BROMOscan displacement titrations by SIM1 and (R,S)-SIM1 from BRD4 (1,2). The amount of bromodomain protein measured by qPCR (Signal; y-axis) is plotted against the corresponding compound concentration in log10 scale (nM, x-axis). Dissociation constants Kd from curve fitting are tabulated. Error values are generated by the GraphPad Prism program and reflect the quality of the fit between the nonlinear least-squares curve and the experimental data. b) AlphaLISA titrations of SIM1 in duplicates against biotin-JQ1:BRD4 BD2 in the absence (red) or presence (blue) of VCB protein. IC50 values from curve fitting are tabulated, together with the resulting cooperativity . Error values are generated by the GraphPad Prism program and reflect the quality of the fit between the nonlinear least-squares curve and the experimental data. c) Fitted curves from FP competition assays measuring displacement of a FAMlabelled HIF-1α peptide from VCB by SIM1 titrated in triplicates, in the presence or absence of tandem BD1-BD2 proteins from BRD2 or BRD4. Kd values from curve fitting are tabulated, together with the resulting cooperativity . d) SPR sensograms to monitor in real-time the interaction of binary complexes SIM1:BRD2(1,2) and SIM1:BRD4(1,2) with immobilized biotin-VCB protein. Sensorgrams shown are for a ternary single-cycle kinetic (SCK) experiments as representative to at least three independent experiments. Values shown are mean ± s.d. from the data fitting of each replicate. Cooperativity is calculated using dissociation constant Kd of SIM1 binary binding to VCB (Extended Data Fig. ). Multiple-cycle kinetic (MCK) ternary data are also shown in Extended Data Fig. . e) Live cell kinetic residence time experiments with BRD2 and BRD4 as measured by NanoBRET target engagement. CRISPR HiBiT-BRD2 or HiBiT-BRD4 cells were incubated with each of the indicated compounds at their pre-determined EC80 values (listed in Methods) followed by addition of a competitive fluorescentlylabeled BET tracer. NanoBRET was measured and is plotted as fractional occupancy over time. From these graphs, residence time rates (Kobs (h -1 ) and half-life (t½ (h)) were determined. Data are presented as mean values with error bars representing the SD of technical triplicates. f) SIM1 exhibits excellent availability and pharmacokinetics exposure in vivo. Mean plasma concentration-time profiles of SIM1 after single intravenous (IV) or subcutaneous (SC) administration (5 mg/kg) to male C57BL/6 mice (n =3) are shown. Further details are in the associated Supplementary Table and Supplementary Data Set 2. Figure . Models of trivalent ternary complex formations and advantages over monovalent and bivalent compounds. a) Proposed mechanism for the formation of a 1:1:1 ternary complex between trivalent PROTAC, VHL and BET protein. Preferential initial binding of the PROTAC to BD2 of BRD4 is followed by conformational change and bidentate binding to BD1. Avidity and cooperativity contribute to formation of a highly stable ternary complex with enhanced residence time at extraordinarily low concentrations of SIM1. b) Shown are different types of degrader-induced ternary complexes, depicted at their varying extents as a function of degrader concentration. A trivalent complex combining avidity with cooperativity shows the highest and most sustained levels of ternary complex formation, with a minimized hook effect. A cooperative bivalent PROTAC complex is next, followed by a non-cooperative bivalent complex. Lastly, the ternary complex induced by molecular glue compounds is shown, which reaches a plateau and unlike PROTACs are not predicted to experience the competitive hook effect at higher concentrations. c) A general model for trifunctional compound-induced ternary complex utilizing a compound with three different warheads (or ligands) to recruit together three distinct protein.