Molecular glue CELMoD compounds are regulators of cereblon conformation

Abstract

Cereblon (CRBN) is a ubiquitin ligase (E3) substrate receptor protein co-opted by CRBN E3 ligase modulatory drug (CELMoD) agents that target therapeutically relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBN's thalidomide-binding domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We performed cryo-electron microscopy analyses of the DNA damage-binding protein 1 (DDB1)-CRBN apo complex and compared these structures with DDB1-CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an open conformation to the canonical closed conformation. The neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy and informing structure-guided design strategies to improve therapeutic efficacy.

Fig. 1.. CRBN^open is allosterically modulated to CRBN^closed by pomalidomide.

(A) ~3.5-Å-resolution cryo-EM reconstruction of CRBN-DDB1 in the unliganded apo form. The Lon domain (tan) is separated from the TBD (red), while the helical bundle (HB, light red) mediates interaction with DDB1, composed of beta propeller A (BPA, light blue), beta propeller B (BPB, medium blue), and beta propeller C (BPC, steel blue). CRBN and DDB1 are thresholded independently to illustrate important features. To the right, a ribbon representation of the CRBN-DDB1 complex modeled from density is shown (same color scheme as on the left). The sensor loop is denoted as a dotted magenta line extending away from the TBD domain. (B) The unsharpened cryo-EM map shows the path of the sensor loop, which interacts with the HB and BPA of DDB1. (C) Surface representation of the ~3.9-Å-resolution cryo-EM reconstruction of CRBN-DDB1 in the closed form in complex with pomalidomide. The N-terminal belt (orange) wraps around the TBD domain. To the right, a ribbon representation of CRBN-DDB1^ΔBPB in complex with pomalidomide is shown, with the sensor loop (magenta) adopting a β-hairpin organization that is tightly packed between the TBD and Lon domains. (D) A detailed view of the density corresponding to pomalidomide (green) and the sensor loop (magenta) in CRBN^closed.

Fig. 2.. Improved CELMoD compounds drive CRBN^closed and recruit Ikaros.

(A) The CRBN^open transition to CRBN^closed is differentially regulated between pomalidomide (~20% of particles adopt CRBN^closed) and the CELMoD agent iberdomide (~50% of particles adopt CRBN^closed). (Top right) The ~3.9-Å-resolution cryo-EM structure of pomalidomide-induced CRBN^closed. (Bottom right) The ~3.7-Å-resolution reconstruction of iberdomide-induced CRBN^closed. The inset panels on the far right depict surface representations of published crystal structures illustrating the ligand (green) interactions with sensor loop (magenta). (B) Colored space-filling representation of CRBN models with residue-specific coloring according to changes in solvency upon addition of iberdomide as detected by HDX-MS. Orange residues correspond to peptides with mild HDX differential, and red residues correspond to peptides with large HDX differential. Mixed conformations are expected during the experiment, so CRBN^open and CRBN^closed are both shown. (C) The ~3.6-Å-resolution reconstruction of iberdomide-induced CRBN^closed in complex with Ikaros ZF1-ZF2-ZF3 (gold). DDB1 and CRBN are thresholded independently to illustrate important features.

Fig. 3.. DDB1 and next-generation CELMoD agents further poise CRBN substrates for ubiquitination in disease contexts.

(A) Three-dimensional classification of unliganded CRBN-DDB1 yields three discrete positions of DDB1’s mobile BPB propeller (colored blue). (Left) ~3.5-Å-resolution cryo-EM reconstructions of CRBN-DDB1 with BPB in a linear (left), hinged (middle), or twisted (right) position. (Right) Structural models in similar orientations for reference, adopting a linear (PDB ID 4CI1, left), hinged (PDB ID 5HXB, middle), or twisted (PDB ID 2HYE, right) position. (B) The ~3.2-Å-resolution cryo-EM reconstructions of CRBN-DDB1 complexed with mezigdomide with a linear (left), hinged (middle), or twisted (right) position of BPB. (Inset) ~3.1-Å-resolution focused refinement of particles from all three orientations reveal a connection between mezigdomide and the Lon domain, “stapling” the CRBN^closed conformation. (C) The ~3.1-Å-resolution reconstruction of mezigdomide-induced CRBN^closed bound to Ikaros ZF1-ZF2-ZF3. The inset shows a focused view of connection between mezigdomide and the Lon domain, stapling CRBN^closed in the presence of Ikaros. (D) In vitro ubiquitination assay tracking MBP-Ikaros ubiquitination in the presence of different molecules used in this study (labeled on the right). (E) Mechanistic model illustrating pathway of assembly. First, unliganded CRBN is open with the sensor loop attached to HB. Second, ligand is added and associates with TBD in the hydrophobic pocket. Third, binding of ligand in the tri-Trp pocket stabilizes the sensor loop as a β-hairpin detached from the HB. Fourth, sensor loop refolding promotes transition of CRBN^open to CRBN^closed without an observed intermediate, and the N-terminal belt is seated (orange). Fifth, substrate is recruited to the CRBN^closed conformation for subsequent ubiquitination by CRL4.