Magnetic Bead-Based Ligand Fishing Identifies SARS-CoV-2 Main Protease Inhibitors From Reynoutria japonica

Traditional Chinese medicines (TCMs) are rich sources of structurally diverse bioactive compounds, yet rapid identification of protease inhibitors from complex botanical matrices remains challenging. Here, a magnetic bead-based affinity-fishing platform coupled with UPLC-Q-Exactive-Orbitrap-MS/MS was applied to identify SARS-CoV-2 main protease (M^pro) inhibitors from Reynoutria japonica. The crude ethanolic extract inhibited M^pro with an IC₅₀ of 44.92 ± 2.31 µg/mL. M^pro-functionalized magnetic beads enriched eight constituents, which were identified by high-resolution mass spectrometry and evaluated using a FRET-based inhibition assay. Six compounds showed concentration-dependent activity, with resveratrol being the most potent (IC₅₀ = 34.01 ± 1.67 µmol/L), followed by glycitein, genistein, emodin, apigenin-7-O-glucoside, and biochanin A. HPLC-DAD further quantified the principal active constituents. Molecular docking suggested interactions with residues in the Mpro substrate-binding pocket, while network pharmacology predicted associations with host-related targets involving IL6, TNF, and AKT1. Overall, this workflow enables efficient enrichment, structural identification, and functional prioritization of M^pro-inhibitory constituents from complex botanical extracts, providing a practical strategy for target-directed natural product discovery.