Unveil the Cereblon Target Atlas and Accelerate Molecular Glue Degrader Discovery

Project Summary/Abstract Clinically credentialed immunomodulatory drugs bind to the substrate receptor cereblon (CRBN) within the CRL4CRBN E3 ubiquitin ligase complex, creating a ‘neo-epitope’ on the CRBN surface that recruits non- endogenous substrates (neosubstrates) with an 8-amino acid G-loop degron motif for subsequent ubiquitination and proteasomal degradation. Such a drug modality is known as molecular glue degraders (MGDs), a subclass of molecular glues (MGs) that are characterized by their ability to induce protein-protein interactions. A significant gap remains in understanding the full scope of CRBN neosubstrates beyond the known G-loop-containing zinc finger proteins in the human proteome, often leading to new CRBN interactors being discovered serendipitously. Using bioinformatic analysis, we have identified 2,458 canonical G-loop targets as potential CRBN interactors and aim to create a more comprehensive atlas of CRBN interactome. The overarching objectives of the proposed work are: 1) to create a comprehensive atlas of CRBN interactome, encompassing canonical G-loop, mutant G- loop, and even the hidden non-G-loop targets, and 2) to further validate these uncharted proteins as bona fide CRBN interactors in the presence of novel MGDs. To achieve objective 1, we will leverage our expertise in bioinformatic analysis, while realizing objective 2 will require the development of efficient target-based MGD discovery methods, building on our knowledge of the CRBN interactome. We plan to employ not only a degradation activity-first, protein-protein docking-guided rational design and screening approach, but also utilize our DNA-encoded chemical library (DEL) platform to develop a ternary complex binding-first, unbiased DEL screening approach. To showcase our strategy, we have prioritized several uncharted G-loop targets, aiming to set compelling examples that will inspire future research into targeting the remaining predicted CRBN interactors with the MGD modality. Upon completion, this proposed work will significantly expand our understanding of the CRBN target landscape, and the molecular mechanisms and cellular pathways influenced by both existing and future CRBN ligand-based MGDs. Given MGD’s unique proximity-inducing mechanism and chemical knockdown pharmacology, our work is also poised to catalyze the development of numerous new CRBN-targeting MGD chemical probes or drugs that address many previously ‘undruggable’ wild-type proteins and a significant number of recalcitrant mutant proteins, thereby significantly advancing biomedical research and ultimately making a difference in public health.