A novel small-molecule compound disrupts influenza A virus PB2 cap-binding and inhibits viral replication

The conserved residues 318-483 in the PB2 subunit of influenza A polymerase is an independently folded cap-binding domain (PB2(cap)) that exhibits a distinct binding mode from other host cap-binding proteins, which suggests that PB2(cap) might be an ideal drug target. This study aimed to identify a new class of anti-influenza inhibitors that specifically disrupts the interaction between PB2(cap) and host cap structures. An innovative fluorescence polarization assay was established for primary screening, followed by cap-binding inhibitory activity, antiviral efficacy and cytotoxicity evaluations of the selected compounds. The best compound was characterized by multi-cycle virus growth assay, cross-protection test, synergism evaluation, mini-replicon assay, binding affinity analysis, docking simulation and mouse study. Several PB2 cap-binding inhibitors were discovered. The compound 7-(4-hydroxy-2-oxo-2H-chromen-3-yl)-6H,7H,8H-chromeno[3',4':5,6]pyrano[3,2-c]chromene-6,8-dione, designated PB2-39, was identified as a potent inhibitor of replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 in vitro and H1N1, H5N1 and H7N9 in vivo. Combinational treatment with the influenza virus release inhibitor zanamivir and PB2-39 exerted a synergistic anti-influenza effect. Mechanistic experiments supported that PB2-39 suppressed viral polymerase activity. Docking and binding affinity analyses demonstrated that PB2-39 interacted with the PB2 cap-binding pocket, suggesting its role as a cap-binding competitor. Our study provides new insights for the strategic development of novel cap-binding inhibitors of influenza A viruses.