2.5. RdRp assay

The SARS-CoV-2 RdRp homogeneous assay kit was used to assess the possible inhibitory effect of PF₅₀ on the activity of SARS-CoV-2 RdRp. This kit comes in a convenient AlphaLISA format, with biotinylated ATP, digoxigenin-labeled RNA duplex, purified RdRp, and RdRp assay buffer. Following the manufacturer's instructions, the enzyme (67 ng/μl) was preincubated with PF₅₀ (120–0.002 μM, 3-fold dilution in PBS) for 30 min at room temperature before initiating the reaction (60 min, 37 °C). During the reaction, RdRp directly incorporates the ATP into the double-stranded RNA chain, which was measured by the increase in Alpha counts (the signal-to-noise ratio) after the addition of the diluted anti-digoxigenin acceptor beads and AlphaScreen Streptavidin-conjugated donor beads. Both the positive (without PF₅₀, 100 % activity “C_e”) and negative (without enzyme, 0 % activity “C₀”) controls, as well as the reference compound (6-Chloropurine-ribose-5′-triphosphate “CPR-P”, 40–0.002 μM) were included in the assay. The % activity at each concentration was calculated using the equation: (C—C₀)/(C_e —C₀), where C is the AlphaScreen intensity in the presence of the PF₅₀ or the reference compound. The % activity values versus a series of compound concentrations were then plotted using non-linear regression analysis of the sigmoidal dose-response curve using Graphpad Prism software to calculate the IC₅₀ value.