HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2
et al., Scientific Reports, doi:10.1038/s41598-025-31755-y, Dec 2025
In vitro study showing that compound C19 significantly reduces SARS-CoV-2 replication by targeting the interaction between viral E protein and host ZO-1 PDZ2 domain.
Alvarez et al., 28 Dec 2025, France, peer-reviewed, 14 authors.
Contact: a.flavio2cg@gmail.com.
In vitro studies are an important part of preclinical research, however results may be very different in vivo.
HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2
Scientific Reports, doi:10.1038/s41598-025-31755-y
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.
Author contributions
Declarations
Competing interests The authors declare no competing interests.
Additional information
Supplementary Information The online version contains supplementary material available at h t t p s : / / d o i . o r g / 1 0 . 1 0 3 8 / s 4 1 5 9 8 -0 2 5 -3 1 7 5 5 -y . Correspondence and requests for materials should be addressed to F.A. Reprints and permissions information is available at www.nature.com/reprints . Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from..
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