SARS-CoV-2 3CL Protease Inhibition Assay

A slightly modified
protocol from the commercially available assay (BPS Bioscience) was
used. Dithiothreitol was substituted with tris(2-carboxyethyl)phosphine
(TCEP), the latter of which was found not to alter the activity of
the enzyme in the assay. The 3CL^pro protease was thawed
on ice and activated by dilution to 10.0 ng/μL with assay buffer.
The enzyme solution was further diluted with assay buffer to 0.5 ng/μL.
Twenty microliters of the enzyme solution was mixed with 5 μL
of increasing concentrations of the complex [2% (v/v) DMSO] diluted
in assay buffer in the dark. The mixture was incubated for 30 min
at 37 °C with slow shaking. The substrate [Dabcyl-KTSAVLQSGFRKM-E(Edans)-NH₂] was diluted to 50 μM, and 25 μL was added to
the enzyme mixture. The mixture was incubated for 4 h at 37 °C
with slow shaking. The generated fluorescence signal (λ_ex = 360 nm; λ_em = 460 nm) was recorded with
a Synergy H4 (BioTek) microplate reader. As a positive control, the
known inhibitor GC376 (IC₅₀ = 140 ± 20 nM) was used.