and VHL/CRBN, we monitored live cell ternary complex formation using NanoBRET (Figure ). 23 In this assay, the endogenously tagged HiBiT-BRD4 complemented with LgBiT served as the energy donor and transiently expressed HaloTag-CRBN or HaloTag-VHL served as the energy acceptor. A NanoBRET signal is observed when the donor and acceptor are in close proximity, making it ideal to measure cellular ternary complex formation and stability. 11, 23 We treated HEK293 HiBiT-BRD4 (LgBiT stable) cells that were transiently expressing either HaloTag-VHL or HaloTag-CRBN with a pre-treatment of proteasome inhibitor, MG132, 39 followed by varying concentrations of 26 and 27, and monitored the NanoBRET signal over 3.5 h (Figure ). Encouragingly, both 26 and 27 can engage and form ternary complexes between BRD4 and either VHL or CRBN, with each compound showing slightly faster association kinetics for CRBN, plateauing after just 30 min. Interestingly, 27 gave more robust dose-response with both VHL and CRBN than 26, suggesting that 27 may form a more stable and/or more highly populated ternary complex. This is likely to be one of the reasons why 27 is a more rapid and potent BRD4 degrader, evidencing that increased ternary complex population and stability positively correlates with the amount of ubiquitination and subsequent degradation. 23 To further understand whether these improvements may be attributed to enhanced intracellular availability of 27 relative to 26, we assessed the binary target engagement of 26 and 27 to either CRBN or VHL using a lytic and live cell NanoBRET target engagement assay (Figure ). 23, 40 190 nM and 412 nM, respectively) in lytic cell format (Figure ). In live cell format, 27 engages CRBN 2.5fold more strongly over 26, again indicated that 27 is more cell permeable than 26.