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. 2022 Nov 3:eabq4064. doi: 10.1126/scitranslmed.abq4064

Fig. 1. S-217622 inhibits SARS-CoV-2 infection in vitro at the post-entry stage.

Fig. 1.

(A)Chemical structure formula of S-217622. (B and C) Immunofluorescence staining of SARS-CoV-2-infected cells. Vero-TMPRSS2 (B) and Calu-3 (C) cells were inoculated with a SARS-CoV-2 Delta variant at MOIs of 0.1 or 10, respectively, and then cultured in the presence of S-217622 for 24 hours and 72 hours, respectively. Cells were stained with anti-SARS-CoV-2 nucleocapsid antibody (green) and Hoechst 33342 (magenta). Scale bars, 100 μm. (D) Viral RNA concentrations were measured in the culture supernatant of Vero-TMPRSS2 cells at 24 hours post-infection (hpi) with a SARS-CoV-2 Delta variant at an MOI of 0.01. (E) Viral RNA concentrations were measured in the culture supernatant of Calu-3 cells at 72 hpi with SARS-CoV-2 Delta variant at an MOI of 0.1. (F) Schematic representation of the SARS-CoV-2 infection experiment in a human airway tissue model. Cells were inoculated at the apical surface with 5,000 pfu of SARS-CoV-2. Viral growth was monitored by titration of progeny virus in the mucus layer on apical surface. The tissue damage by viral infection was estimated by measurement of lactate dehydrogenase (LDH) released from cells into the basal culture medium. (G) Growth of the SARS-CoV-2 Delta variant in a human airway tissue model. Basal culture medium was supplemented with the indicated concentration of S-217622. (H) The abundance of LDH in the basal culture medium was measured by LDH-Glo luminescent assay. (I) A time of the addition assay was conducted using S-217622. Calu-3 cells were treated with S-217622 during inoculation (entry stage), after inoculation (post-entry stage), or both entry and post-entry stages of SARS-CoV-2 at an MOI of 3. The inhibitory effect of each treatment was evaluated by measurement of intracellular viral RNA concentrations at 7 hpi. Camostat and anti-SARS-CoV-2 spike protein antibody were used as entry inhibitors for assay controls. The values shown are mean ± standard deviation (SD) of triplicate samples. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Dunnett’s test (D, E, H, I) or Kruskal-Wallis test with Dunn’s test (G).