Protsil2 1kt

To determine whether the phenothiazine would affect the cleavage of SARS-CoV-2 S by the CTSL protein (Sino Biological, Beijing, China, 10486-H08H), about 2 μg recombinant SARS-CoV-2 S protein (Sino Biological, 40589-V08B1) was incubated with the candidate compound on ice for 15 min. Additionally, about 10 ng recombinant CTSL protein was incubated with or without teicoplanin in the CTSL activation buffer (400 mM sodium acetate, 4 mM EDTA, 8 mM DTT and pH 5.5), and the mixture was incubated on ice for 15 min for pre-activation. After incubation, the phenothiazine-S mixture was added to the pre-activated CTSL protein solution and incubated at 37 °C for 1 h. Finally, all of the samples were proceeded to the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed with silver staining (PROTSIL2-1KT, Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions.

To evaluate the CTSL enzymatic activity upon teicoplanin treatment in vitro, the purified 250 ng CTSL proteins (Sino Biological Inc., 10486-H08H, China) were added into the CTSL assay buffer (400 mM NaAc, 4 mM EDTA, 8 mM DTT, and pH 5.5) and incubated in ice for 15 min for CTSL activation. To evaluate the inhibition of teicoplanin on CTSL activity, the purified 250 ng CTSL proteins were also co-incubated with 50 μM teicoplanin (Selleck, S1399, United States). After activation, about 2 μg in vitro purified SARS-CoV S (Sino Biological Inc., 40634-V08B, China) or SARS-CoV-2 S (Sino Biological Inc., 40589-V08B1, China) were added into each group. S protein only group (without CTSL and teicoplanin) was set as control group. The S-CTSL-teicoplanin mixtures were incubated at 37°C for 1.5 h. The enzymatic reaction was stopped by adding with SDS-PAGE loading buffer and followed by boiling at 100°C for 10 min. Digested proteins were proceeded to SDS-PAGE and analyzed by silver staining (Sigma-Aldrich, PROTSIL2-1KT, United States).