TR-FRET binding assay

SNAP-tagged proteins (ACE2, LepR, VEGFR2) were fluorescently labeled by incubating cells transfected with the corresponding expression vectors with an SNAP suicide substrate conjugated to the long-lived fluorophore Terbium cryptate (Tb; Lumi4-Tb, 100 nM; Cisbio Bioassays) in Tag-lite labeling medium (1 h, on ice) (^{Keppler et al., 2003}). After several washes, cells were collected using enzyme-free cell dissociation buffer (Sigma-Aldrich), resuspended in Tag-lite buffer and distributed into a 384-well plate. Efficient fluorescent labeling of SNAP was verified by reading fluorescence signal at 620 nm. Experiments were then conducted according to the different modes described below. All reagents were diluted in Tag-lite buffer (final reaction volume of 14 μL) and incubations were performed at room temperature; except in the case of compounds Fenbendazole and Elaidic Acid, where incubation was performed at 37°C to avoid precipitation of compounds. TR-FRET signals were detected using a plate reader (Tecan F500; Tecan, Männedorf, Switzerland) with the following settings: excitation at 340 nm (Tb, energy donor), emission at 665 nm (d2, acceptor) and 620 nm (donor); delay of 150 μs; and integration time of 500 μs. Data is expressed as TR-FRET ratio (acceptor/donor). When indicated, TR-FRET ratio was normalized to % of basal or % of maximal binding (Bmax).

Cells expressing Lumi4-Tb-labelled SNAP-tagged proteins were incubated with different concentrations of RBD-d2 and TR-FRET ratios were determined after 2 h incubation (at room temperature).

Cells expressing Lumi4-Tb-labelled SNAP-tagged proteins were mixed with mock-transfected cells or cells expressing non-labelled SNAP-ACE2 (1:1 ratio of cells) and the RBD-d2 tracer (5 nM) and TR-FRET signals were determined after 2 h incubation (at room temperature).

Association kinetics were determined by continuously monitoring TR-FRET signal for 1 h after addition of RBD-d2 (5nM) or S1-S2-d2 (20 nM). For dissociation kinetics, cells were incubated for 1 h with RBD-d2 (5 nM) or S1-S2-d2 (20 nM) to reach equilibrium, followed by the addition of saturating concentrations of non-labelled RBD (1 μM) or S1-S2 (200 nM). TR-FRET signals were then immediately and continuously monitored for 1 h at room temperature.

Lumi4-Tb-labelled ACE2 cells were pre-incubated with competitors (either single or increasing concentrations) for 1 h before addition of RBD-d2 (5 nM). Data are expressed as the acceptor/donor ratio or normalized as % when indicated (maximal TR-FRET ratio = 100%, non-specific binding = 0%). Obtained IC₅₀ values from each experiment (performed in triplicates) were converted into K_i using the Cheng-Prussof equation using the mean of the individual K_d values derived from the saturation experiments (^{Cheng and Prusoff, 1973}). The Z′ value (^{Zhang et al., 1999}) of the TR-FRET assay was determined using total and non-specific (excess of non-labelled RBD) TR-FRET ratios as positive and negative controls, respectively, as follow:

where σp = standard deviation of positive control; σn = standard deviation of negative control; μp = mean of positive control; μn = mean of negative control.

Lumi4-Tb-labelled ACE2 cells, co-expressing TMPRSS2 when indicated, were incubated with d2-labelled anti-FLAG tag antibody (2 μg/mL, 1 h at room temperature; 61FG2DLF, Cisbio Bioassays), followed by addition of non-labelled RBD (5 nM) and heparin (0.3 or 3 mg/mL) when indicated. TR-FRET signal was read after 2 h incubation (room temperature). Non-specific signal was defined in the presence of excess of non-labelled anti-FLAG antibody (20 μg/mL; F7425; Sigma-Aldrich).