SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET

Cell Chem Biol. 2022 Jan 20;29(1):74-83.e4. doi: 10.1016/j.chembiol.2021.06.008. Epub 2021 Jul 2.

Abstract

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.

Keywords: 2019-nCoV; ACE2; COVID-19; HTRF; HTS; SARS-CoV; TMPRSS2; TR-FRET; heparan sulfate; spike protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiotensin-Converting Enzyme 2 / chemistry*
  • Cells, Cultured
  • Fluorescence Resonance Energy Transfer*
  • HEK293 Cells
  • Humans
  • Protein Binding
  • Spike Glycoprotein, Coronavirus / chemistry*
  • Time Factors

Substances

  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2
  • ACE2 protein, human
  • Angiotensin-Converting Enzyme 2