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NanoBRET™ in Live Cells as a Method to Assess E3 Ligase and Target Protein Occupancy for PROTACs

Vasta, J. D. et al., Promega Corporation


AACR 2020 Abstract #6407

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 cell permeability and E3 ligase occupancy in living cells, we have developed a panel of NanoBRET™ target engagement (TE) assays for key E3 ligase including CRBN, VHL, XIAP, cIAP, and MDM2. NanoBRETtarget engagement (TE) 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 using bioluminescent resonance energy transfer (BRET). This method has previously been applied to several other protein classes including kinases, bromodomains, and HDACs.

Here, we demonstrate that the NanoBRET platform allowed assessment of PROTAC cell permeability & E3 ligase occupancy. Using NanoBRET TE assays for proteins targeted by the E3 ligases, we obtained intracellular PROTAC binding kinetics. We further extend the analysis of PROTAC permeability as a dynamic process in real-time, using BRD-targeting MZ1 and dBET1 as a model system. Together these approaches allow a mechanistic interrogation of intracellular PROTAC permeability, E3 ligase occupancy, target occupancy, and target residence time.

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