Figure 4.

Phenothiazines bound to the spike of SARS-CoV-2 and inhibited its cleavage. (A–E) Purified SARS-CoV-2 S proteins were immobilized on the CM7 sensor chip, and the candidate compounds including perphenazine (A), fluphenazine decanoate (B), acepromazine maleate (C), prochlorperazine maleate (D), and alimemazine hemitartrate (E) with different concentrations (from 7.18 to 250 μM) flowed over it for the SPR. The K_D values were analyzed by Biocore Insight Evaluation Software. (F) The inhibitory activities of phenothiazines on CTSL-mediated S protein cleavage. Purified CTSL protein was incubated with or without candidate compounds on ice for 15 min, then S protein was added and incubated at 37 °C for 1 h. Proteins were subjected to silver staining. The expression of the full-length protein was quantitated by measuring band intensities using ImageJ software (version 1). Teicoplanin was used as a positive control. (G,H) Prochlorperazine maleate or teicoplanin was co-incubated with SARS-CoV-2 PsV at 37 °C for 1 h prior to being added to the HEK293T-hACE2 cells. The spike protein level was detected by Western blotting (G). The expression of the full-length protein was quantitated by measuring band intensities using ImageJ software (H). The values were normalized to β-actin. Data are presented as means ± SD. p-values below 0.05 (p < 0.05) were considered to demonstrate statistically significant differences. Statistical significance was defined as * p < 0.05, ** p < 0.01, *** p < 0.001.