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 journalArticlepeer-review

Authors

Colleges, School and Institutes

External organisations

  • Keele University

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.

Bibliographic note

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)).

Details

Original languageEnglish
Pages (from-to)485-490
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume534
Early online date17 Nov 2020
Publication statusPublished - 1 Jan 2021

Keywords

  • 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