A Novel Cell-Based High-Throughput Screening Model for Inhibitors Targeting Influenza Virus Hemagglutinin–α-2,6-Sialic Acid Interaction

Abstract

Rising drug resistance undermines current anti-influenza virus therapies. Although targeting the hemagglutinin (HA)–sialic acid receptor interaction is a promising strategy, progress is impeded by the lack of subtype-independent screening models. Herein, we established a fluorescence-based cell high-throughput model using fluorescein isothiocyanate-conjugated Sambucus Nigra Lectin (FITC-SNA) as a stable HA surrogate and α-2,6-sialyltransferase (ST6GAL1)-overexpressing MDCK cells to mimic the HA–receptor interface. This platform was designed to serve as an efficient primary screening tool to rapidly filter large compound libraries for potential binders to the receptor-binding interface. Screening 10,000 compounds identified Obatoclax Mesylate and Ethylparaben as primary hits. Both exhibited broad-spectrum HA inhibition activity, validating the model’s capability to identify compounds interfering with viral attachment. Further cellular antiviral assays revealed cytotoxicity for both compounds, resulting in low selectivity indexes (SIs), indicating that while these molecules effectively target the interaction site, they require substantial structural optimization for therapeutic use. Molecular docking confirmed their binding to type A H1N1, H3N2, and B/Victoria HA proteins, while ADMET predictions highlighted specific structural optimization needs to mitigate toxicity. In conclusion, this subtype-independent, highly specific high-throughput screening (HTS) model provides an efficient and reliable platform for early-stage influenza drug discovery and lead compound development.