We next tested if intracellular compound efficacy was comparable to the established IC50 concentrations by determining the intracellular potency of our inhibitors. A live-cell nano-luciferase bioluminescence resonance energy transfer (NanoBRET) system was employed for initial assays. In this assay, a transiently expressed luciferase-tagged BRD4-D1 fusion (NLuc-BRD4-D1) was used to monitor the competitive displacement of a fluorescently-labelled ligand upon treatment with inhibitor, providing a live-cell competition experiment to determine the intracellular binding of the compounds of interest. As shown in Figure , compound 3 displayed comparable efficacy to I-BET151 with low nanomolar EC50 values of 0.14 and 0.16 μM, respectively. Using the kinetic affinity of the commercial tracer, these were converted to Ki,app, as described previously, supporting low nanomolar binding affinity in cells (Figure ). In contrast, both 1 and 2 were observed to be 10-fold and 20-fold less efficacious than I-BET151 with EC50 = 1.3 μM, Ki, app = 0.50 μM and EC50 = 2.5 μM, Ki, app = 0.94 μM, respectively. Given that this assay monitors cellular BRD4-D1 engagement, the marked difference in the observed affinities of these compounds suggests that 1 and 2 have reduced cell penetrance for engaging BRD4-D1. To test this hypothesis, we used the NanoBRET system to qualitatively assess the binding kinetics of our inhibitors. In this assay format, HEK293T cells expressing Nluc-BRD4-D1 are preincubated with a saturating dose of inhibitor before the tracer is added, and the resulting change in BRET signal is monitored over time. In turn, a longer inhibitor resonance time translates to a slower return of BRET signal when the fluorescent tracer is then added. At saturating doses of both 2 and 3, kinetic traces characteristic of inhibitors with long resonance times were observed compared to DMSO, consistent with their potency in the AlphaScreen assay (Figure ). Saturating concentrations of 1 (10 μM) showed limiting toxicity to cells, similar to TSEC viability, and was not evaluated.