In forming ternary complexes with VHL and BET proteins, SIM1 exhibited a positive cooperativity α value of 3.5 as shown in competitive AlphaLISA assays (Fig. ), measuring binding of SIM1 alone (IC50 = 205nM) or SIM1:VCB binary complex (IC50 = 58nM) via the displacement of biotin-JQ1 41 from BRD4. As a cross-validation, we evaluated cooperativity in a competitive FP assay measuring binding at the VHL end of the PROTAC molecule via the displacement of a fluorescent HIF-1α peptide probe . In this experiment too SIM1 exhibited positive cooperativity from enhanced affinity of its competitive displacement once pre-engaged with BRD2 or BRD4 tandem ( = 4.4 or 7.3, respectively) compared to SIM1 alone (Fig. ).