Extended Data Fig. 6: Rational design of improved intramolecular bivalent glue BET degraders.

a, Structure of double JQ1 containing intramolecular bivalent glue degrader IBG2. b, HiBiT degradation assay. HEK293 HiBiT knock-in cells were treated with IBG1, IBG2 or IBG3 for 5 h and levels of BRD2-, BRD3- and BRD4-HiBiT proteins were quantified via HiBiT lytic detection system. Data, n = 3 independent experiments, mean +/− s.d. c, BET protein degradation specificity. KBM7 cells expressing BRD2^Tandem or BRD3^Tandem dual fluorescence reporters were treated with increasing concentrations of IBG1, IBG3 or dBET6 for 6 h and BET protein levels were quantified via flow cytometry. d, Size exclusion chromatograms of BRD4^Tandem incubated with DMSO, MT1, IBG1 or IBG3. Data for DMSO and IBG1 as in Fig. 4c, data representative of n = 2 independent experiments. e, Bromodomain tandem selectivity. KBM7 cells expressing isolated BRD4 bromodomains or mutated BRD4^Tandem constructs were treated with IBG1 (1 nM), IBG3 (0.1 nM) or dBET6 (10 nM) for 6 h and protein levels were evaluated via flow cytometry. f, BRD4 stability CRISPR screen. KBM7 iCas9 BRD4 dual fluorescence reporter cells expressing a CRL-focused sgRNA library were treated with IBG3 (0.1 nM) for 6 h before flow cytometric cell sorting as in Fig. 2b. 20 S proteasome subunits (blue), COP9 signalosome subunits (cyan) and E1 or E2 ubiquitin enzymes (purple) inside the scoring window (one-sided MAGeCK p-value < 0.01, fold-change > 1.5; dashed lines) are highlighted. g, DCAF16 dependency. BRD4(S) dual fluorescence reporter KBM7 iCas9 cells were lentivirally transduced with a DCAF16-targeting sgRNA and 3 days post Cas9 induction cells were treated with DMSO, IBG1 (1 nM) or IBG3 (0.1 nM) for 6 h before FACS-based quantification of BRD4 levels. h, Bromodomain arrangement. KBM7 cells expressing dual fluorescence reporters harbouring tandems of either BD1 or BD2 of BRD4 were treated with DMSO, IBG1 (1 nM), IBG3 (0.1 nM) or dBET6 (10 nM) for 6 h and analysed by flow cytometry. i, j, Structures (i) and HiBiT-BRD4 degradation activity (j) of bivalent BET inhibitors MT1 and MS645 after treatment for 24 h. Data for c,e,g,h, n = 3 independent experiments, mean +/− s.d. Data in j, mean of n = 2 independent experiments.