FIGURE 2.

The in vitro antiviral dose‐response screening results of the EIF2AK2 inhibitor compound C16. Inhibition of SARS‐CoV‐2 replication was tested in various cells by developing CPE via ATP luminescence at 72H (solid shapes) or direct virus inhibition at 48 h (empty shapes). For the 48 h direct virus inhibition, %virus growth was normalised to the infected + DMSO only wells (max) and uninfected + DMSO only wells (min). For the 72 h CPE/ATP Luminescence assay, cell growth was normalised to the cells+DMSO control wells (Max) and Cells+DMSO+virus only (Min) wells for EC₅₀ calculations (black triangles) and Cells+DMSO control wells (Max) and Media+DMSO control wells (Min) for the CC₅₀ calculations (red squares, cytotoxicity). The 48H and 72H EC₅₀ assays were performed at the CDI biosafety level 3 laboratory. (A–C) A549 human lung cells overexpressing ACE2 were treated with either C16 or remdesivir and infected with SARS‐CoV‐2 infectious clone reporter strain. C16 by itself was found to have relatively high potency in the 48H direct virus inhibition assay (EC₅₀ = 1.25 μM) and the 72H ATP luminescence assay measuring cytopathic effect (EC₅₀ = 2.96 μM) with low cytoxocity (CC₅₀ = 26.2 μM). (D–F) VeroE6 African Green monkey kidney cells overexpressing TMPRSS2 were treated with either C16 or remdesivir and infected with SARS‐CoV‐2 infectious clone reporter strain. The 48H direct virus inhibition EC₅₀ was 1.65 μM and the 72H CPE EC₅₀ was 2.06 μM in C16 treated VeroE6+TMPRSS2 cells with low cytoxocity (CC₅₀ = 14.4 μM). Similar results were observed in remdesivir treated cells. (G–J) Treatment of C16 on VeroE6 cells infected with SARS‐CoV‐2 shows C16 had improved potencies relative to remdesivir. VeroE6 cells treated with C16 had EC₅₀ values of 0.568 and 1.06 μM in the 48H direct virus inhibition and 72H CPE assays, respectively. The combination treatment with C16 and remdesivir was also evaluated in VeroE6 cells. The EC₅₀ was 650 nM in the 48H direct virus inhibition assay and 1.52 μM in the 72H CPE assay. In the remdesivir treated VeroE6 cells, the EC₅₀ was 1.39 and 1.86 μM in the same assays.