| Assay Method Information | |
| | HCoV-OC43 3CLpro inhibition assay |
| Description: | For the HCoV-OC433CLpro assay, 2 μL of 0.02 μM recombinant HCoV-OC433CL protease were mixed with serial dilutions of each compound in 4 μL assay buffer containing 40mM HEPES, pH = 8.0, 1mM CHAPS, 150mM NaCl, 1mM EDTA, 1mM TCEP in a well of a 384-well plate and pre-incubated at rt for 1 hr. The custom-synthesized fluorogenic 3CLpro peptide substrate used in the assay is as follows: FAM-KTSAVLQSGFRKMEK-TAMRA (peptide sequence according to SEQ ID NO: 3). This FRET -based substrate contains a FAMfluorophore attached at the N terminus of a canonical 3CLpro peptide substrate. The fluorophore is internally quenched by the TAMRA. The reaction was initiated by the addition of 10 μL of the substrate with a final concentration of 15 μM and each well was incubated at RT for 30 mins. The final concentration of the protease used at the assay was 25 nM and each compound was at a final concentration range of 100 0.0016 μM. The IC50 value which is the value causing 50% inhibition of the catalytic activity of the HCoV-OC433CLpro was calculated by four parameters equation analysis. |
| Affinity data for this assay | |
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