A Cell-Based ELISA for High-Throughput Screening of Inhibitors Blocking Influenza Virus Attachment to the Subtype-Independent α2,6-Sialic Acid Receptor

Rising drug resistance underscores the urgent need for novel anti-influenza strategies. Targeting the hemagglutinin (HA)–sialic acid receptor interaction offers a promising broad-spectrum approach, yet progress is impeded by the lack of screening models that report the receptor-recognition step in a subtype-independent manner. Herein, we established a robust cell-based ELISA platform for high-throughput screening (HTS) that operates on the subtype-independent receptor-recognition step. The model utilizes ST6GAL1-overexpressing MDCK cells to enrich human-type α2,6-sialic acid receptors and employs horseradish peroxidase (HRP)-conjugated Sambucus nigra lectin (SNA) as a functional, though not structural, surrogate for the receptor-recognition step, yielding a high Z’ factor of 0.78. Given that SNA is a lectin rather than HA, the assay cannot by itself distinguish HA-specific blockade from membrane-mediated or virucidal effects. Orthogonal validation is therefore required for every hit. Screening a library of 2600 compounds identified two hits, Hypericin and protoporphyrin IX (PPIX), which effectively blocked SNA–receptor recognition with IC50 values of 18.39 ± 6.23 μM and 9.77 ± 3.05 μM, respectively. An orthogonal flow cytometry assay with FITC-conjugated SNA reproduced this inhibition on intact cells in a dose-dependent manner (IC50 = 17.68 ± 9.54 μM for Hypericin and 25.31 ± 19.47 μM for PPIX), confirming that the inhibition is not an artifact of the colorimetric readout. Hemagglutination inhibition (HA) assays with distinct pre-incubation strategies indicated that both compounds act on the viral particle rather than on the host receptor. Surface plasmon resonance (SPR) and molecular docking studies further indicated direct, dose-dependent but low-affinity binding to HA proteins. Notably, cytopathic effect (CPE) assays demonstrated their potent antiviral efficacy across H1N1 and H3N2 subtypes, with PPIX exhibiting exceptionally potent activity against H3N2 (EC50 = 0.50 ± 0.17 μM). Taken together, these data identify Hypericin and PPIX as viral-side attachment inhibitors with weak HA binding and multimodal antiviral activity. Because neither a virucidal nor a fusion-blocking control was included, the relative contribution of HA engagement to their overall antiviral effect remains to be established. In conclusion, this BSL-1-compatible ELISA platform, which probes the subtype-independent receptor-recognition step, provides an efficient tool for discovering broad-spectrum influenza entry inhibitors, and the identified hits offer novel pharmacological scaffolds for further structural optimization.