Fig. 3: IBG1 enhances the intrinsic interaction between the tandem bromodomain region of BRD4 and DCAF16.
From: Targeted protein degradation via intramolecular bivalent glues
a, ITC measurement of DCAF16–DDB1(ΔBPB)–DDA1 binding to pre-incubated BRD4Tandem–IBG1 complex (1:1.1 molar ratio). Data representative of n = 2 independent experiments. DP, differential power. b, TR-FRET ternary complex-formation assay. Europium-labelled anti-His bound to BRD4Tandem was incubated with equimolar Cy5-labelled DCAF16–DDB1(ΔBPB)–DDA1 and increasing concentrations of IBG1 or JQ1. Mean ± s.d. of n = 3 technical replicates. c, TR-FRET complex-stabilization assay. His-tagged BRD4Tandem- or BRD4BD1 (200 nM) bound to anti-His–europium was incubated with increasing concentrations of Cy5-labelled DCAF16–DDB1(ΔBPB)–DDA1 in the presence or absence of 1 µM IBG1. Mean ± s.d. of n = 2 independent experiments, each with 2 technical replicates. The asterisk denotes a datapoint that was excluded from non-linear regression fitting. d,e, UV chromatograms from SEC analysis. DCAF16–DDB1(ΔBPB)–DDA1 and BRD4Tandem alone or mixed at a 2:1 molar ratio in the presence of excess IBG1 (d), DCAF16–DDB1(ΔBPB)–DDA1 and BRD4Tandem mixed at a 1:1 molar ratio in the absence or presence of excess IBG1 (e), or DCAF16–DDB1(ΔBPB)–DDA1 mixed with BRD4BD1 and BRD4BD2 at a molar ratio of 1:1:1 with excess IBG1 (e) were run on an S200 10/300 column. Data representative of n = 2 independent experiments. mAU, milli-absorbance units. f, BET protein stability reporter assay. Tandem mTagBFP fusions with BRD2, BRD3 or BRD4 bromodomains, isolated BRD4 bromodomains or bromodomain chimeras were expressed in KBM7 cells and protein stability was quantified by FACS following treatment with DMSO, IBG1 (1 nM) or dBET6 (10 nM) for 6 h. Mean ± s.d. of n = 3 independent experiments.
