Inhibitor potency and assay conditions: A case study on SARS-CoV-2 main protease

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  Fig. 1.. (A) List of buffers, EDTA is ethylenediaminetetraacetic acid. (B) Processing of the substrate Abz-VVTLQ/SGDap(Dnp)R-OH (20 µM) by SARS-CoV-2 Mpro (0.5 µM) in different buffers as relative fluorescence units per second (RFU/s). (C) Inhibitory activity of boceprevir and atazanavir (50 µM) in different buffers, [substrate] = 20 µM, [enzyme] = 0.5 µM. (D) Dose–response curves for Mpro inhibition by boceprevir in buffers A and H, [substrate] = 20 µM, [enzyme] = 0.5 µM, IC50 stands for half-maximal inhibitory concentration. (E) The pH profile of the substrate Abz-VVTLQ/SGDap(Dnp)R-OH in buffers with variable additives. Buffer concentration is 50 mM except for buffers with 20% glycerol, where the concentration is 40 mM. Bis-Tris buffer for pH 6 and 6.5; Tris buffer for pH 7, 7.5, and 8. (F) Michaelis−Menten curves for the substrate Abz-VVTLQ/SGDap(Dnp)R-OH in buffers A and H, v stands for reaction rate.