Rapid screening and identification of influenza virus polymerase inhibitors from Rubus idaeus L. by at-line nanofractionation

• Eight PAN inhibitors were successfully screened out from Rubus idaeus L. • Ellagic acid and luteoloside can bind to PAN and exhibit satisfactory in-vitro inhibitory activity. • The binding mechanisms of ellagic acid and luteoloside with PAN were clarified by molecular docking and dynamic simulations. The N-terminal region of the polymerase subunit (PAN) is considered as the most promising target for the development of broad-spectrum antivirals, since it is highly conserved in structure and sequences among different influenza strains. In this study, a PAN based at-line nanofractionation (ANF) platform was applied to rapidly screen for PAN inhibitors from Rubus idaeus L. extract which presented good inhibitory effect on PAN with a half maximal inhibitory concentration (IC 50 ) value of 11 μg/mL. Eight PAN inhibitors were successfully screened out, comprising four organic acids and four flavones. In parallel, magnetic ligand fishing was used to isolate the PAN ligands from the extract, and finally four compounds were fished out and considered as PAN ligands (including avicularin, quercetin-3-O-rutinoside, ellagic acid and luteoloside) which were also identified by the ANF platform. Among them, ellagic acid and luteoloside displayed satisfactory in vitro inhibitory activity against PAN, with IC 50 values of 1.31 ± 0.33 and 6.23 ± 0.97 μM, respectively. The molecular docking and dynamic simulations revealed that ellagic acid and luteoloside bind to PAN in a distinctly different mode compared to baloxavir. These compounds formed direct interactions with catalytic pocket residues and adjacent amino acids in PAN, establishing hydrogen bonds and water-mediated bridges that modulated the protein's secondary structure and stability. The result from the study suggests that ellagic acid and luteoloside could serve as promising anti-influenza molecules. Furthermore, the ANF platform was proved effective for high-throughput screening of PAN inhibitors from complex herbal extracts, highlighting its significant potential for future anti-influenza drug development.