Fig. 5: Evaluation of anti-viral activity in cell-based models.

a, b CETSA assays of HEK293-NSP14 cells treated with DMSO or 20 μM C10 at various heating temperatures (a), and with varying concentrations of C10 at 54 °C (b). Three times experiments were repeated independently with similar results. c Schematic diagram outlining the anti-viral activity assays conducted in cell-based assays. Created in BioRender. Che, J. (2025) https://BioRender.com/0f4t8er. d Histogram showing the infection ratios of A549-ACE2 cells treated with the selected C-family compounds at 20 μM. Data are presented as mean ± SD from three independent replicates. e Dose-dependent inhibition curves of remdesivir against SARS-CoV-2 strain (Wuhan-Hu-1), along with the 50% cytotoxic concentration (CC₅₀) values, as measured in A549-ACE2 cells. Data are presented as averages from two independent replicates. Two times, experiments were repeated independently with similar results. f, h Dose-dependent inhibition curves of C3 (f), C7 (g), and C10 (h) against SARS-CoV−2 different strains in A549-ACE2 cells, with the 50% cytotoxic concentration (CC₅₀) values. Data are presented as averages from two independent replicates. The experiment was repeated twice independently with similar results. i Frequency of NSP14 amino acid changes emerging in SARS-CoV-2 trVLP after 5 passages in Vero-N cells challenged with 10 μM or 20 μM C10. j Schematic of the SARS-CoV-2 replicon assay (left) and quantification of replicon activity by luciferase reporter readout (right). Data are mean ± SD from three biological replicates. Statistical significance was determined by two-sided unpaired t-test. k Time-of-addition analysis of C10. For all the experimental groups, cells were infected with SARS-CoV-2 trVLP at an MOI of 0.02, and the luciferase activity was measured. Data are presented as mean ± SD from three independent replicates. Statistical significance was determined by two-sided unpaired t-test. Source data are provided as a Source Data file.