Fig. 3: Target engagement for PROTACs in cells.

a Based on quantitative western blots (Supplementary Fig. 3) and assuming the MOLM-14 cell volume is ~1000 µm³, BTK and CRBN concentrations are 780 and 13 nM, respectively. The weak binding between pomalidomide and CRBN suggests that CRBN binding is the limiting factor for ternary complex formation. b CRBN in-cell target engagement assay. HEK-293 cells were transiently transfected with plasmids expressing a fusion protein of CRBN and nano-luciferase (nLuc) for 24 h and then the cells were treated with a CRBN tracer (0.5 µM), which binds to CRBN to induce bioluminescence resonance energy transfer (BRET). Adding PROTACs to cells would compete the CRBN tracer binding to CRBN, thus reducing the NanoBRET signals. It should be noted that the NanoBRET assay is ratiometric and independent of the expression level of the nLuc fusion protein. The target engagement IC₅₀ values for RC-1 (blue circle), IRC-1 (violet pyramid), and RNC-1 (green inverted pyramid) are 0.25, 0.86, and 1.69 µM, respectively. c BTK in-cell target engagement assay. This assay is the same as in b, except BTK-nLuc fusion plasmid and BTK tracer (1.0 µM) were used. The target engagement IC₅₀ values for RC-1 (blue circle), IRC-1 (violet pyramid), and RNC-1 (green inverted pyramid) are 0.043, 0.13, and 1.27 µM, respectively. d The same BTK in-cell target engagement assay as in c was applied to RC-Ctrl (blue circle), RC-1 (violet pyramid), DD-03-171 (green inverted pyramid), and MT-802 (brown square). The target engagement IC₅₀ values for RC-1 and RC-Ctrl are the same within experimental errors, demonstrating that the intracellular accumulation of RC-1 is similar to its parent warhead molecule. In contrast, the target engagement IC₅₀ values for DD-03-171 and MT-802 are 5 and 11 folds of that of RC-1, respectively. Triplicates were performed with SEM as the error bars. e Chemical structures of IRC-1-DiMe and RNC-1-CN-DiMe. f BTK target engagement for RC-1 (blue circle), IRC-1-DiMe (violet pyramid), and RNC-1-CN-DiMe (green inverted pyramid). The same BTK in-cell target engagement assay as in c was applied to RC-1, IRC-1-DiMe, and RNC-1-CN-DiMe. The IC₅₀ values for RC-1, IRC-1-DiMe, and RNC-1-CN-DiMe are 0.033, 0.38, and 0.93 µM, respectively. g Chemical structures of FLT3 degraders. h CRBN in-cell target engagement assay as in b. The target engagement IC₅₀ values for pomalidomide (blue circle), RC-FLT3 (brown square), IRC-FLT3 (violet pyramid), and RNC-FLT3 (green inverted pyramid) are 0.3, 0.3, 1.5, and 10.1 µM, respectively. Data are presented as mean values ± SEM (n = 3 biologically independent samples). Source data are provided as a Source Data file.