Protease inhibition assay

EV71 3C protease inhibition assays were based on a published procedure⁷ and performed in a buffer containing Tris-HCl (50 mM), NaCl (150 mM), EDTA (1 mM), glycerol (10% v/v) and DTT (2 mM) at pH 7.0. The protease (6 μM) and varying inhibitor concentrations were incubated at 25 °C for 2 h. The final DMSO concentration was maintained at 2%. After that, the chromogenic peptide substrate succinyl-EALFQ-pNA (Peptides International, USA) was added to make a final concentration of 200 μM. The contents were incubated at 25 °C for 2 h. Absorbance at 405 nm was measured with a plate reader at 30 °C. All experiments were conducted in duplicates. IC₅₀ values were derived by fitting the initial velocity against the log [inhibitor] using GraphPad Prism 5 software (USA).