Fig. 4: BRD9 and warhead stereochemistry facilitate covalent modification of Cysteine 58 on CRL4^DCAF16.

a BRD9 degradation in MV4-11 cells following 6 h treatment with AMPTX-1-ent-1 (red) and AMPTX-1-ent-2 (blue). BRD9 levels were quantified by immunofluorescence. Data are represented as mean ± standard deviation from n = 3 biological replicates. b Table showing relative abundance (%) for each species, calculated using the peak area for unmodified DCAF16 (24,919.7 Da, corresponding to the molecular weight of the most abundant post-translationally modified state of DCAF16) as a reference m/z. Relative abundances for 1, 2 and 3 sites were then calculated using 24,919.7 + 692 Da, 24,919.7 + 1384 Da, and 24,919.7 + 2076 Da, respectively. c Intact MS deconvolution spectra of DCAF16 protein from samples prepared using 3.6 µM DCAF16-DDB1 alone; 3.6 µM DCAF16-DDB1 and 18 µM AMPTX-1-ent-1; or 3.6 µM DCAF16-DDB1, 18 µM AMPTX-1-ent-1, and 3.6 µM BRD9^BD. Samples were incubated at room temperature for 2 h prior to MS analysis. The different deconvolution peaks of DCAF16 correspond to multiply-phosphorylated protein species. d Intact MS spectra corresponding to a sample prepared using 3.6 µM DCAF16^C58S-DDB1, 18 µM AMPTX-1-ent-1, 3.6 µM BRD9^BD, 2 h incubation at room temperature prior to MS analysis. e BRD9 degradation following 6 h treatment of AMPTX-1-ent-1 in MCF-7 wildtype (DCAF16-WT, red) or MCF-7 cells containing a C58S mutation in DCAF16 (DCAF16 C58S, green). BRD9 levels were quantified by immunofluorescence. Data from n = 2 biological replicates are shown.