Fig. 2: AZ’6421 degrades ERα through a PROTAC mechanism of action.

a Immunoblots for ERα and vinculin loading control. MCF7 cells were pre-treated with 10 µM MG132 or DMSO control for 1 h before being exposed to DMSO, AZ’6421, or fulvestrant, at the concentrations indicated, for a further 7 h. A representative immunoblot from two biological replicates is shown. b As in a but cells were pre-treated with 10 µM VHL ligand (Ac-(S,R,S)-AHPC) prior to addition of DMSO, AZ’6421 or fulvestrant. c MCF7 cells expressing BFP-Cas9 were reverse transfected with the crRNA:tracrRNA pool targeting VHL for 72 h prior to treatment with increasing concentrations of AZ’6421 for a further 24 h. Cells were then fixed, stained, and imaged to assess ERα and Cas9 expression. ERα levels in Cas9 positive cells were normalised to unedited cells (0) and ESR1 KO cells (-100). Data points are from three independent experiments, points in bold represent the mean. d Structure of AZ’6421 and the (S)-prolinol epimer, 7. e Concentration-response curves for AZ’6421 and 7. ERα levels in MCF7 cells were assessed using an immunofluorescence endpoint after 24 h exposure to compound. Individual data points were normalised to vehicle control (0% inhibition) and 100 nM fulvestrant (100% inhibition). Data points are from 5 (AZ'6421) or 2 (Cpd 7) independent experiments with technical duplicates performed for each experiment, bold points represent the mean. All concentration-response curves were plotted using nonlinear regression analysis in GraphPad Prism.