Figure 4.
Thermal shift binding assay of SARS-CoV-2, EV-A71, and EV-D68 proteases against inhibitors investigated in this study. (A) SARS-CoV-2 Mpro; (B) SARS-CoV-2 PLPro; (C) EV-A71 2Apro; (D) EV-A71 3Cpro; (E) EV-D68 2Apro; and (F) EV-D68 3Cpro. 3 μM protease in its corresponding enzymatic reaction buffer in the presence of 4 mM DTT or in the absence of DTT was pre-incubated with DMSO or 40 μM protease inhibitors (shikonin was tested at 10 μM because 40 μM Shikonin completely quenches SYPRO orange dye fluorescence signal) at 30 °C for 30 min. The melting temperature (Tm) was calculated as the mid log of the transition phase from the native to the denatured protein using a Boltzmann model.32 * indicates that a fluorescence peak was not observed in the melting curve; red dash line shows the protease Tm with DMSO in the presence of 4 mM DTT.