Fig. 1: A cell-based phenotypic screen for inhibitors of the UPS.

A Representation of the screen workflow. Positive hits were defined as compounds that accumulated the UFD reporter in the nucleus above basal levels, defined by the signal in the DMSO-treated wells (negative control). Positive control wells treated with epoxomicin (PI proteasome inhibitor, 100 nM) were also included in each plate of the screen. One positive hit was found in the initial screen, which formed the basis for a structure-activity relationship study (SAR). One active and one inactive structurally related compound were selected for further characterization. B Chemical structure of the hit compound CBK092352. C Summary of structure-activity relationships (SAR). Attenuation of activity is observed when R₃ is substituted, whereas substituents in R₂ are accepted. Removal of the nitro group results in an inactive compound. D Structures of the analogs selected for further characterization, the active compound CBK77 and the inactive compound CBK07. E Representative images of MelJuSo Ub-YFP cells treated for 16 h with the indicated compounds (10 µM). DMSO at 0.1% was used as negative control. The nuclei were counterstained with Hoechst and cells imaged live with an automated widefield microscope. Scale bar = 20 µm. F Concentration-response experiments performed with MelJuSo Ub-YFP cells. Cells were treated for 6 h with a range of compound concentrations. Nuclei were stained with Hoechst and cells were directly imaged live with an automated widefield microscope. Data are represented as mean ± SD of three independent experiments. Nonlinear curve fitting is depicted in red. The half-maximal effective concentration (EC₅₀) upon CBK77 treatment is shown (4.3 µM, 95% confidence interval 3.8–5.0).