Development of a nano-luciferase based assay to measure the binding of SARS-CoV-2 spike receptor binding domain to ACE-2.
Development of a nano-luciferase based assay to measure the binding of SARS-CoV-2 spike receptor binding domain to ACE-2.
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DOI：
10.1016/j.bbrc.2020.11.055
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发表时间：
2021-01-01
影响因子：
3.1
通讯作者：
Richardson A
中科院分区：
文献类型：
--
作者：
Lima MA;Skidmore M;Khanim F;Richardson A
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蛋白的受体结合结构域（RBD）与其主要受体血管紧张素转换酶2（ACE 2）的结合。将RBD与HiBIT标签和IL 6分泌信号融合以使得能够从细胞培养基容易地收集。向表达ACE 2的细胞中加入含有这种蛋白质的培养基（称为HiBIT-RBD），导致对ACE 2具有特异性的结合，并且具有时间和浓度依赖性。大肠杆菌中表达的RBD和真核细胞中表达的全长S1 - Fc融合蛋白（Fc融合S1）均可抑制结合。已知在RBD与ACE 2相互作用中起作用的残基的突变也降低了结合。该测定可用于鉴定抑制通过与ACE 2结合介导的病毒摄取到细胞中的药物。
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.
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DOI：
10.1016/s2589-7500(20)30192-8
发表时间：
2020-12
期刊：
The Lancet. Digital health
影响因子：
--
作者：
Zhou Y;Wang F;Tang J;Nussinov R;Cheng F
通讯作者：
Cheng F
影响因子：
56.9
作者：
Yan, Renhong;Zhang, Yuanyuan;Zhou, Qiang
通讯作者：
Zhou, Qiang
影响因子：
12.8
作者：
Khanim, F. L.;Merrick, B. A. M. E.;Giles, H. V.;Jankute, M.;Jackson, J. B.;Giles, L. J.;Birtwistle, J.;Bunce, C. M.;Drayson, M. T.
通讯作者：
Drayson, M. T.
影响因子：
5.3
作者：
Richardson, A;Malik, RK;Parsons, JT
通讯作者：
Parsons, JT
影响因子：
2.7
作者：
Oh-Hashi K;Furuta E;Fujimura K;Hirata Y
通讯作者：
Hirata Y