Mechanistic insights into the small-molecule inhibition of influenza A virus entry

SignificanceInfluenza A virus (IAV) poses a persistent global health threat due to its rapid evolution and immune evasion, limiting the effectiveness of existing vaccines and treatments. Targeting hemagglutinin (HA), the key viral entry protein, offers a promising antiviral approach, yet developing broad-spectrum inhibitors is hindered by structural differences between HA subtypes and their susceptibility to mutations. Using cryo-EM, we uncover how two small molecules inhibit group 2 IAV by stabilizing HA in its prefusion state, blocking viral entry. Our study highlights the challenges of universal inhibition and introduces a divide and conquer strategy designing group-specific inhibitors and combination therapies to achieve broader protection. These insights advance antiviral drug development against IAV and emerging viral threats.Influenza A virus (IAV) is a zoonotic pathogen responsible for seasonal and pandemic flu. The extensive genetic and antigenic diversity within and between IAV phylogenetic groups presents major challenges for developing universal vaccines and broad-spectrum antiviral therapies. Current interventions provide limited protection due to the virus s high mutation rate and capacity for immune evasion. Recent advancements in viral hemagglutinin (HA)-targeting small-molecule entry inhibitors offer a promising avenue to overcome these limitations. Here, we present structural and functional analyses of two group 2 HA-specific small-molecule inhibitors recently identified by our team. Cryogenic electron microscopy (cryo-EM) structures revealed that these inhibitors bind a conserved pocket within the HA stalk, likely interfering with the conformational rearrangements necessary for membrane fusion and viral entry. Structure-guided mutagenesis confirmed the critical roles of key interacting residues and uncovered distinct resistance profiles between the two compounds, as well as in comparison to Arbidol, a previously reported HA inhibitor. Notably, our structural analysis highlights intrinsic barriers to achieving cross-group inhibition with current small-molecule designs. To address this, we propose an alternative strategy for broadening antiviral coverage. Together, these findings provide mechanistic insights into IAV entry inhibition and a foundation for the rational design of next-generation anti-influenza therapeutics.