Optimization of the HTRF-based spike protein/ACE2 inhibitor screening system

To optimize the interaction between the spike protein (S protein) and ACE2, we performed cross-titration experiments to determine the maximal effect. In brief, ACE2 and SARS-Cov-2 S-protein were prepared at multiple concentrations with a PBS containing 0.1% BSA, 5 μl ACE2 (Mammalian, C-Fc, DRA36, Novoprotein) and 5 μl SARS-Cov-2 S-protein RBD (Mammalian, C-6His, C05Y, Novoprotein). Each concentration was added into each well of the 384-microplate (ProxiPlate™ 384-shallow well Microplates, 66PLP96025, CISBIO) and the mixture incubated at 37 °C for 1 h. Next, 5 μl PAb Anti Human IgG-d2 (61HFCDAB) and 5 μl MAb Anti-6HIS-Tb cryptate Gold (61HI2TLB) were added to each well with the ACE2/S-protein RBD mixture (final reactive volume of 20 μl) following the supplier’s protocols. After 30 min final incubation at room temperature, HTRF signals were measured using a Multimode Reader (Spark 10M, Tecan) equipped with an excitation filter of 340 nm, and fluorescence detected at 620 and 665 nm with a lag time of 100 μs and an integration time of 200 μs. The results were analyzed using a two-wavelength signal ratio: [intensity (665 nm)/intensity (620 nm)] × 10⁴ (HTRF Ratio). The Z factor was calculated using the following equation:

Standard deviation (SD).

The initial screening assay was repeated twice and the hits confirmed by the determination of IC₅₀ (HTRF) in quadruplicates. IC₅₀ (HTRF) was defined as the compound concentration at which the combination of ACE2 and S-RBD decreased by 50%.