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

Abstract

BACKGROUND: The conserved residues 318−483 in PB2 subunit of influenza A polymerase is an independently folded cap-binding domain (PB2cap) that exhibits a distinct binding mode from other host cap-binding proteins, which suggests that PB2cap might be an ideal drug target. This study aims to identify a new class of anti-influenza inhibitors that specifically disrupts the interaction between PB2cap and host cap-structures. METHOD: 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, crossprotection test, synergism evaluation, mini-replicon assay, binding affinity analysis, docking simulation and mouse study. RESULTS: Several PB2 cap-binding inhibitors were discovered. One of the best compounds, designated PB2-39, was identified as a potent inhibitor against the replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9, H9N2 in vitro and H1N1, H5N1, H7N9 in vivo. Combinational treatment of the influenza virus release inhibitor zanamivir and PB2-39 exerted 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. CONCLUSION: Our study provides new insights for the development of influenza PB2 cap-binding inhibitors through reverse chemical genetics. To the best of our knowledge, this is the first biochemical screening system that utilizes PB2 cap-binding domain as a selection target.