Molecular & Structural Bioscience, School of Life Sciences, Keele University Staffordshire, ST5 5BG, United Kingdom.
School of Biomedical Sciences, Institute of Clinical Sciences Medical School University of Birmingham, Birmingham, B15 2TT, United Kingdom.
Biochem Biophys Res Commun. 2021 Jan 1;534:485-490. doi: 10.1016/j.bbrc.2020.11.055. Epub 2020 Nov 17.
To identify drugs that could potentially be used to treat infection with SARS-CoV-2, a high throughput 384-well assay was developed to measure the binding of the receptor binding domain (RBD) of the viral S1 protein to its main receptor, angiotensin converting enzyme 2 (ACE2). The RBD was fused to both a HiBIT tag and an IL6 secretion signal to enable facile collection from the cell culture media. The addition of culture media containing this protein, termed HiBIT-RBD, to cells expressing ACE2 led to binding that was specific to ACE2 and both time and concentration dependant, Binding could be inhibited by both RBD expressed in E. coli and by a full length S1 - Fc fusion protein (Fc-fused S1) expressed in eukaryotic cells. The mutation of residues that are known to play a role in the interaction of RBD with ACE2 also reduced binding. This assay may be used to identify drugs which inhibit the viral uptake into cells mediated by binding to ACE2.
为了鉴定可能用于治疗 SARS-CoV-2 感染的药物，开发了一种高通量 384 孔测定法来测量病毒 S1 蛋白的受体结合域（RBD）与其主要受体血管紧张素转换酶 2（ACE2）的结合。RBD 融合到 HiBIT 标签和 IL6 分泌信号上，以便从细胞培养液中轻松收集。将含有这种蛋白质的培养基（称为 HiBIT-RBD）添加到表达 ACE2 的细胞中，导致与 ACE2 特异性结合，且具有时间和浓度依赖性，结合可以被在大肠杆菌中表达的 RBD 和在真核细胞中表达的全长 S1-Fc 融合蛋白（Fc 融合 S1）抑制。突变已知在 RBD 与 ACE2 相互作用中起作用的残基也会降低结合。该测定法可用于鉴定通过与 ACE2 结合抑制病毒进入细胞的药物。
