Interestingly cis-SIM1 and SIM1 showed higher EC50 values for BRD4 binding than MT1 (Fig. ). To determine if this is due to a reduced binding affinity of BRD4 and/or reduced permeability, NanoBRET target engagement assays were performed measuring displacement of a fluorescent BET tracer molecule bound to HiBiT-BRD4 . In permeabilized cells, we observed binding of SIM1, cis-SIM1, and MT1 to endogenous HiBiT-BRD4 with near-identical binding affinities and IC50 values (Extended Data Fig. ). However, in live cells, SIM1 showed reduced binding affinity to BRD4 compared to MT1 (Extended Data Fig. ), suggesting the EC50 shift observed in the conformational sensor assay reflects reduced permeability of the trivalent PROTAC relative to MT1. Reduced permeability was also observed relative to VH298, using NanoBRET target engagement with NanoLuc-VHL, however cellular permeability of SIM1 was within 2-fold lower than MZ1 (Fig. ) .