Ahmed Oraby, University of Alberta

Ahmed K. Oraby1, Hesham Saafan2, Rafael Dias2, Arlo Loutan1, Matthias Götte1, Frederick G. West2, and David J. Marchant1 Affiliation: 1 Department of Medical Microbiology & Immunology, Faculty of Medicine and Dentistry, University of Alberta 2 Department of Chemistry, Faculty of Science, University of Alberta

Expressing & Multiplying

Influenza A virus (IAV) remains a major global health threat due to its genetic variability and pandemic potential. Antiviral options are limited and prone to resistance, underscoring the need for new strategies targeting essential replication machinery. The viral RNA-dependent RNA polymerase (RdRp) is required for genome replication and cap-snatching-mediated transcription. Within this complex, the PB2 cap-binding domain binds the 5' cap of host mRNAs and is essential for viral mRNA synthesis, making it an attractive yet underexplored antiviral target. We employed a structure-guided approach to identify novel small-molecule inhibitors targeting the PB2 cap-binding pocket of Influenza A. An integrated workflow combining ensemble docking, molecular dynamics simulations, and hydration-site thermodynamic analysis prioritized scaffolds predicted to reinforce conserved cap-binding interactions and displace high-energy water molecules. Guided by these insights, we synthesized diverse analogues that demonstrated potent inhibition, with IC50 values as low as 3 nM in biochemical polymerase assays. Inhibition was maintained across representative Influenza A subtypes, including seasonal H3N2, avian H5N1, and bat-derived H17N10. The compounds also retained potency in influenza A minigenome assays, confirming functional inhibition of the viral polymerase complex in cells. These results demonstrate robust and conserved engagement of the PB2 cap-binding pocket across divergent Influenza A subtypes and provide chemically validated scaffolds for further PB2-directed antiviral development.