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    New method to detect SARS-CoV-2 binding to ACE2 by TR-FRET in living cells

    A study of the Ralf Jockers team

    New method to detect SARS-CoV-2 binding to ACE2 by TR-FRET in living cells


    The COVID-19 pandemic, caused by the SARS-COV-2 virus infection, constitutes a real therapeutic challenge given that no effective treatment is available yet. The identification of drugs targeting the interaction between the Spike protein of SARS-CoV-2 and its entry receptor in human cells, the ACE2 protein, is a promising therapeutic strategy. The team of Ralf Jockers has developed a new method to detect the physical interaction between the Spike protein and ACE2 in living cells. The assay is based on the highly sensitive time-resolved (TR)-FRET technique and allows to investigate the Spike-ACE2 interaction under ‘real-life’ conditions taking into account the important impact of cellular components on this interaction. The assay is quantitative, high-throughput compatible and is suitable for drug screening and for mechanistic studies. This study is published in Cell Chemical Biology.


    COVID-19 is triggered by infection with the SARS-CoV-2 virus. A critical step in viral infection is the entry of SARS-CoV-2 into host cells, initiated by the interaction between the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, present on the surface of the viral particle, and its receptor present at the cell surface of human cells, the angiotensin I converting enzyme 2 (ACE2). In vitro assays exist to measure the interaction between the purified ACE2 extracellular domain and RBD in a cell-free setting and simple environment. Means to probe the ACE2/RBD interaction under ‘real-life’ conditions, in a more complex cellular context, were lacking.


    We have developed here a cellular binding assay measuring the time-resolved FRET signal reflecting the proximity between fluorescent RBD (RBD-d2) and fluorescent SNAP-ACE2 fusion protein anchored in the plasma membrane. The influence of cellular components, such as proximal membrane proteins (TMPRSS2, CD4, heparan sulfate proteoglycans (HSPG)) on the ACE2/RBD interaction were then investigated and revealed their impact on binding kinetics and conformational changes within the ACE2/RBD complex. The cell binding assay not only allows the quantitative detection of the binding of RBD-d2 to SNAP-ACE2 with high sensitivity providing measurements of binding affinity and kinetics at the cell surface, but also allows the detection of inhibitory molecules (small chemical compounds, peptides, nanobodies, antibodies).

    Being compatible with high-throughput screening (HTS), the SNAP-ACE2/RBD-d2 binding assay is well suited to identify novel inhibitors interfering with the formation of ACE2/RBD complex in living cells. It can also be applied to characterize therapeutic neutralizing antibodies or vaccine efficacy in a cellular environment.


    Legend: Quantitative TR-FRET assay detecting SARS-CoV-2 spike (d2 fluorophore) interaction to ACE2 (SNAP-lumi4-tb) in living cells. HTS-compatible, the assay is suitable for detection of inhibitory molecules by screening. The assay is performed in living cells, unveiling of the impact of membrane components such as the protease TMPRSS2, heparan sulfate proteoglycans (HSPG), or co-receptors (CD4) on ACE2/Spike complex.


    This study was supported by the RA-Covid-19 projet “MELATOVID” (ANR-20-COV4-0001).



    Cecon E, Burridge M, Cao L, Carter L, Ravichandran R, Dam J, Jockers R. SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET. Cell Chem Biol. 2021 Jul 2:S2451-9456(21)00307-X. doi: 10.1016/j.chembiol.2021.06.008.


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