Extended Data Fig. 5 ∣. Characterization of the IMiDs and the indicated peptides in HEK293T cells and multiple myeloma MM.1S cells.
(a) Model of dipeptide degraders with CRBN built from 6BOY and 6H0F. Left: Model of JQ1-FcQ (yellow) by molecular modeling of dBET6 (orange) in the ternary complex with CRBN (grey) and BRD4 (blue). Middle: zoom-in of the CRBN binding pocket with thalidomide analog (orange), Ac-FcQ (yellow), or Ac-FcN (green). Right: Model of pomalidomide (orange), FcQ (yellow), and FcN (green) in the ternary complex with CRBN (grey) and IKZF1 (blue, space-filling mode). (b) Structures of Boc-cQ, Me-FcQ, Ac-FcQ, H2N-FcN or H2N-FcQ, Boc-FcN or Boc-FcQ, and GGGFcQ. (c–d) Quantitative proteomics of co-immunoprecipitated proteins from lysates of HEK-CRBN cells overexpressing IKZF1 after 2 h incubation with 1 μM (c) FcN or (d) GGGFcQ. Protein levels were normalized to the amount of CRBN in each channel. P-values for the abundance ratios were calculated by one-way ANOVA with TukeyHSD post-hoc test. (e) Competitive inhibition of BRD4 degradation by the indicated dipeptide degrader (JQ1-FcN, JQ1-FcQ) with the indicated compounds in HEK293T cells for 4 h. All western blot data are representative of 2 independent replicates. (f) Quantitative proteomics of MM.1S cells after treatment with 10 μM of Boc-FcN for 10 h. P-values for the abundance ratios were calculated using the t-test (background) method. (g) Protein expression levels of IKZF1 after treatment with the indicated compounds in MM.1S cells for 10 h. Data are presented as mean ± SD (n = 3 biologically independent samples). (h) Quantitative proteomics of HEK293T cells after treatment with 0.1 μM of JQ1-FcQ or dBET6 for 2 h. P-values for the abundance ratios were calculated using the t-test (background) method. (i) Protein expression levels of BRD2 and BRD4 after treatment with the indicated compounds in HEK293T cells for 2 h. Data are presented as mean ± SD (n = 3 biologically independent samples). All proteomics experiments were performed in biological triplicates. For uncropped western blot images, see Supplementary Fig. 10.