A Cell-Based ELISA for High-Throughput Screening of Inhibitors Blocking Influenza Virus Attachment to the Subtype-Independent α2,6-Sialic Acid Receptor
- 1Beijing Key Laboratory of Technology and Application for Anti-Infective New Drugs Research and Development, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
Abstract
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.
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BibTeX
@article{guo2026cellbased,
title = {{A Cell-Based ELISA for High-Throughput Screening of Inhibitors Blocking Influenza Virus Attachment to the Subtype-Independent α2,6-Sialic Acid Receptor}},
author = {Keyu Guo and Jingshuai Wu and Yaru Liu and Jiahui Luo and Weilian Jiang and Chenyin Wang and Ren Sheng and Shunwang Li and Xiuyong Fan and Yexiang Wu and Yanni Xu and Shuyi Si and Yongxin Zhang and Jing Zhang},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27209205},
url = {https://www.mdpi.com/1422-0067/27/20/9205},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A Cell-Based ELISA for High-Throughput Screening of Inhibitors Blocking Influenza Virus Attachment to the Subtype-Independent α2,6-Sialic Acid Receptor
AU - Keyu Guo
AU - Jingshuai Wu
AU - Yaru Liu
AU - Jiahui Luo
AU - Weilian Jiang
AU - Chenyin Wang
AU - Ren Sheng
AU - Shunwang Li
AU - Xiuyong Fan
AU - Yexiang Wu
AU - Yanni Xu
AU - Shuyi Si
AU - Yongxin Zhang
AU - Jing Zhang
PY - 2026
DA - 2026/10/16
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27209205
UR - https://www.mdpi.com/1422-0067/27/20/9205
AB - 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.
SN - 1422-0067
ER -