Figure 4. SIM1 induces a conformation change in BRD4 intramolecularly engaging its BD1 and BD2 to form a 1:1:1 ternary complex with VHL.

a) Size exclusion chromatography of complex formation after incubation of SIM1 (red), MZ1 or cis-SIM1 (orange), MT1 (green) or DMSO (cyan) with BD1-BD2 tandem domain from BRD4 (left panel: wild type, middle panel: N140F mutant, right panel: wild type with VCB protein). Intensity of peaks is absorbance at 280 nm. b) NanoBRET conformational biosensor assay consisting of either the BD1-BD2 tandem domain of BRD4 wild-type (WT) or containing the BD2 N433F mutation flanked by NanoLuc donor and HaloTag acceptor fusion tags. HEK293 cells were transiently transfected with either the WT or N433F mutant biosensor and treated with a serial dilution of SIM1, cis-SIM1, or MT1 compounds. NanoBRET was measured to determine a change in tag proximity indicative of a conformational change. Data are presented as mean values with error bars representing the SD of technical quadruplicates. For treatments which showed a conformational change, EC₅₀ values were calculated and are shown. c) NanoBRET target engagement assays of HEK293 cells transiently transfected with the VHL-NanoLuc fusion in permeabilized and live cell formats. Cells were treated with a fluorescent VHL tracer then incubated with the indicated compounds across the indicated concentration range to measure competitive displacement. Fractional occupancy is plotted against concentration and from these graphs, IC₅₀ values for each compound are shown for both permeabilized and live cells. Data are presented as mean values with error bars representing the SD of technical triplicates. d) ITC titrations of BRD4 BD1-BD2 tandem proteins (loaded in the syringe, N-to-F mutants at 300μM, WT 200μM) into a 1:1 mixture of SIM1 (16μM) and VCB (32μM) pre-incubated into the sample cell. Binding parameters from data fit are shown for each titration. The high binding affinity to WT was not resolvable due to competing equilibria during the titration. e) NanoBRET kinetic ternary complex formation in HEK293 cells transiently expressing HaloTag-VHL paired with either full-length BRD4 WT, N140F or N433F mutants treated with SIM1, cis-SIM1, MT1 or DMSO control. NanoBRET was continuously monitored for 2h after compound addition and showed differential levels of ternary complex formation for each BRD4 variant. Data are presented as mean values with error bars representing the SD of technical quadruplicates.