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

Papain-like protease (PL) 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, revealing an inspiring strategy for COVID-19 treatment. This study aimed to discover PL 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 inhibitors with IC values ranging from 9.5 to 43.2 μM. Pro-ISG15 cleavage assays further demonstrated that several biflavones exhibited potent inhibitory effects against PL-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.