SARS-CoV-2 3CLpro was incubated with drugs at 0–100 µM for an hour at 37 °C. Then, 1.25 µM IQF peptide substrate was added to the mixture to a final volume of 100 µL and incubated at 37 °C for another three hours, prior to detection. With the same parameters applied in protease activity assays, the RFU readouts obtained from the SPARK® multimode microplate reader (TECAN, Switzerland) were normalised to the negative control (vehicle only) in each assay plate. After drug treatment at a concentration between 0–100 µM, points of relative protease activity were fitted to a normalised dose-response model in GraphPad Prism 7.03 for IC50 characterisation, where Y=Bottom+TopBottom1+10(LogIC50X)·HillSlope .

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