Fig. 5.

Antiviral effect of alimemazine on SARS-CoV-2 multiplication. (A) Effect of alimemazine on SARS2-S-mediated membrane fusion. HEK293T cells transfected with a plasmid expressing SARS2-S (left) or SARS2-S containing 69–70 deletion and the N501Y and E484K substitutions in addition to the D614G substitution (right) together with a plasmid expressing ZsGreen (ZsG) were overlaid on HEK293T/ACE2 cells that had been pretreated with 0.1% DMSO or 10 μM alimemazine for 90 min. DMSO and alimemazine were present throughout the experimental period. After a 4-h incubation, fluorescent images of the cells were captured (left), and the average number (ave. #) of nuclei in each syncytium was determined with a high-content imaging system (right). The presented data are the mean ± SD of the results of four independent experiments. Statistical significance was determined by a Student's t-test. **p < 0.01. (B and C) Effect of alimemazine on SARS2-S binding to ACE2 (B) or to NRP-1 (C). ELISA-based SARS2-S–ACE2 and SARS2-S–NRP-1 binding assays were performed in the presence of DMSO (0.1%), the neutralizing antibody anti-SARS2-S S1 domain (anti-S1 Ab; 5 μg/mL, used only in B), or alimemazine (10 μM or 100 μM). The mean chemiluminescence (B) and absorbance (C) values of DMSO-treated samples were each set to 1. The presented data are the mean ± SD of the results of three replicates. Statistical significance was determined by comparing the chemiluminescence (B) and absorbance (C) values of the experimentally treated samples with the values for the DMSO-treated samples. ns, p > 0.05, not significant; **p < 0.01; ND, not detected. (D) Effect of alimemazine on SARS-CoV-2 multiplication in cultured cells. HEK293T/ACE2 cells that had been pretreated with 0.1% DMSO or 10 μM alimemazine for 90 min were inoculated (MOI = 0.1) with SARS-CoV-2 TY7-501 strain. After 24 h, the cells in the tissue culture supernatant were collected, and the virus titers were determined by a TCID₅₀ assay. The presented data are the mean and SD of the results of three independent experiments. Statistical significance was determined by a Student's t-test. **p < 0.01. (E) Effect of alimemazine on SARS-CoV-2 multiplication in mice. Eight-week-old BALB/c mice were treated with alimemazine (10 mg/kg) or saline by oral gavage 1 h prior to and 24 h after intranasal inoculation with SARS-CoV-2 TY7-501 strain (2 × 10⁴ TCID₅₀ per mouse). At 2 days post-viral inoculation, lung tissues were harvested, and the tissue viral titers were determined by a TCID₅₀ assay. The presented data are the mean and SD of the results from four mice per group. Statistical significance was determined by a Student's t-test.