Fig. 1. Library design, TR-FRET assay, screen data, and hit validation.

(A) Schematic of CDC34A/SCF-mediated substrate ubiquitination. CC0651 inhibits ubiquitin (Ub) transfer by stabilizing the noncovalent CDC34A-donor ubiquitin complex. (B) Selection of compounds for custom CDC34A screening library. (C) Schematic of TR-FRET assay for detection of the CDC34A-ubiquitin interaction. (D) Bioactivity results from screen of CDC34A custom library (blue) with CC0651-positive controls (red) and DMSO-negative controls (black). Inhibition values from emission ratio measurements (520 nm/490 nm) were normalized to CC0651 (100%) and DMSO controls (0%). (E) Dose-response inhibition by CC0651 and hit 1 in TR-FRET assay. Median effective concentration (EC₅₀) values represent the mean ± SD (n = 3). (F) Dose-response inhibition by CC0651 and hit 1 in ubiquitination assay. Concentrations tested were 1.1, 3.3, 11.1, 33.3, 100, and 300 μM for each compound.