Figure 8

CRBN binding and E3 inhibition by thalidomide derivatives. (a) Competitive elution assay with racemic thalidomide-immobilized beads. Glutarimide binds FLAG-CRBN with an affinity similar to that of (R)-thalidomide, but weaker than that of (S)-thalidomide. Phthalimide exhibits no binding. FLAG-CRBN was processed as in Fig. 1b. (b) Effect of glutarimide binding on CRBN auto-ubiquitylation by CRBN-containing E3 ubiquitin ligase CRL4. The method used was that described in Fig. 1c. (c) Competition binding assay using thalidomide-immobilized beads. The eluate with 1 mM thalidomide and glutarimide yielded bands, but not with other compounds. The intact glutarimide ring in a relaxed six-membered ring conformation is important for CRBN binding with the imide group serving as both a hydrogen donor and acceptor. Correspondingly, glutarimide still binds CRBN, although phthalimide and glutaric anhydride, which lack the ring amide of glutarimide, exhibited no binding. Both carbonyl groups of glutarimide are important, and the absence of one carbonyl group results in loss of binding (δ-valerolactam). Bulky modification of the glutarimide C4-carbon atom also results in loss of CRBN binding due to steric clash (cycloheximide). (d) Chemical structures of compounds utilized in the binding assay. Full-length blots in (a), (b) and (c) are presented in Supplementary Fig. 10.