A High-Throughput Method to Prioritize PROTAC Intracellular Target Engagement and Cell Permeability Using NanoBRET.

Target engagement and cell permeation are important parameters that may limit the efficacy of proteolysis-targeting chimeras (PROTACs). Here, we present an approach that facilitates both the quantitation of PROTAC binding affinity for an E3 ligase of interest, as well as the assessment of relative intracellular availability. We present a panel of E3 ligase target engagement assays based upon the NanoBRET Target Engagement platform. Querying E3 ligase engagement under live-cell and permeabilized-cell conditions allow calculation of an availability index that can be used to rank order the intracellular availability of PROTACs. Here we present examples where the cellular availability of PROTACs and their monovalent precursors are prioritized using NanoBRET assays for CRBN or VHL E3 ligases.