Fig. 4. Bifunctional compounds bind covalently to DCAF11 and display antiproliferative activity.

a Structures of 15 and bifunctional compounds 16 and 17 with saturated double bond. b Structure of probe 18 with BODIPY as fluorophore. c BRD2 levels in KBM7 cells treated with compounds 9 and 16 for 4 h. Representative result of n = 3. d BRD2 levels in Jurkat cells treated with PROTAC MZ1 or 9 (10 μM, 5 h) prior to washout and further incubation for the indicated time. Representative results of n = 3. e BRD2 levels in Hep G2 cells pretreated with 30 μM 15 for 1 h followed by 10 μM of compound 9 for another 4 h. Representative result of n = 2. f Fluorescence labelling of DCAF11. Purified DCAF11 protein was incubated with 18 for 40 min followed by analysis of in-gel fluorescence. Representative result of n = 3. g In-Cell Western for BRD2 levels in Hep G2 cells treated with 9 for 4 h. Representative results (n = 4 biological replicates). h Relative BRD2 protein levels as detected using In-Cell Western in Hep G2 cells pretreated with compounds 2, 19-47 or JQ1 (structure shown in Supplementary Table 1) at 30 μM or MLN4924 at 1 μM for 1 h, followed by 9 (10 μM, 4 h) or DMSO addition. Data are mean values ± SD (n = 3 for JQ1 and MLN4924, n = 4 for 19–45, n = 5 for DMSO, 2, 46–47; n = biological replicates). i Structures of representative indolinones (46, 47) and corresponding bifunctional compounds (48, 49). j BRD2 levels in Jurkat cells treated with 9, 48 or 49 at 3 μM for 6 h. Data are mean values ± SD (n = 4 biological replicates). Statistical significance was calculated with unpaired two-tailed Student’s t-tests comparing 10 to 48 or 49 (P = 0.0002 or 0.0003 respectively; ***P < 0.001). k Influence on cell viability. Data are mean values ± SD (n = 3 biological replicates). The data of GI₅₀ are shown in Supplementary Fig. 7h. l Caspase-3/7 activity for apoptosis detection. Data are mean values ± SD (n = 3 biological replicates).