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<p>The SARS-CoV-2 E protein through its C-terminal PDZ-binding motif (PBM) interacts with several host PDZ-containing proteins, including Zonula occludens-1 (ZO-1) protein via its PDZ2 domain, thereby contributing to viral pathogenesis. Targeting this interaction represents a potential therapeutic strategy. In this study, we determined the X-ray structure of the E PBM peptide in complex with the ZO-1 PDZ2 domain at 1.7 Å resolution. The structure revealed a domain-swapped dimer conformation of ZO-1 PDZ2, with the E PBM peptide conventionally bound within the PDZ domain’s canonical binding groove, exhibiting key interactions characteristic of type II PBM/PDZ interactions. To identify potential inhibitors of the E PBM/ZO-1 PDZ2 interaction, we performed a Homogeneous Time-Resolved Fluorescence (HTRF) screening using a protein-protein interaction-focused library of 1000 compounds. This led to the identification of 36 hits that disrupted this interaction. Subsequent cytotoxicity and dose-response assays narrowed the selection to 14 promising compounds. Docking simulations showed that some compounds bind within or near the PBM-binding pocket, supporting a competitive mechanism of interaction inhibition, while others bind at a central interface between the two PDZ monomers, suggesting an inhibition of dimerization, which in turn prevents PBM binding. Thus, the E PBM/ZO-1 PDZ2 interaction can be inhibited through both direct and indirect mechanisms. Finally, antiviral assays using a NanoLuciferase-expressing recombinant SARS-CoV-2 demonstrated that one compound, C19, significantly reduced viral replication, highlighting its potential as a candidate for further therapeutic development.</p>

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HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月28日

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  • Flavio Alvarez,
  • Florence Larrous,
  • Ariel Mechaly,
  • Benjamin Bardiaux,
  • Quentin Goor,
  • Ahmed Haouz,
  • Blandine Séon-Méniel,
  • Rodolphe Alves de Sousa,
  • Stéphane Bourg,
  • Didier Desmaële,
  • Bruno Figadère,
  • Nicolas Wolff,
  • Hélène Munier-Lehmann,
  • Célia Caillet-Saguy

摘要

The SARS-CoV-2 E protein through its C-terminal PDZ-binding motif (PBM) interacts with several host PDZ-containing proteins, including Zonula occludens-1 (ZO-1) protein via its PDZ2 domain, thereby contributing to viral pathogenesis. Targeting this interaction represents a potential therapeutic strategy. In this study, we determined the X-ray structure of the E PBM peptide in complex with the ZO-1 PDZ2 domain at 1.7 Å resolution. The structure revealed a domain-swapped dimer conformation of ZO-1 PDZ2, with the E PBM peptide conventionally bound within the PDZ domain’s canonical binding groove, exhibiting key interactions characteristic of type II PBM/PDZ interactions. To identify potential inhibitors of the E PBM/ZO-1 PDZ2 interaction, we performed a Homogeneous Time-Resolved Fluorescence (HTRF) screening using a protein-protein interaction-focused library of 1000 compounds. This led to the identification of 36 hits that disrupted this interaction. Subsequent cytotoxicity and dose-response assays narrowed the selection to 14 promising compounds. Docking simulations showed that some compounds bind within or near the PBM-binding pocket, supporting a competitive mechanism of interaction inhibition, while others bind at a central interface between the two PDZ monomers, suggesting an inhibition of dimerization, which in turn prevents PBM binding. Thus, the E PBM/ZO-1 PDZ2 interaction can be inhibited through both direct and indirect mechanisms. Finally, antiviral assays using a NanoLuciferase-expressing recombinant SARS-CoV-2 demonstrated that one compound, C19, significantly reduced viral replication, highlighting its potential as a candidate for further therapeutic development.