Fig. 2: In vitro resistance selection of EGT710 and characterization of selected viruses.

A Concentration of EGT710 in serial passaging experiments performed with Δ3678 mNG SARS-CoV-2 in VeroE6-TMPRSS2 cells for selections 1, 2, and 4, reaching a maximum concentration of 8 µM. Selection 3 reached a maximum concentration of 4 µM. B Susceptibility of the selected virus population to EGT710 after 13 passages determined in VeroE6-TMPRSS2 cells using qRT-PCR at 24 h post-infection. Geometric mean EC₅₀ values from n = 2 independent experiments. C Prevalence of nucleic acid substitutions present in the nsp5 gene identified by next generation sequencing (NGS) at passage 13, and prevalence in GISAID sequences. Nt, nucleotide; nt ref, nucleotide reference; nt sub, nucleotide substitution; nt freq, nucleotide frequency by NGS D Location of amino acid substitutions relative to the EGT710 binding site. E, F Susceptibility of recombinant Δ3678 mNG SARS-CoV-2 containing nsp5 mutations to EGT710 determined in VeroE6-TMPRSS2 cells using high-content imaging at 24 h post-infection: E representative concentration-response curves from a single experiment and F geometric mean EC₅₀ values from n = 2 independent experiments. G Fitness of recombinant Δ3678 mNG SARS-CoV-2 containing nsp5 mutations. VeroE6-TMPRSS2 cells were infected with 0.1 MOI of recombinant viruses, and qRT-PCR was used to quantify RNA copies/mL in the supernatant at 12, 24, and 36 h post-infection. Bars are mean value from n = 3 experiments ± standard deviation; *, p < 0.05; **p < 0.005. Figures generated using PyMol and GraphPad Prism software.