Effect of pH on small-molecule inhibitor binding to influenza virus hemagglutinin

Influenza A viruses (IAVs) impose a tremendous socio-economic burden and the mainstay preventative strategy of using vaccines faces challenges related to annual reformulation and variable efficacy (30-70%). The occurrence of antiviral resistance to the current FDA-approved anti-influenza drugs further highlights the urgent need for novel therapeutics. Our research group previously identified and optimized potent small-molecule inhibitors targeting IAV's hemagglutinin (HA), a surface glycoprotein crucial for viral entry and membrane fusion. Fusion occurs after the virus is taken up by endocytosis in the late endosomes under acidic conditions (pH ∼4.9-5.5). In this study, we report the biophysical characterization of two small-molecule inhibitors that binds to recombinant H3 and H7 HA proteins (phylogenetic group 2). These two compounds exhibited binding affinities (K D ) ranging from ∼0.4 to 18.6 μM and significantly stabilized H7 HA based on thermal shift assay. Remarkably, lowering the pH from 7.2 to 6.2 resulted in up to a ∼267-fold increase in binding strength. Detailed analysis of the compound binding site suggested a potential role of the E97 sidechain in enhancing affinity at lower pH. On the other hand, re-modeling of the compound binding site due to propagated structural changes appears to be the most likely explanation. Collectively, these findings elucidate a pH-dependent mechanism of action for HA-targeting antivirals and underscore the importance of evaluating protein-ligand interactions under physiologically relevant conditions. This consideration is particularly important for viral proteins such as IAV HA that undergo pH-triggered conformational changes during the endosome-dependent viral entry.