Chemoproteomics-Enabled Discovery of a Covalent Molecular Glue Degrader Targeting NF-κB

Abstract

Targeted protein degradation using heterobifunctional Proteolysis-Targeting Chimeras (PROTACs) or molecular glues has arisen as a powerful therapeutic modality for degrading disease targets. While PROTAC design is becoming more modular and straightforward, the discovery of novel molecular glue degraders has been more challenging. While several recent studies have showcased phenotypic screening and counter-screening approaches to discover new molecular glue degraders, mechanistically elucidating the ternary complex induced by the small molecule that led to the initial phenotype, i.e. identifying the degraded target and relevant components of the ubiquitin-proteasome system, has remained cumbersome and laborious. To overcome these obstacles, we have coupled the screening of a covalent ligand library for anti-proliferative effects in leukemia cells with quantitative proteomic and chemoproteomic approaches to rapidly discover both novel covalent molecular glue degraders and their associated ternary complex components and anti-proliferative mechanisms. We have identified a cysteine-reactive covalent ligand EN450 that impairs leukemia cell viability in a NEDDylation and proteasome-dependent manner. Chemoproteomic profiling revealed covalent interaction of EN450 with an allosteric C111 in the E2 ubiquitin ligase UBE2D. Follow-up quantitative proteomic profiling revealed the proteasome-mediated degradation of the oncogenic transcription factor NFKB1 as a putative degradation target. Subsequent validation studies demonstrated that EN450 induced the ternary complex formation between UBE2D and NFKB1 and that both UBE2D and NFKB1 were important for the anti-proliferative mechanisms of EN450. Our study thus puts forth the discovery of a novel molecular glue degrader that uniquely induced the proximity of an E2 ligase with a transcription factor to induce its degradation and anti-proliferative effects in cancer cells. Taken more broadly, our study showcases a rapid and modular approach for discovering novel covalent molecular glue degraders and their respective ternary complex components in an unbiased fashion.

Proteome-scale induced proximity screens reveal highly potent protein degraders and stabilizers

“…UBE2B was the most potent inducer of degradation together with its highly similar paralog UBE2A ( Figure 3D ). In addition, two related E2s UBE2D1 and UBE2D4 were also highly active degradation inducers, consistent with the recent discovery of a UBE2D-dependent molecular glue targeting NF-kB (King et al, 2022). Thus, despite the highly conserved E2 fold, there are clear differences in the functionality of E2s in mediating proximity-dependent degradation.…”

“…One of the most potent degradation inducers that we identified in both ORFeome screens was the E2 conjugating enzyme UBE2B (also known as Rad6B)( Figure 1E and 1F ). UBE2B was a particularly interesting effector, because until recently, E2 enzymes had not been harnessed in targeted protein degradation (King et al, 2022). To further characterize UBE2B, we first tested if its catalytic activity is required for degradation.…”

“…The promiscuous activity of UBE2B towards diverse and unrelated targets indicates that it could be a powerful effector for targeted protein degradation applications. While E2s have not been considered easily druggable due to the lack of deep pockets, recent reports have demonstrated that they can be targeted at allosteric sites or by molecular glues that promote their interactions with other proteins (Hewitt et al, 2016; Huang et al, 2014; King et al, 2022; Morreale et al, 2017; St-Cyr et al, 2021). These results suggest that hijacking E2s for targeted protein degradation may not be as challenging as previously thought.…”

Chasing molecular glue degraders: screening approaches

“…The fragment EN450 was reported to have a differential effect in WT vs. siUBE2M cells and proposed to act as a MG that binds covalently to the E2 UBE2D to induce degradation of NFKB1. 157…”

Rational Chemical Design of Molecular Glue Degraders

“…Chemical screening for anti-cancer phenotypes coupled with counter-screening in hyponeddylated lines impaired in Cullin E3 ligase activity with subsequent multi-omic mechanistic deconvolution has led to the discovery of novel molecular glue degraders that act through interactions of CDK12-cyclin K with a CRL4B ligase complex to induce cyclin K degradation 15 . Phenotypic screening of covalent ligands for NEDDylation- and proteasome-dependent anti-cancer activity coupled with chemoproteomic approaches were also used to discover a covalent molecular glue between the E2 ubiquitin conjugating enzyme UBE2D and the transcription factor NFKB1 to ubiquitinate and degrade NFKB1 leading to impaired cancer cell viability 16 . These cell-based phenotypic screening approaches can also be deployed to perform target-based molecular glue degrader screens using cell lines expressing target proteins bearing high-throughput screening-detectable tags (e.g.…”