We have successfully delivered a proof of concept that chemical profiling in engineered cell systems can lead to the identification of novel molecular glue degraders. We have identified that cells mutant for the gene UBE2M feature impaired activity of up to two hundred different E3 ligases. Based on this finding, we have devised comparative chemical profiling in UBE2Mmut cells to find compounds that require a CRL ligase to convey their mechanism. Coupling this screening paradigm to orthogonal target ID methods, this led to the identification of novel glue degraders that destabilize the protein Cyclin K (Mayor Ruiz, Nat Chem Biol 2020). Based on this concept, we have in the meantime identified a suite of additional small molecules that might act as MGDs, and that we are mechanistically following up in the meantime.
In addition, we have developed an additional discovery approach for novel MGDs. This rests on time-resolved probing of impaired ligase dynamics in intact cells following drug treatment. Based on a variety of different control experiments, we could validate this approach, leading us to identify novel molecular glue degraders that re-program the activity of the E3 ligase DCAF15. The description of this strategy as well as the chemical and mechanistic characterization of the identified, novel glue degraders was published in the Journal of American Chemical Society (JACS) (Hanzl et al., JACS 2022).
In addition, we developed methodologies to characterize functional hotspots on E3 ligases that are hijacked for neosubstrates degradation via molecular glue degraders and PROTACs. In brief, we devised a multi-layered functional genomics approach that allows us to probe, in cellulo, how E3 ligases recognize neosubstrates at a single amino acid resolution. These results were published in Nature Chemical Biology (Hanzl, 2022).
In addition, using FACS-based CRISPR/Cas9 screens, we have identified a novel molecular-glue like modality. In a collaboration with Alessio Ciulli’s lab, we identify and characterize the first of its kind “intramolecular glue degrader”. This bifunctional molecule acts in cis, meaning it binds two adjacent protein domains on the neosubstrate BRD4 and rearranges them. This rearrangement causes the stabilization of a pre-existing interaction between BRD4 and the E3 ligase DCAF16, leading to BRD4 ubiquitination and degradation in a DCAF16-dependent manner (Hsia, Hinterndorfer, Cowan, Nature 2024). Moreover, we have identified novel molecular glues that induce the selective degradation of the elongation factor GSPT2 by leveraging a phenotypic profiling strategy in isogenic cells (Ng, ACS Chem Biol 2024). We also have identified that merely tethering a known small-molecule binder with a flexible alkylamine tail can be sufficient to convert a binder into a degrader that mechanistically works by co-opting the E3 ligase FBXO22 (Kagiou, Nature Communications 2024).
Lastly, we have co-developed a novel methodology to profile degraders that is based on nascent proteomics and uncouples primary degradation events from secondary consequences (Jochem, Cell Chem Bio 2024). We have also developed the first trivalent PROTAC that engages CRBN and VHL in parallel (Bond, JACS 2024). Enabled by the knowledge we could gain through this grant, we have also launched several related projects that will be funded via different sources moving forward.
