A Live Cell Method To Assess E3 Ligase And Target Protein Occupancy For Protacs

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that hijack ubiquitin E3 ligases and induce degradation of intracellular proteins through a tightly regulated proteosomal mechanism. Although several successful PROTACs have been developed against key intracellular target classes including bromodomains, kinases, and nuclear hormone receptors, these bivalent molecules often suffer from poor cell permeability due to high molecular weight. To enable a high-throughput, quantitative readout for PROTAC permeability and E3 ligase occupancy in living cells, we have developed a panel of target engagement (TE) assays for key E3 ligase including CRBN, VHL, XIAP, cIAP, and MDM2. These intracellular assays are the first biophysical method to enable the quantitative determination of compound occupancy, potency, and residence time for specific target proteins inside living cells. The assays provide a direct measure of compound binding or occupancy to a target under physiological conditions using bioluminescent resonance energy transfer (BRET). Citation Format: James Vasta, Cesear Corona, Jennifer Wilkinson, Morgan R. Ingold, Chad Zimprich, Marie Schwinn, Thomas Machleidt, Jim Hartnett, Mei Cong, Frank Fan, Richard L. Somberg, Matthew Robers. A live cell method to assess E3 ligase and target protein occupancy for PROTACs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6407.