Our experiments corroborated the previously published binding affinities of pan-BET inhibitor (+)-JQ1, with IC50 values 0.046 and 0.024 μM for BRD4-D1 and -D2, respectively, compared to the respective published values of 0.077 and 0.033 μM. In the case of I-BET151, TR-FRET was previously used to determine IC50 values of 0.036 μM and 0.329 μM for BRD4-D1 and -D2, respectively. We obtained similar values in our AlphaScreen assay (see Table ). iBETBD2 was first disclosed by Gilan et al. in 2020, where they reported nanomolar affinity for BRD4-D2 with approximately 1000fold selectivity for BRD4-D2 over BRD4-D1. We recapitulated these results in our assay platform as well. In contrast, ZL0454 and ZL0590 were reported to have nanomolar affinities against BRD4-D1 in a TR-FRET assay, but we were unable to replicate this activity in our AlphaScreen assay. For ZL0590, we saw no binding at micromolar concentrations, while the authors report a BRD4-D1 IC50 of 90 ± 9 nM. This may be the result of a reportedly novel binding site, which is outside of the canonical acetylated lysine binding site. Due to this binding pose, ZL0590 may bind to the bromodomain without displacing the acetylated histone peptide used in AlphaScreen. However, this hypothesis does not explain why ZL0590 could displace a similar peptide in the reported TR-FRET assay. We were unable to replicate the reported binding affinity of the BRD4 selective inhibitor, ZL0454, which was reported to have BRD4-D1 and BRD4-D2 affinities of 49 and 32 nM, respectively. Interestingly, we did measure submicromolar affinity for ZL0454 against BRD2-D2,