Fig. 2
Selective degradation of HCV NS3. a Quantitative assessment of cellular degradation. A schematic depiction of the protein expressed in cells is shown. Expression of NS3-eGFP-2A-mCherry was induced, and cells were treated for 4 h with increasing concentrations of the indicated small molecule. eGFP and mCherry fluorescence were quantified by flow cytometry. The concentration that led to a 50% decrease in NS3-eGFP fluorescence (DC50) was determined by nonlinear regression. One representative experiment is shown, and the DC50 values were averaged from n ≥ 2 independent experiments. Source data are provided as a Source Data file. b Quantitative proteomics analysis of cellular degradation. The scatter plot depicts the log2 fold change (FC) in protein abundance in induced cells treated for 4 h with 1000 nM DGY-08-097 compared to the DMSO control. Data shown are of a single quantitative TMT 10-plex experiment (showing ~8700 proteins, each quantified by ≥ 2 unique peptides). Significant changes were assessed by a moderated t-test as implemented in the limma package53. The log2 fold change is shown on the y-axis and negative log10 p-values on the x-axis (n = 3 independent biological replicates). HCV NS3 is significantly downregulated with a log2 fold change of 1.5, and p-value of 3.08 × 10-10. c Evaluation of NS3 degradation by negative control compounds that cannot engage CRBN. Expression of NS3-eGFP-2A-mCherry was induced, and cells were treated for 4 h with increasing concentrations of the indicated small molecule. eGFP and mCherry fluorescence were quantified by flow cytometry. One representative experiment is shown from n = 2 independent experiments. d Lenalidomide competition assay. Expression of NS3-eGFP-2A-mCherry was induced, and cells were treated for 4 h with 1000 nM of each telaprevir-derived compound, and co-treated with either DMSO or 5000 nM lenalidomide. The eGFP and mCherry signals were quantified by flow cytometry. Data are presented as means normalized to DMSO ± standard deviation of n = 2 independent experiments. Asterisks indicate that the differences between samples are statistically significant, using the unpaired t-test (**0.001 < p < 0.01; not significant, p > 0.05). Source data are provided as a Source Data file
