Rational discovery of Cyclin K molecular glue degraders via scalable chemical profiling

Targeted protein degradation is a new paradigm in drug development that promises to overcome limitations of traditional pharmacology. It is based on small molecules that induce proximity of a target protein and an E3 ubiquitin ligase, leading to target ubiquitination and proteasomal degradation1. Much focus is currently placed on heterobifunctional degraders, which simultaneously bind target and ligase with dedicated warheads that are connected by a flexible linker2-4. While this design principle is straightforward, the associated degradable space is intrinsically limited to ligandable proteins. Conversely, molecular glue degraders were shown to degrade unligandable proteins by orchestrating protein-protein interactions involving ligase and neo-substrate. The best-characterized examples are the clinically approved thalidomide analogs, which degrade zinc finger transcription factors by redirecting CRL4CRBN ligase activity5-8. However, the discovery of the few described molecular glue degraders has been serendipitous, thus hampering broad translational efforts. To address this limitation, we here describe a scalable strategy towards glue degrader discovery by comparative chemical screening in hypo-neddylated cellular models with broadly abrogated ligase activity. In a proof of concept, this led to the identification of a novel glue degrader that reprograms the CRL4DCAF15 ligase and enabled the discovery of three undescribed small molecules that induce the destabilization of cyclin K (CCNK). Integrating orthogonal functional genomics approaches with proteomics and drug-affinity chromatography strategies revealed an unprecedented mechanism of action where drug binding stabilizes an interaction of CDK12/13-CCNK with a CUL4B:DDB1 ligase complex that results in ubiquitination and degradation of CCNK. Notably, this interaction is independent of a dedicated substrate receptor, thus functionally segregating this mechanism from all described glue degraders. Collectively, our data outline a versatile and broadly applicable strategy to identify and characterize novel degraders with non-obvious mechanisms and thus empower future drug discovery efforts.