library comprising over 9000 TCM-derived compounds. Through virtual screening and enzymatic evaluations, nine natural biflavones were confirmed to be effective PL^pro inhibitors with IC₅₀ values ranging from 9.5 to 43.2 μM. Pro-ISG15 cleavage assays further demonstrated that several biflavones exhibited potent inhibitory effects against PL^pro-mediated deISGylation, a key process involved in viral immune evasion. Herein, we report the discovery, antiviral evaluation, structure-activity relationship elucidation and molecular docking investigation of biflavones as potent inhibitors of SARS-CoV-2 PL^pro.