Unlocking Targeted Protein Degradation

Targeted protein degradation is a promising new therapeutic strategy consisting of small molecules, most commonly molecular glues or Proteolysis Targeting Chimeras (PROTACs), which elicit degradation of a target protein.  These compounds function to bring into proximity the target protein with an E3 ligase complex component.  This results in formation of a ternary complex which serves to ubiquitinate and degrade the target protein via the ubiquitin proteasomal pathway. Significant challenges persist to characterize the cellular mechanism of action and the highly dynamic degradation responses induced by these compounds. Here we present a live-cell, luminescence-based technology platform, combined with CRISPR/Cas9 endogenous tagging, with these capabilities.  Tagging of target proteins with the small peptide, HiBiT, which has high affinity for and can complement with the LgBiT protein to produce NanoBiT luminescence, allows for sensitive detection of endogenous protein levels in living cells and can also serve as a BRET energy donor to study protein:protein or protein:small molecule interactions required for successful degradation.  We demonstrate the ability to quantitate key degradation parameters for compound triaging and ranking including rate, Dmax, and Dmax50. We further confirm mechanism by monitoring the kinetics of induced ternary complex formation and target ubiquitination using either a PROTAC or molecular glue.  Finally, we highlight the ability of these tools to identify the key cellular mechanisms underpinning the dramatic improvement in degradation potency and kinetics of SIM1, a novel trivalent degrader, compared to the parent bivalent MZ1 PROTAC. Together, these approaches expand the capabilities for understanding degrader efficacy in live cells as well as identifying next generation protein degradation therapeutic targets.