Department of Biochemistry, German University in Cairo, Main Entrance of Al Tagamoa Al Khames, New Cairo, 11835, Egypt.
Division of Bioinformatics, Institute for Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Virol J. 2023 Jul 8;20(1):142. doi: 10.1186/s12985-023-02095-y.
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.
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.
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.
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.
自 2019 年 12 月以来，SARS-CoV-2 引发了全球大流行，针对 COVID-19 的药物靶点研究仍然是一个重要的挑战。在这里，我们研究了 SARS-CoV 和 SARS-CoV-2 的包膜蛋白 E，这是一种高度保守的 75-76 个氨基酸的病毒孔蛋白，对病毒的组装和释放至关重要。E 蛋白在 HEK293 细胞中通过重组表达，一个膜定向信号肽确保其转运到质膜。
使用膜片钳电生理学结合细胞活力测定法研究了两种 E 蛋白的病毒孔蛋白通道活性。我们验证了经典病毒孔蛋白抑制剂金刚烷胺、金刚乙胺和 5-（N，N-己二亚甲基）阿米洛利的抑制作用，并测试了四种伊维菌素衍生物。
经典抑制剂在膜片钳记录和活力测定中表现出很强的活性。相比之下，伊维菌素和米尔贝肟在膜片钳记录中抑制了 E 通道，但在细胞活力测定中对 E 蛋白仅显示出中等活性，这也对测试化合物的一般细胞毒性活性敏感。nemadectin 和伊维菌素糖苷无活性。所有伊维菌素衍生物在浓度>5µM 时均具有细胞毒性，即低于抑制 E 蛋白所需的水平。
本研究证明了经典病毒孔蛋白抑制剂直接抑制 SARS-CoV-2 的 E 蛋白。伊维菌素和米尔贝肟抑制 E 蛋白通道，但它们的细胞毒性表明其不适合临床应用。
