Lima, MA, Skidmore, MA ORCID: https://orcid.org/0000-0002-0287-5594, Khanim, F and Richardson, A ORCID: https://orcid.org/0000-0003-1825-3375 (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.

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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.

Item Type: Article
Additional Information: The final version of this article and all relevant information related to it can be found online at; https://www.sciencedirect.com/science/article/pii/S0006291X20320994?via%3Dihub
Uncontrolled Keywords: SARS-CoV-2; ACE2; Binding assay; HiBIT tag; Mutation; Nano-luciferase
Subjects: Q Science > Q Science (General)
R Medicine > R Medicine (General)
R Medicine > R Medicine (General) > R735 Medical education. Medical schools. Research
Divisions: Faculty of Natural Sciences > School of Life Sciences
Depositing User: Symplectic
Date Deposited: 26 Nov 2020 10:04
Last Modified: 02 Jan 2021 01:02
URI: https://eprints.keele.ac.uk/id/eprint/8934

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