Development of a nano-luciferase based assay to measure the binding of SARS-CoV-2 spike receptor binding domain to ACE-2
Research output: Contribution to journal › Article › peer-review
Colleges, School and Institutes
- Keele University
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.
Funding Information: This research was funded by Keele University (AR) and the BBSRC ( UK ( BB/L023717/1 (MAS); BIV-HVB-2020/07/SKIDMORE (MAS & MAL); BB/S009787/1 (MAS & MAL)).
|Number of pages||6|
|Journal||Biochemical and Biophysical Research Communications|
|Early online date||17 Nov 2020|
|Publication status||Published - 1 Jan 2021|
- ACE2, Binding assay, HiBIT tag, Mutation, Nano-luciferase, SARS-CoV-2, COVID-19/drug therapy, Humans, SARS-CoV-2/drug effects, Angiotensin-Converting Enzyme 2/chemistry, Binding Sites/genetics, Spike Glycoprotein, Coronavirus/genetics, Nanotechnology/methods, Receptors, Virus/genetics, Protein Binding, Protein Domains, Luciferases/genetics, Antiviral Agents/metabolism