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Real-Time Monitoring of PROTAC and Molecular Glue Targeted Degradation in Living Cells

Riching, K. M. et al., Promega Corporation


AACR 2020 Abstract #5230

The emergence of targeted protein degradation as a broad, new therapeutic modality has greatly expanded the opportunities for treatments of many diseases. Currently, small molecule degrader compounds fall under two major categories; molecular glues and heterobifunctional PROTACs. The two types of compounds both facilitate and induce a ternary complex consisting of an E3 ligase-degrader-target protein, bringing into proximity the machinery proteins required to ubiquitinate and ultimately degrade the target protein. Significant challenges exist in characterization and rank-ordering of degradation compounds in live cells given the differences in dynamics of protein loss and recovery among compounds. Currently, the availability of technologies to interrogate real-time protein degradation is severely lacking.

Here, we present a live-cell, luminescence-based technology platform with these capabilities. We employ CRISPR/Cas9 endogenous 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. This 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. We demonstrate the power of this technology in continuous 24-hour monitoring of endogenous target protein levels, and the ability to quantify key degradation parameters for compound ranking including rate, Dmax, and DMax50. We further show the ability to measure the mechanistic steps important for the degradation including kinetics of PROTAC- or molecular glue-induced ternary complex formation and target ubiquitination. These studies facilitate discernment of individual parameters required for successful degradation, ultimately enabling chemical design strategies for optimization and rank ordering of therapeutic degradation compounds.

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