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Patch-clamp studies and cell viability assays suggest a distinct site for viroporin inhibitors on the E protein of SARS-CoV-2

Breitinger et al., Virology Journal, doi:10.1186/s12985-023-02095-y

Jul 2023

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In Vitro analysis of inhibitors against the SARS-CoV-2 E ion channel.

- The E protein of SARS-CoV-2 is a viroporin that forms ion channels important for viral replication. The E proteins from SARS-CoV and SARS-CoV-2 are highly similar.
- The study expressed recombinant E proteins from SARS-CoV (1-E) and SARS-CoV-2 (2-E) in human embryonic kidney (HEK293) cells.
- Cell viability assays showed 1-E and 2-E proteins reduced cell viability to a comparable level, suggesting similar ion channel activity.
- Patch clamp electrophysiology directly measured and confirmed 1-E and 2-E have similar ion channel function.
- Known viroporin inhibitors (amantadine, rimantadine, HMA) inhibited 1-E and 2-E channels similarly in cell viability and patch clamp assays.
- Ivermectin and derivatives (milbemycin, ivermectin aglycon) inhibited 2-E channel activity in patch clamp assays, but were less effective at inhibiting 2-E in cell viability assays, likely due to intracellular access limitations.
- Ivermectin was the most potent 2-E inhibitor in patch clamp assays (IC₅₀ 0.88 nM), comparable to rimantadine. Milbemycin and ivermectin aglycon had lower potency.
- Ivermectin derivatives demonstrated cytotoxicity at concentrations ≥5 μM, below their inhibitory concentrations against the E protein.
- While the very low patch clamp IC₅₀ suggests ivermectin can directly inhibit the E ion channel at non-cytotoxic levels in vitro, it is unclear whether safe and effective intracellular concentrations may be achieved in human tissues. The cell viability assays and cytotoxicity results indicate limitations for clinical use of ivermectin against this specific viral target. Further research is needed to determine if this specific mechanism of action could contribute to the efficacy seen in studies.
- Authors note the positive efficacy seen in several studies and greater efficacy seen with prophylaxis, although authors cite only a small fraction of the research. However, authors discussion appears biased especially in terms of the extensive safety data available from real-world human use and in view of the dosage used in COVID-19 studies. Further, authors appear to extend conclusions from the mechanism of action studied, however the current proposed mechanisms of action are different (for example, inhibition of importin-α/β-dependent nuclear import of viral proteins Götz, Kosyna, Wagstaff, Wagstaff (B), 3CL^pro inhibition Mody, binding to glycan sites on the SARS-CoV-2 spike protein preventing interaction with blood and epithelial cells and inhibiting hemagglutination Boschi, inhibiting SARS-CoV-2 induced formation of fibrin clots resistant to degradation Vottero, and cardioprotective Liu, immunomodulatory Munson and anti-inflammatory DiNicolantonio properties).

1. Boschi et al., SARS-CoV-2 Spike Protein Induces Hemagglutination: Implications for COVID-19 Morbidities and Therapeutics and for Vaccine Adverse Effects, bioRxiv, doi:10.1101/2022.11.24.517882.biorxiv.org, doi.org.

2. DiNicolantonio et al., Anti-inflammatory activity of ivermectin in late-stage COVID-19 may reflect activation of systemic glycine receptors, Open Heart, doi:10.1136/openhrt-2021-001655.bmj.com, doi.org.

3. Götz et al., Influenza A viruses escape from MxA restriction at the expense of efficient nuclear vRNP import, Scientific Reports, doi:10.1038/srep23138.nature.com, doi.org.

4. Kosyna et al., The importin α/β-specific inhibitor Ivermectin affects HIF-dependent hypoxia response pathways, Biological Chemistry, doi:10.1515/hsz-2015-0171.degruyter.com, doi.org.

5. Liu et al., Genome-wide analyses reveal the detrimental impacts of SARS-CoV-2 viral gene Orf9c on human pluripotent stem cell-derived cardiomyocytes, Stem Cell Reports, doi:10.1016/j.stemcr.2022.01.014.sciencedirect.com, doi.org.

6. Mody et al., Identification of 3-chymotrypsin like protease (3CLPro) inhibitors as potential anti-SARS-CoV-2 agents, Communications Biology, doi:10.1038/s42003-020-01577-x.nature.com, doi.org.

7. Munson et al., Niclosamide and ivermectin modulate caspase-1 activity and proinflammatory cytokine secretion in a monocytic cell line, British Society For Nanomedicine Early Career Researcher Summer Meeting, 2021, web.archive.org/web/20230401070026/https://michealmunson.github.io/COVID.pdf.archive.org.

8. Vottero et al., Computational Prediction of the Interaction of Ivermectin with Fibrinogen, Molecular Sciences, doi:10.3390/ijms241411449.mdpi.com, doi.org.

9. Wagstaff et al., Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus, Biochemical Journal, doi:10.1042/BJ20120150.portlandpress.com, doi.org.

10. Wagstaff (B) et al., An AlphaScreen®-Based Assay for High-Throughput Screening for Specific Inhibitors of Nuclear Import, SLAS Discovery, doi:10.1177/1087057110390360.sciencedirect.com, doi.org.

Important missing comments or errors? Let us know.

Breitinger et al., 8 Jul 2023, Germany, peer-reviewed, 4 authors. Contact: ulrike.breitinger@guc.edu.eg.

In Vitro studies are an important part of preclinical research, however results may be very different in vivo.

This Paper Ivermectin All

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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