P07-19

Exploring the Power of Structural Biology on Degrader Discovery

Yifan HU *, Wenjun GUI, Jiaquan WU

Biortus Biosciences Co. Ltd, Biortus Biosciences Co. Ltd
( * E-mail: yifan.hu@biortus.bio )

The emergence of protein degradation as a novel therapeutic modality has garnered significant attention within the drug discovery community. Unlike traditional small molecule drug discovery, where structure-based drug design (SBDD) is extensively utilized for the rational design of hit and lead compounds, the application of structural biology to degrader discovery has thus far been limited to elucidating the binding modes of selected lead PROTACs or molecular glues. This limitation primarily stems from two challenges: the lack of robust protocols for assembling stable degradation complexes, and the suboptimal resolution of these complex structures as determined by either X-ray crystallography or cryo-electron microscopy (cryo-EM).

In this study, we presented a comprehensive analysis of the CRBN E3 ligase-based PROTAC/molecular glue system using both cryo-EM and X-ray crystallography. We successfully resolved the cryo-EM structure of the CRBN-DDB1-PROTAC-target ternary complex at a resolution of 3.1 Å. Additionally, we resolved the X-ray crystallography structure of the CRBN-DDB1-molecular glue-target ternary complex at a resolution of 2.7 Å. Given that the target protein is identical, with the only variable being the compound (PROTAC or molecular glue), we conducted a detailed comparative analysis of the cryo-EM and X-ray crystallography structures, particularly focusing on the binding interfaces of these ternary complexes.

This study highlights several key findings. First, while the high-resolution cryo-EM and X-ray crystallography structures show considerable similarity in the DDB1 domain, they exhibit significant differences in the conformation of the target protein. Second, the binding poses of the PROTAC and molecular glue in both structures provided atomic-level insights into their interactions with CRBN and the target protein. Third, we identified a novel interface between CRBN and the target in the cryo-EM structure that enhances the cooperativity of ternary complex formation; this interface was absent in the X-ray crystallography structure, potentially due to the shorter length of the molecular glue compared to the PROTAC. Finally, we demonstrated the importance of selecting appropriate DDB1 truncations for achieving high-resolution structures. Specifically, the cryo-EM structure benefited from the deletion of the BPB domain in DDB1, which stabilized the ternary complex, while the full-length DDB1 used in X-ray crystallography improved crystallization success.

Our findings establish cryo-EM and X-ray crystallography as complementary techniques for the structural determination of E3 ligase-PROTAC/molecular glue-target ternary complexes, offering advantages in both resolution and speed.