UC Berkeley

The human proteome is vast and complex – comprised of at least 17,000 encoded proteins with varying degrees of expression depending on cell and tissue type – but an estimated 85% of encoded proteins are considered “undruggable” by traditional occupancy-driven pharmacology. Undruggable protein classes are typically characterized by highly dynamic or complex protein structure, intrinsic disorder, lack of well-defined active or allosteric pockets, or extensive interfaces with other biomolecules. Fortunately, many historically undruggable protein classes such as GTPases and transcription factors have been effectively targeted through recent advances in covalent drug development, which utilizes electrophilic reactive functional groups to engage with nucleophilic components of proteins including protein termini and side chains. Furthermore, induced proximity, which is broadly defined within drug discovery as the utilization of multispecific small molecules or biologics to facilitate biomolecule interactions resulting in co-optation of native biological pathways, has also enabled access to undruggable targets. Particularly of note are protein degraders in which small molecule-mediated induced proximity results in clearance of a desired protein target using native degradation machinery. Therefore, advancements in covalent approaches to induced proximity are an attractive area of investigation with potential widespread applications in therapeutics and are discussed in detail in the following dissertation.This work also describes the combination of covalent chemoproteomics with phenotypic screening to develop a workflow for discovery of covalent small molecule protein degraders, termed molecular glue degraders. Utilizing this methodology, we screen a library of covalent fragments for anti-proliferative effects and identify a proteasome-dependent compound, EN450. Cysteine-reactive chemoproteomics identify E2 ubiquitin-conjugating enzyme UBE2D as a covalent protein target of EN450, and quantitative proteomics reveal oncogenic transcription factor N as a downregulated protein target. Further in vitro assays provide evidence towards EN450-mediated ternary complex formation between UBE2D and NF-κB and subsequent ubiquitination of NF-κB, supporting an induced proximity mechanism of degradation. In summary, we have developed a scalable screening method and applied it to identify a covalent molecular glue degrader targeting a high value oncoprotein.