In our prior report, 1 engaged BRD4 at lower concentrations in MM.1S cells through a cellular thermal shift assay (CETSA). We thus used the CETSA assay in HEK293T cells to assess if there was a cell line-dependence in our assays and to validate our NanoBRET assay. Compound 3 stabilized BRD4 at comparable concentrations to I-BET151, whereas both 1 and 2 required micromolar concentrations to significantly stabilize BRD4 (Figure ). Together, these data are consistent with the notion that the imidazole-based inhibitors 1 and 2 have reduced cell permeability following a Caco2 permeability analysis for compound 2 (Supplementary For evaluation of our inhibitors, 1, 2, and 3 as in vivo probes for BET function, we also measured their liver microsome stability, tissue distribution, and pharmacokinetic properties (Figure ). Our first set of experiments measured mouse liver microsome stability of all three compounds, while compound 3 was previously characterized in human liver microsomes. In the imidazole series, both compounds were sufficiently stable with half-lives greater than 50 minutes for mouse liver microsomes. However, 1 remained 2.7-fold more stable compared to 2, indicating a liability from incorporating the pendant ethanol group. The triazole-based inhibitor 3, on the other hand, was more stable in human liver microsomes compared to mouse liver microsomes (117 min versus 10 min, respectively). This result supports further optimization of this scaffold will be needed in future designs, including adding stabilizing isosteres or removing methyl groups which are susceptible to p450-mediated oxidation or demethylation. After assessing first past metabolism, we next turned to assessing in vivo stability and tissue distribution. Due to an observed sex difference in pharmacokinetics, where compounds 1 and 3 displayed lower plasma exposure in female mice as compared to male mice (Supplementary Table ), we mainly limited our analysis and subsequent therapeutic dosing to male mice. All compounds were administered via intraperitoneal (IP) injection at 10 mg/kg. Inhibitor 1 yielded the longest half-life in the liver of 6.8 hours with good liver exposure, while 2 had an intermediate half-life in the liver of 3.1 hours (Figure ).