Validation of broad-spectrum activity and wide therapeutic window of the novel antiviral compounds. (A) Vero E6 cells were infected with SARS-CoV-2 (MOI 0.05), Huh7 cells were infected with CoV 229E (MOI 5), Vero cells were infected with EBOV (MOI 0.5), CCHFV (MOI 1) and SW13 cells were infected with HAZV (MOI 1). Infection was followed by treatment with 10 μM TH3289 for 24 h (SARS-CoV-2, CoV 229E, HAZV) or 48 h (EBOV, CCHFV) and end-point dilution assay was performed on Vero E6 (SARS-CoV-2), Vero cells (CCHFV, EBOV) or Huh7 cells (CoV 229E). Data are presented as mean ± SD from n = 2 (SARS-CoV-2, CoV 229E, CCHFV, HAZV) and n = 1 (EBOV) biological replicates. Statistical significance was determined using unpaired two-tailed t-tests. *** p < 0.001. (B) Representative images from Vero E6 cells infected with SARS-CoV-2 (MOI 0.05) and treated with 10 μM TH3289 or DMSO. Cells were stained for SARS-CoV-2 Spike (green) and DAPI (blue). Scale bars equal 100 μm. (C) Huh7 cells were infected with CoV 229E (MOI 5). Infection was followed by treatment with 10 μM TH3289 or 10 μM TH6744 for 24 h and end-point dilution assay was performed on Huh7 cells. Data are presented as mean ± SD from n = 2 biological replicates. Statistical significance was determined using unpaired two-tailed t-tests. *** p < 0.001. (D) Representative images from Huh7 cells infected with CoV 229E (MOI 5) and treated with 10 μM TH3289, 10 μM TH6744 or DMSO. Cells were stained for Pan Coronavirus (green) and DAPI (blue). Scale bars equal 100 μm. (E,F) SW13 cells infected with HAZV (MOI 1) and treated with indicated doses of TH3289 (E) or TH6744 (F). Cell viability was determined by nuclei count (blue line), infected cells by HAZV-NP staining (orange line) and virus titer by end-point dilution assay (black line). Data are presented as Mean ± SD from n = 2 biological replicates. Curve fitting was performed to determine IC50.