SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET. Issue 1 (20th January 2022)

• Record Type: Journal Article
• Authors: Cecon, Erika; Burridge, Matilda; Cao, Longxing; Carter, Lauren; Ravichandran, Rashmi; Dam, Julie; Jockers, Ralf
• Is Part Of: Cell chemical biology. Volume 29:Issue 1(2022)
• Page Start: 74
• Page End: 83.e4
• Publication Date: 2022-01-20
• Subjects: SARS-CoV -- spike protein -- ACE2 -- TR-FRET -- HTRF -- COVID-19 -- 2019-nCoV -- HTS -- TMPRSS2 -- heparan sulfate; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2021.06.008
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: Targeting the interaction between the SARS-CoV-2 spike protein and human ACE2, its primary cell membrane receptor, is a promising therapeutic strategy to prevent viral entry. Recent in vitro studies revealed that the receptor binding domain (RBD) of the spike protein plays a prominent role in ACE2 binding, yet a simple and quantitative assay for monitoring this interaction in a cellular environment is lacking. Here, we developed an RBD-ACE2 binding assay that is based on time-resolved FRET, which reliably monitors the interaction in a physiologically relevant and cellular context. Because it is modular, the assay can monitor the impact of different cellular components, such as heparan sulfate, lipids, and membrane proteins on the RBD-ACE2 interaction and it can be extended to the full-length spike protein. The assay is HTS compatible and can detect small-molecule competitive and allosteric modulators of the RBD-ACE2 interaction with high relevance for SARS-CoV-2 therapeutics. Graphical abstract: Highlights: Quantitative assay detecting SARS-CoV-2 spike interactions to ACE2 in living cells HTS-compatible assay suitable for detection of inhibitory molecules Unveiling of the impact of membrane components TMPRSS2/HSPG/CD4 on ACE2/RBD complex Detection of conformational changes within ACE2/RBD complex Abstract : Cecon et al. describe a quantitative, time-resolved FRET assay capable of detecting SARS-CoV-2 spike interactions with ACE2 in living cells. The assay monitors the interaction in a physiologically relevant cellular environment and is suitable for diverse applications, including mechanistic studies, drug screening, and characterization of neutralizing antibodies or vaccine efficacy.