Figure 6. ZL lead compounds are potent and highly specific BRD4 inhibitors.

(A). Specificity of BRD4 inhibitors to BRD4 BDs was determined using TR-FRET assays. Data shown as the IC₅₀ values of tested BRD4 inhibitors to the BRD2 and BRD4 BDs separately. (B). IC₅₀ values of ZL lead compounds. hSAECs were pretreated with indicated series of BRD4 inhibitors from 0.01 nM to 100 µM final concentrations overnight prior to poly(I:C) stimulation (10 µg/ml for 4 h). The IC₅₀ values of each inhibitor were estimated by poly(I:C) induced expression of innate immune genes IL6 and CIG5. (C). BRD4 inhibitors block IL6 expression. Shown as % inhibition of JQ1, MS436, RVX208, ZL0420, and ZL0454 inhibitors on poly(I:C)-induced IL6 expression. The results are from three independent experiments (n=3). (D) hSAECs were preincubated with ZL0420, ZL0454, and JQ1 overnight respectively before poly(I:C) stimulation. After fixation, cells were stained with anti-H3K122ac and phospho-Ser 2 CTD RNA Pol II Abs. (E). Quantifications of total fluorescence intensities on images of immunostaining of H3K122ac and phospho-Ser 2 CTDRNA Pol II in hSAECs. * p<0.01, n = 5. (F). Left, schematic diagram of the IP-SID-SRM-MS assay. Stable isotope labelled internal control peptides are used to quantify target proteins in a triple quadrupole MS. Right, SID-SRM-MS assay for two BRD4 peptides (BRD4-638 peptide and 717 peptide). (G). IP-SID-SRM-MS for BRD4 associated Pol II, H3, and H4 proteins were normalized by the input protein concentration and presented as native/aqua values. *: p < 0.01 compared to poly(I:C) only and #: p < 0.01 compared to mock treatment, n=3.