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. 2022 Jun 1;23(11):6206. doi: 10.3390/ijms23116206

Figure 9.

Figure 9

(A) Cartoon models of CDC34A and E3 ligase-mediated substrate ubiquitination being blocked by MG CC0651 (i.e., inhibition of ubiquitin transfer by stabilizing the noncovalent CDC34A-donor ubiquitin complex); (B) schematic of TR-FRET assay to detect CDC34A/UbE2R1–ubiquitin interactions. The assay used an N-terminal His-tagged CDC34A for recognition by an anti-His6 antibody coupled to Tb3+ and an N-terminal cysteine mutant of ubiquitin (denoted UbCys0) stoichiometrically labeled with 5′-iodoacetamide-fluorescein. In response to titration with CC0651, excitation of Tb3+ at 340 nm resulted in fluorescence energy transfer to the fluorescein moiety and emission at 520 nm; (C) diagram to explain the led compounds 2ab, 2cb, 2db, 2gb, and 2aη generated by medicinal chemistry hybridization of the prototype compound CC0651 and the isonipecotamide hit BDC22455743; (D) Ub IC50 (µM), TR-FRET EC50 (µM), and p27-Ub(n) IC50 (µM) for the prototype compound CC0651 and the led compounds 2ab, 2cb, 2db, 2gb, and 2aη were shown. IC50 values represent the mean +/− variance, n = 2. EC50 values represent the mean +/− SD, n = 3. (B,D) are adapted from an original paper by St-Cyr et al. (some information is also from their provided supplemental materials) [37].