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Development of a nano-luciferase based assay to measure the binding of SARS-CoV-2 spike receptor binding domain to ACE-2

Andrade De Lima, M; Skidmore, M; Khanim, F; Richardson, A

Authors

F Khanim



Abstract

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.

Citation

Andrade De Lima, M., Skidmore, M., Khanim, F., & Richardson, A. (2020). Development of a nano-luciferase based assay to measure the binding of SARS-CoV-2 spike receptor binding domain to ACE-2. Biochemical and Biophysical Research Communications, 534, 485--490. https://doi.org/10.1016/j.bbrc.2020.11.055

Journal Article Type Article
Acceptance Date Nov 11, 2020
Publication Date Nov 11, 2020
Journal Biochemical and Biophysical Research Communications
Print ISSN 0006-291X
Electronic ISSN 1090-2104
Publisher Elsevier
Volume 534
Pages 485--490
DOI https://doi.org/10.1016/j.bbrc.2020.11.055
Keywords ACE2, Binding assay, HiBIT tag, Mutation, Nano-luciferase, SARS-CoV-2, Angiotensin-Converting Enzyme 2, Antiviral Agents, Binding Sites, COVID-19, Humans, Luciferases, Nanotechnology, Protein Binding, Protein Domains, Receptors, Virus, SARS-CoV-2, Spike G
Publisher URL https://www.sciencedirect.com/science/article/pii/S0006291X20320994?via%3Dihub#!