Fig. 5: UNI418 protects against SARS-CoV-2 infection.

a Antiviral activity and cytotoxicity of UNI418. Vero cells mock-infected or infected with SARS-CoV-2 at an MOI of 0.01 or 0.001 were individually treated with increasing concentrations of UNI418, with apilimod, UNC3230 and remdesivir used as controls. On Day 2 (for UNI418, apilimod and remdesivir) or Day 1 (for UNC3230) postinfection, the number of viral spike protein-positive cells was determined by immunofluorescence microscopy using an anti-spike antibody. The antiviral activity (EC₅₀) was calculated as the concentration inhibiting the spike-positive cell number by 50% (red line). In parallel, the cytotoxicity (CC₅₀) was determined as the concentration that reduced the viability of mock-infected cells by 50% using MTT (black line). The selectivity index (SI) is the ratio of the CC₅₀ to the EC₅₀. The values are expressed as the means ± SEMs from three independent experiments. b Reduction of SARS-CoV-2 genomic RNA as determined by quantitative RT‒PCR. Vero cells were infected with SARS-CoV-2 (MOI, 0.001) for 1 h at 37 °C. After removal of the unabsorbed virus, the cells were treated with increasing concentrations of UNI418 or apilimod for 24 h. Viral RNA was purified from culture supernatants and subjected to one-step quantitative RT‒PCR to measure relative amounts of the viral NP gene. The values are expressed as the means ± SEMs of three samples. Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparison test; *p < 0.05; ***p < 0.001; ****p < 0.0001. c Reduction in SARS-CoV-2 NP expression by UNI418. SARS-CoV-2-infected Vero cells were treated with increasing concentrations of UNI418 or remdesivir (RDV). On Day 1 postinfection, cell lysates were harvested to measure viral nucleocapsid protein (NP) expression by Western blot analysis. β-Actin was used as a loading control. The marker size is labeled on the left side of the gels. d Blockade of the endocytic pathway by UNI418 in SARS-CoV-2-infected cells. Vero cells were pretreated with DMSO as a mock control (top), 1 µM UNI418 (middle) or 1 µM apilimod (bottom) for 1 h at 37 °C. To synchronize viral infection, the cells were infected with highly purified SARS-CoV-2 (MOI, 10) for 1 h at 4 °C in the presence of each compound. After shifting to 37 °C again, the cells were fixed at 1, 2, 4, and 8 h postinfection for immunolabeling with antibodies specific for viral NP (green) and cellular EEA1 (red). Nuclei were counterstained with DAPI (blue) for all samples. Original magnification, 630×.