Fig. 6: Repurposing etacrynic acid as an E3 recruiter for targeted protein degradation.
From: Proteome-wide ligandability maps of drugs with diverse cysteine-reactive chemotypes
a Heat map showing RH/L values of representative cysteines engaged onto E3 ubiquitinating enzymes. NA: not detected. b Chemical structure of EtA-(S)-JQ1. c Representative western blots showing the effect of EtA-(S)-JQ1 treatment on BRD4 expression in HEK293T cells. d Bar chart showing the effect of EtA-(S)-JQ1 (100 nM) on BRD4 mRNA expression levels in HEK293T cells over the indicated time points. Data are mean ± s.d. (n  =  3, two-sided Student’s t-test, p  <  0.05 considered significant). e Representative western blots showing that bortezomib or etacrynic acid attenuated EtA-(S)-JQ1-mediated BRD4 degradation. HEK293T cells were pre-treated with DMSO, the proteasome inhibitor bortezomib (BTZ) (1 μM, 2 h) or etacrynic acid (20 μM, 2 h), followed by EtA-(S)-JQ1 treatment (100 nM, 4 h). f Representative western blots showing that EtA-(S)-JQ1, but not JQ1 (20 μM, 4 h), induced BRD4 degradation in HEK293T cells. MZ1 was used as a positive control. g Chemical structures of 11, a non-covalent version of EtA-(S)-JQ1, and 12, the same molecule as EtA-JQ1 but with the (R)-(-)-JQ1 enantiomer as the binding moiety. h Representative western blots showing that EtA-(S)-JQ1, but not JQ1 (20 μM, 4 h), nor 11/12, induced BRD4 degradation in HEK293T cells. i Volcano plot showing the log2 ratio of the EtA and EtA-(S)-JQ1 (5 μM) treated channels and the -log10(adj. pvalue) for all quantified proteins (n  =  3, LIMMA-test). j Mapping of EtA-(S)-JQ1-engaged cysteine (C1368) and active site cysteine (C2579) on the 3D structure of HECTD1, predicted by AlphaFold2. k Representative western blots showing the effect of hectd1 knockdown on EtA-(S)-JQ1 (100 nM, 4 h)-induced BRD4 degradation in HEK293T cells. l Representative western blots showing the effect of dcaf16 knockdown on EtA-(S)-JQ1 (100 nM, 4 h)-induced BRD4 degradation in HEK293T cells. m Representative western blots showing that the C1368S mutation of HECTD1 abolished EtA-(S)-JQ1 (100 nM, 4 h)-induced BRD4 degradation in HEK293T cells (100 nM, 4 h). Quantification data from three independent experiments for panels (c, e, f, h, k–m) are provided in Supplementary Fig. 17. Source data are provided as a Source Data file.
