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

## TL;DR

This study shows how pH affects the binding of small-molecule inhibitors to influenza virus proteins, which could lead to better antiviral drugs.

## Contribution

The study reveals a pH-dependent mechanism for small-molecule binding to influenza HA and identifies a potential role of E97 in this process.

## Key findings

- Binding strength of inhibitors increased up to 267-fold when pH was lowered from 7.2 to 6.2.
- Thermal shift assays showed that inhibitors significantly stabilized H7 HA.
- Structural changes in the binding site may explain the pH-dependent binding enhancement.

## Abstract

Influenza A viruses (IAVs) impose a tremendous socioeconomic 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 Food and Drug Administration–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 bind 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 side chain in enhancing affinity at lower pH. On the other hand, remodeling of the compound binding site because of 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.