Fig 1: Designed Raman-active CRBN based PROTACs are potent BET degraders. (a) Chemical structures of BET bromodomain inhibitor (+)-JQ1, CRBN ligands lenalidomide and 1, and Raman-active probe BADY. (b) Designed Raman-active PROTACs LS1–LS4 bearing CRBN E3 ligase ligands. (c) Representative western blot after 24 h treatment of HeLa cells with DMSO, MZ1, cisMZ1, LS1, LS2, LS3 and LS4. (d) BRD4 quantification by western blot following the treatment conditions described in (c). Bars represent the mean ± SD from n = 3 biological replicates. Statistical analysis was performed using one-way ANOVA compared to DMSO; **** p < 0.0001. (e) Quantitative whole proteome analysis of HeLa cells after 6 h treatment with 30 nM of LS1 or LS2, compared to DMSO treatment (n = 4 biological replicates).
Fig 2: LS1 and LS2 form stable ternary complexes. (a) Modelled structure of the ternary complex formed between BRD4BD1 (green), LS1 (cyan) and CRBN (magenta). The structure shown is a representative snapshot taken from a 50 ns molecular dynamics simulation. (b) Schematic representation of the SPR binding assay used to measure the kinetics of ternary complex formation. (c) Representative SPR sensorgrams depicting the ternary complex binding kinetics for LS1:BRD4BD1 (left) and LS2:BRD4BD1 (right). (LS1: n = 2 independent experiments, LS2: n = 1).
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