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

Part # PS374

Abstract

Kristin M. Riching, Sarah Mahan, Elizabeth A. Caine, James Vasta, Cesear Corona, Matt Robers, Danette L. Daniels, and Marjeta Urh
Promega Corporation

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. 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 use 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. 

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