Fig. 5 |. Degradation score as metric to nominate the imide analogues with reduced off-targets and cellular target engagement studies.
a, Plot showing pair-wise comparison of GFP degradation levels induced by pairs of SNAr pomalidomide analogues with C4 and C5 modifications (Wilcoxon matchedpairs signed-rank test; P = 0.0029) at the same dose. Data are the mean of at least three independent replicates. Centre of the box plot is median. Minima and maxima are 1.5 times the interquartile range over the 75th and under the 25th percentile, respectively. b, Degradation of pomalidomide-sensitive ZF degrons inside cells by pomalidomide analogues with and without H-bond donor(s) immediately adjacent to the phthalimide ring. Data are the mean of at least three independent replicates. (two tailed Welch’s t-test; P < 0.0001). Centre of the box plot is median. Minima and maxima are 1.5 times the interquartile range over the 75th and under the 25th percentile, respectively. c, Representative immunoblots (from at least two independent replicates) for endogenous ZF proteins ZFP91 and IKZF3 in Jurkat cells treated with pomalidomide analogues (21 and 22) with and without a H-bond donor (NH) immediately attached to the phthalimide ring. d, Scatter dot plot showing ZF degradation scores (that is, −log(degradation sum + 1)) for individual pomalidomide analogues and pomalidomide-based PROTACs investigated in this study. Data are the mean of at least three independent replicates. (C5 -F indicate fluoro substitution on C5) e, A competition-based NanoBRET assay for CRBN engagement in cells by the pomalidomide analogues. f, Dose-dependent CRBN binding curves of selected analogues determined in U2OS cells using NanoBRET-based intracellular CRBN binding assay. Data are the mean of at least four independent replicates. g, BRET ratio for selected analogues treated at 1 μM dose in a NanoBRET based ternary complex formation assay (for the ternary complex analysis of all the imide analogues generated in this study, see Supplementary Fig. 9). Data are the mean two independent replicates.