Measurement of SARS-CoV-2 3CL protease inhibition

Inhibition of the biochemical activity of the SARS-CoV-2 3CL protease was quantified as previously described with modifications⁵. Serial dilutions of the test compound were prepared in the assay buffer, and then incubated with 0.2 µM of the protease for 10 min at 37 °C. The substrate was then added at 20 µM per well, and then fluorescence was continuously measured on a plate reader for 3 min. Inhibition was then calculated by comparison to control wells with no inhibitor added. IC₅₀ values were determined by nonlinear regression (GraphPad Prism). For calculations, a 100% active enzyme was assumed.

Kinetic parameters were determined as previously described¹⁴. Compounds were pre-incubated with the protease at differing timepoints at various concentrations to derive k_obs, which were then used for the calculation of k_inact and K_i by nonlinear regression (GraphPad Prism).