Development of a magnetic beads-based ligand fishing method for screening SARS-CoV-2 NSP5 inhibitors from complex herbal mixtures: Rosmarinus officinalis as a case study.

SARS-CoV-2 NSP5, a conserved protease essential for viral replication, represents a critical therapeutic target. Natural products provide diverse chemical scaffolds for antiviral development but remain underexploited. Traditional screening methods struggle to resolve specific inhibitors in complex herbal matrices due to selectivity and throughput limitations. To address these challenges, we developed an innovative magnetic beads-based ligand fishing strategy coupled with UPLC-Q-Exactive-Orbitrap-MS/MS for rapid discovery of SARS-CoV-2 NSP5 inhibitors from complex herbal matrices. Screening of 44 traditional Chinese medicinal extracts identified Rosmarinus officinalis as a potent inhibitor, exhibiting an IC50 of 19.90 ± 3.16 μg/mL against NSP5. Through ligand fishing and mass spectrometry analysis, 15 bioactive compounds were identified, including 8 diterpenoids (predominantly abietane-type) and 3 phenylpropanoids. Notably, rosmarinic acid demonstrated the strongest inhibition (IC50 = 5.784 ± 0.20 μM), surpassing the positive control ebselen in molecular docking studies. Structural characterization revealed that hydroxyl and carboxyl groups in diterpenoids formed critical hydrogen bonds with catalytic residues (Cys145, Ser144) of NSP5. This methodological advancement not only improved the accuracy of natural product screening but also facilitated the development of targeted antiviral strategies against SARS-CoV-2 and other emerging viral pathogens.