Assay Method Information

Assay Name:  FRET enzyme assay
Description:  The expression and purification of the 3CLpro of MERS-CoV and SARS-CoV were performed by a standard method described previously by our lab (11, 19, 20). We also cloned and expressed the 3CLpro of SARS-CoV-2. The codon-optimized complementary DNA (cDNA) of the full length of 3CLpro of SARS-CoV-2 (GenBank accession number MN908947.3) fused with sequences encoding six histidine at the N terminus was synthesized by Integrated DNA (Coralville, IA). The synthesized gene was subcloned into the pET-28a(+) vector. The expression and purification of SARS-CoV-2 3CLpro were conducted after a standard procedure described by our lab (19). Briefly, stock solutions of compounds 6a to 6k and 7a to 7k were prepared in dimethyl sulfoxide (DMSO) and diluted in assay buffer, which was composed of 20 mM Hepes buffer (pH 8) containing NaCl (200 mM), EDTA (0.4 mM), glycerol (60%), and 6 mM dithiothreitol (DTT). The protease (3CLpro of MERS-CoV, SARS-CoV, or SARS-CoV-2) was mixed with serial dilutions of each compound or with DMSO in 25 μl of assay buffer and incubated at 37C for 30 min (MERS-CoV) or at room temperature for 1 hour (SARS-CoV and SARS-CoV-2), followed by the addition of 25 μl of assay buffer containing substrate (FAM-SAVLQ/SG-QXL 520, AnaSpec, Fremont, CA). The substrate was derived from the cleavage sites on the viral polyproteins of SARS-CoV. Fluorescence readings were obtained using an excitation wavelength of 480 nm and an emission wavelength of 520 nm on a fluorescence microplate reader (FLx800, BioTek, Winooski, VT) 1 hour after the addition of substrate. Relative fluorescence units (RFU) were determined by subtracting background values (substrate-containing well without protease) from the raw fluorescence values, as described previously (19). The dose-dependent FRET inhibition curves were fitted with a variable slope by using GraphPad Prism software (GraphPad, La Jolla, CA) to determine the IC50 values of the compounds.
Affinity data for this assay

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