Ester 23 gave a 3.4-fold increase in the degradation of BRD4 than amide 1 (Dmax 50 = 11 vs 37 nM, respectively) (Table ). BRD4 degradation was increased by a further 3.7-fold when further switching from To determine whether our heterotrivalent PROTACs could drive antiproliferative effects, we evaluated the cytotoxicity of compounds 23 -32 in cell viability assays performed in three different cell lines: RKO, KBM7 and K562 (Supplementary Figure & Supplementary Table ). The results of this assay shows that the cytotoxicity of the compounds, as measured by their EC50 values, follows the same trends observed in the western blot and HiBiT data. This further indicates that degradation is the major driver of cytotoxicity for these compounds, and that most of the second generation heterotrivalent PROTACS are potent cytotoxic compounds, with 26 and 27 standing out as the most cytotoxic across cell lines (Supplementary Figure & Supplementary Table ). The degradation profiles from both western blot and live cell HiBiT assay and the cell viability data indicate that compounds 26 and 27 are the most potent BET degraders of the series. We therefore wanted to further discriminate between these two compounds by evaluating their cell antiproliferation activity in BET sensitive RKO wild-type (WT) and CRBN and/or VHL KO/dKO cell lines (Figure & Supplementary Table ).