Biotinylated ace2

AlphaScreen assays were performed similar to a previously reported protocol for Spike:ACE2 interactions (CITE). Final concentrations in the assays are as follows: 4 nM Spike-RBD, 4 nM ACE2, 10 μg/mL protein A acceptor beads, 10 μg/mL streptavidin donor beads, and varying concentrations of inhibitor. All proteins were prepared in assay buffer (PBS + 0.05 mg/mL BSA). The peptides were serially diluted in assay buffer (PBS + 0.05 mg/mL BSA) that contained 0.16% TEA to appropriate concentrations and were preincubated with Fc-Spike-RBD (R&D Systems, Cat. No. 10542-CV-100) at 37 °C for 1 h (12.5 μL total volume). These were then added to AlphaScreen plates (Perkin Elmer, 384-well) that had been preincubated for 30 min with 12.5 μL of Biotinylated-ACE2 (Sino Biological, Cat. No. 10108-H27B-B). After a 30-min incubation at 37 °C, 25 μL of premixed acceptor/donor beads were then added to the plates and incubated for 30 min at 37 °C. After incubation, the Alpha signal was read using a Neo2 plate reader (Biotek). Data was then normalized, with 100% being the positive control (no peptides added) and 0% being just ACE2 added. The normalized data was plotted and fitted using a nonlinear regression model (4 parameters) on GraphPad Prism.