Figure 1.

Schematic illustration of the RBD-ACE2 binding assay. Ninety-six-well plates were coated with ACE2 (100 ng/well) and blocked with TSTA buffer. Each compound (10 µM) from the libraries was incubated with Fc-RBD (1 µg/mL) for 1 h at 25 °C. Following incubation, mixtures were added to the ACE2-coated plates and incubated for 1 h at 37 °C. After washing, the plates were incubated for 1 h at 37 °C with the alkaline phosphatase-conjugated goat anti-human Fc fragment. The plates were then washed, and a colorimetric reaction was developed by the addition of pNPP (measured at 405 nm). Left panel: a maximum signal of an uninterrupted RBD-ACE2 interaction was obtained in the presence of noninhibitory compounds. Right panel: a reduced signal of an inhibited RBD-ACE2 interaction was obtained in the presence of inhibitory compounds.