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Effect of pH on small-molecule inhibitor binding to influenza virus hemagglutinin
Journal article   Open access   Peer reviewed

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

Varada Anirudhan, Irina Gaisina, Amir Shimon, Hyun Lee, Saad Alqarni, Balaji Manicassamy, Terry W. Moore, Kai Xu, Michael Caffrey and Lijun Rong
The Journal of biological chemistry, Vol.302(3), 111150
03/2026
DOI: 10.1016/j.jbc.2026.111150
PMCID: PMC12887400
PMID: 41525845
url
https://doi.org/10.1016/j.jbc.2026.111150View
Published (Version of record) Open Access

Abstract

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 (KD) 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.
 antivirals fusion inhibitors hemagglutinin influenza virus mechanism of action surface plasmon resonance thermal shift assay

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