Initial work with multi-specific drug modalities has been transformative for drug discovery and greatly expanded the therapeutic landscape . This trivalent compound concept is not limited to E3 ligase recruitment, and could find utility beyond PROTACs for emerging approaches for small-molecule induced proximity . We thus anticipate broad applicability of the approach to improve performance of a wide range of multi-specific agents and modalities for chemical biology and pharmaceutical development. 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.