Patch-clamp studies and cell viability assays suggest a distinct site for viroporin inhibitors on the E protein of SARS-CoV-2
Ulrike Breitinger, Christine Adel Sedky, Heinrich Sticht, Hans-Georg Breitinger
Virology Journal, doi:10.1186/s12985-023-02095-y
Background SARS-CoV-2 has caused a worldwide pandemic since December 2019 and the search for pharmaceutical targets against COVID-19 remains an important challenge. Here, we studied the envelope protein E of SARS-CoV and SARS-CoV-2, a highly conserved 75-76 amino acid viroporin that is crucial for virus assembly and release. E protein channels were recombinantly expressed in HEK293 cells, a membrane-directing signal peptide ensured transfer to the plasma membrane. Methods Viroporin channel activity of both E proteins was investigated using patch-clamp electrophysiology in combination with a cell viability assay. We verified inhibition by classical viroporin inhibitors amantadine, rimantadine and 5-(N,N-hexamethylene)-amiloride, and tested four ivermectin derivatives.
Results Classical inhibitors showed potent activity in patch-clamp recordings and viability assays. In contrast, ivermectin and milbemycin inhibited the E channel in patch-clamp recordings but displayed only moderate activity on the E protein in the cell viability assay, which is also sensitive to general cytotoxic activity of the tested compounds. Nemadectin and ivermectin aglycon were inactive. All ivermectin derivatives were cytotoxic at concentrations > 5 µM, i.e. below the level required for E protein inhibition.
Conclusions This study demonstrates direct inhibition of the SARS-CoV-2 E protein by classical viroporin inhibitors. Ivermectin and milbemycin inhibit the E protein channel but their cytotoxicity argues against clinical application.
Author contributions UB: conception, design of work, data acquisition and analysis, interpretation of data, drafting, writing and revising the manuscript. CS: data acquisition and analysis, interpretation of data, revising the manuscript. HS: conception, data analysis, interpretation of data, revising the manuscript. HGB: conception, design of work, data acquisition and analysis, interpretation of data, drafting, writing and revising the manuscript. All authors have approved the submitted version (and any substantially modified version that involves the author's contribution to the study); all authors have agreed both to be personally accountable for their own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.
Declarations Ethics approval and consent to participate Not applicable.
Consent for publication Not applicable.
Competing interests The authors declare that they have no competing interests.
Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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