5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid

Compounds in Table 1 were diluted in 25 mM Tris buffer (pH = 8.0) to a final concentration of 100 μM for screen. DMSO was used as a solvent control. In all, 10 μL compound solution was add to black 96-well plate (Greiner). In total, 30 μL of 2 μM GM^pro was added to the plate and incubated with the compounds at 37 °C incubator for 30 min. In total, 330 μL of 25 mM Tris buffer was also added as blank control. Then 10 μL of 20 μM peptide substrate (Dabcyl-TSAVLQ↓SGFRKMK-Edans) solution (Genscript) in DMSO was added. The RFU value was measured with an excitation wavelength of 340 nm and emission wavelength of 490 nm at 37 °C for 1 h by using a microplate reader (TECAN Infinite 200 Pro, Switzerland). The RFU change curves vs time with or without inhibitors were plotted by GraphPad Prism 8.0.
Because GC376 and Boceprevir are time-dependent covalent inhibitors, we evaluated the enzyme inhibitory activity without any preincubation. In all, 20 mM GC376 and Boceprevir in DMSO were diluted to 60 μM to 0.015 μM and 120 μM to 0.03 μM by 25 mM Tris buffer (pH = 8.0) respectively. In total, 30 μL inhibitor solution with a series of concentration in 25 mM Tris buffer (pH = 8.0) was mixed with 10 μL of 100 μM peptide substrate firstly. In total, 30 μL Tris buffer was also mixed with 10 μL of 100 μM peptide substrate as negative control. Then, 10 μL of 200 nM final concentration of M^pro was added to the plate. The RFU value was measured with an excitation wavelength of 360 nm and emission wavelength of 490 nm at 37 °C for 1 h by using SpectraMax Paradigm Muti-Mode Detection Platform (Molecular Devices, USA)^{39 (link)}. Experiments were performed in triplicate. First 1200 s change of fluorescence value was used to calculate the reaction rate v₀ by SoftMax Pro 7.1. The reaction rate of different inhibitor concentration is divided by the reaction rate of the negative control to calculate the inhibition rate with Microsoft Excel 2016. Inhibition curve was plotted by GraphPad Prism 8.0.

ALG-097161, nirmatrelvir, and PF-00835231 were synthesized by Aligos Therapeutics and purified to >95% purity. The synthesis of ALG-097161 is described in Text S1. GS-441524 was obtained from MedChem Express (catalog [cat.] no. HY-103586). The African green monkey kidney VeroE6 cell line was purchased from ATCC (cat. no. CRL-1586) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco cat. no. 41965-039) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (FCS).
The SARS-CoV-2 GHB-03021 (EPI ISL407976|2020-02-03) isolate was obtained from a Belgian patient returning from Wuhan in February 2020. The isolate was passaged 7 times on VeroE6 cells, which introduced two series of amino acid deletions in the spike protein (37 (link)).
SARS-CoV-2 3CLpro wild-type and mutant enzymes were produced as previously described (38 (link)). Peptide substrate (Dabcyl-KTSAVLQSGFRKM-E(Edans)-NH₂) for FRET was sourced from Biopeptide (San Diego, CA) at >95% purity.

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.