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. Author manuscript; available in PMC: 2024 Mar 5.
Published in final edited form as: Nat Chem. 2023 Dec 18;16(2):218–228. doi: 10.1038/s41557-023-01379-8

Fig. 2 |. Validation of image-based results using NanoBRET and immunoblotting.

Fig. 2 |

Structural insights and degradation potential of the thalidomide analogues with C4 and C5 amino groups on the phthalimide ring. a, A NanoBRET assay to quantify ternary complex formation between ZFP91, CRBN and imide analogues or PROTACs. b, BRET ratio of reported PROTACs treated at 1 μM dose in a NanoBRET assay. Data are the mean of two independent replicates. c,d, Representative immunoblots (from at least two independent replicates) demonstrating off-target degradation of endogenous ZF protein ZFP91 by MS4078 (ALK PROTAC) (c) and dTAG-13 (FKBP12F36V PROTAC) (d) in a dose-dependent manner in Jurkat cells. e, Crystal structure showing the glutarimide ring deeply buried in the CRBN exposing the 4-amino group of pomalidomide, which makes a crucial water-mediated hydrogen bond between CRBN (E377) and IKZF1 (Q147). Modification on C5 position would potentially bump off the ZF degrons (PDB: 6H0F). f, Structures of pomalidomide (1) and 5-aminothalidomide (8). g, Representative immunoblots (from at least two independent replicates) of endogenous ZF proteins ZFP91 and IKZF3 in MM1.S cells treated with pairs of imide-based analogues with C4 (pomalidomide) and C5 amino modifications on the phthalimide ring.