{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "Beijing 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"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 14,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Keyu Guo"
    },
    {
      "affs": [
        0
      ],
      "name": "Jingshuai Wu"
    },
    {
      "affs": [
        0
      ],
      "name": "Yaru Liu"
    },
    {
      "affs": [
        0
      ],
      "name": "Jiahui Luo"
    },
    {
      "affs": [
        0
      ],
      "name": "Weilian Jiang"
    },
    {
      "affs": [
        0
      ],
      "name": "Chenyin Wang"
    },
    {
      "affs": [
        0
      ],
      "name": "Ren Sheng"
    },
    {
      "affs": [
        0
      ],
      "name": "Shunwang Li"
    },
    {
      "affs": [
        0
      ],
      "name": "Xiuyong Fan"
    },
    {
      "affs": [
        0
      ],
      "name": "Yexiang Wu"
    },
    {
      "affs": [
        0
      ],
      "name": "Yanni Xu"
    },
    {
      "affs": [
        0
      ],
      "name": "Shuyi Si"
    },
    {
      "affs": [
        0
      ],
      "name": "Yongxin Zhang",
      "orcid": "0000-0003-0950-9455"
    },
    {
      "affs": [
        0
      ],
      "name": "Jing Zhang"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/a6jpa3mtnqpda.bib",
  "cited_by": 0,
  "doi": "10.3390/ijms27209205",
  "fulltext_url": null,
  "id": "a6jpa3mtnqpda",
  "issns": [
    "1422-0067"
  ],
  "landing_url": "https://www.mdpi.com/1422-0067/27/20/9205",
  "language": null,
  "licence": "CC-BY-4.0",
  "licence_label": "CC BY 4.0",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "2713|Influenza Virus Research Studies"
    ]
  ],
  "openalex": "W7222720067",
  "pdf_url": null,
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-16",
  "publisher": "MDPI AG",
  "ris": "https://research.draconitic.com/paper/a6jpa3mtnqpda.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:18:57Z",
      "native_id": "10.3390/ijms27209205",
      "source": "crossref",
      "url": "https://www.mdpi.com/1422-0067/27/20/9205"
    }
  ],
  "subjects": [
    {
      "field": "medicine",
      "origin": "openalex:2713|Influenza Virus Research Studies",
      "subfield": "epidemiology-public-health"
    }
  ],
  "title": "A Cell-Based ELISA for High-Throughput Screening of Inhibitors Blocking Influenza Virus Attachment to the Subtype-Independent α2,6-Sialic Acid Receptor",
  "title_text": "A Cell-Based ELISA for High-Throughput Screening of Inhibitors Blocking Influenza Virus Attachment to the Subtype-Independent α2,6-Sialic Acid Receptor",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:18:57Z",
  "url": "https://research.draconitic.com/paper/a6jpa3mtnqpda",
  "venue": "International Journal of Molecular Sciences",
  "versions": []
}