Gc376

GC376 (Selleck, S0475) and Boceprevir (Selleck, S3733) were purchased from Selleck. Compounds 11a and 13b were synthesized following previously reported protocols (5 (link), 11 (link)).

Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS) were purchased from Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from Eurobio. Remdesivir (GS-5734) and GC376 were purchased from Selleck Chemicals (Houston, TX, USA). Camostat mesylate, tariquidar, and dimethylsulfoxide (DMSO) were obtained from Sigma (Saint Louis, MO, USA). The lichen compounds were resuspended in DMSO at 100 mM.

The kinetic assays were implemented using the FRET substrate (FRET-S), Dabcyl-KTSAVLQ↓SGFRKM-E(Edans)-NH₂ (BPS Bioscience, United States), and standard covalent inhibitors GC-376, PF-00835231, boceprevir, telaprevir (Selleckchem, United States), and thimerosal (Serva). FRET-S contains a main-protease cleavage site (indicated by the arrow in the sequence above) and was utilized as the substrate in the FRET-based cleavage assay. Stock solutions of the inhibitors were prepared in DMSO (final concentration 5.0 mM).

VeroE6/TMPRSS2 cells (JCRB #1819) [16 (link)] and Calu-3 cells were cultured in DMEM containing 10% FBS. SARS-CoV-2 Pango lineage A (JPN/TY-WK-521/2020 EPI_ISL_408667), alpha strain (JPN/QK002/2020 EPI_ISL_768526), beta strain (JPN/ TY8-612-P1/2021 EPI_ISL_1123289), gamma strain (JPN/TY7-501/2021 EPI_ISL_833366), and delta strain (JPN/TY11-927-P1/2021 EPI_ISL_2158617) were obtained from NIID, JAPAN, and handled in biosafety level 3 (BSL3). While performing SARS-CoV-2 infection experiments, DMEM containing 2% FBS was used. The chemical compound library was obtained from Tokiwa Phytochemical (Chiba, Japan) and retinoids including ATRA, all-trans retinal, retinol, 9-cis RA, and 13-cis RA were obtained from Sigma Aldrich (St. Louis, MO, USA). GC376 was purchased from Selleck chemicals (Houston, TX, USA).

The enzymatic activities of 3CL^pro WT and P108S were determined using a fluorescent substrate with the cleavage site of SARS CoV-2 3CL^pro (Dabcyl-KTSAVLQ↓SGFRKME-Edans; GL Biochem). 3CL^pro WT or P108S at a final concentration of 5 μmol/l was incubated in a buffer of 20 m mol/l Tris-HCl (pH7.5), 100 m mol/l NaCl, and 5 m mol/l DTT with the addition of the substrate at a final concentration of 3.125, 6.25, 12.5, 25, 50, 100 or 200 μmol/l at room temperature. The change in fluorescence intensity was monitored with a fluorescence spectrophotometer (Cytation 5; BioTek) at an emission wavelength of 460 nm and an excitation wavelength at 340 nm. The kinetic parameters were determined with GraphPad Prism 8 software and the initial rate measurement of the substrate cleavage. For the inhibition assay, the SARS-CoV 3CL^pro inhibitor GC376 (Selleck) at a final concentration of 1, 2, 5, 10, or 20 μmol/l was incubated with 5 μmol/l 3CL^pro WT or P108S and 12.5, 25 or 50 μmol/l of substrate.