2.4. Bio-layer interferometry (BLI) binding assay

The binding affinities between tested compounds and SARS-CoV-2 RdRp were measured using BLI-based Octet Platform (Octet Red 96, Fortebio). A temperature of 30 °C and a stirring speed of 1000 revolutions per minute (rpm) were used on Fortebio Octet Red 96 in black 96 well throughout the experiment. 1 × Phosphate-buffered saline (PBS; pH = 7.4) with 0.01% Tween-20, 0.1% bovine serum albumin (BSA), and 2% DMSO was used as assay buffer for all measurements. The purified His-tagged SARS-CoV-2 RdRp were captured via Ni-NTA (NTA) biosensors (Cat# 18-5101, ForteBio) at a concentration of 150 μg/mL, resulting in a saturation response of 5–6 nm after 300 s. Subsequently, the loaded biosensors were washed for 3 min in buffer to clear up loose nonspecifically bound SARS-CoV-2 RdRp and to establish a stable baseline. For binding kinetic measurements, the association of SARS-CoV-2 RdRp and tested compounds (3.125–100 μmol/L in assay buffer) was measured for 60–180 s and the dissociation of them was measured for 120 s in assay buffer. Reference wells that utilized buffer instead of tested compounds were also included to correct the baseline shift. A parallel set of Ni-NTA sensors that were incubated in buffer-only were prepared as the negative reference controls to correct the non-specific binding of the compounds to the biosensor surface. Raw kinetic data were analyzed using a double reference subtraction approach in which both the background and non-specific binding were subtracted. The binding affinity constant K _D (K _D = k _dis/k _on; k _on is the association rate constant, k _dis is the dissociation rate constant) values were calculated using 1:1 binding model through global fitting of multiple kinetic traces. Data Analysis 9.0 software was used to analyze the real-time monitoring data. All the measurements were performed in three independent experiments.