Temporal and Spatial Characterization of CUL3-Driven Targeted Degradation of BET Family BRD Proteins by the Macrocycle-Based Degrader BTR2004.

Targeted protein degradation (TPD) is a promising modality that leverages the endogenous cellular protein degradation machinery to degrade selected proteins. Recently, we validated CUL3 E3 ligase as a new actionable E3 ligase for TPD application by developing a synthetic macrocycle ligand to engage KLHL20. Linking the KLHL20 ligand to JQ1, we created the PROTAC molecule BTR2004, which exhibited potent degradation of BET family proteins BRD 2, 3, and 4. As CUL3 is new to the TPD field, here we report the first temporal and spatial characterization of CUL3-driven TPD with BTR2004. Our study revealed the target protein degradation kinetics, BTR2004 intracellular activity half-life, and the onset of BTR2004 cell permeabilization. Employing proximity ligation and confocal microscopy techniques, we also illustrate the subcellular location of the ternary complex assembly upon BTR2004 treatment. These characterizations provide further insight into the processes that govern TPD and features that could be incorporated into the design of future macrocyclic PROTAC molecules.