UC Berkeley

Targeted protein therapeutics have garnered significant interest in modern drug discovery, in part due to their ability to target classically “undruggable” proteins. “Undruggable” proteins, which make up the majority of disease-related proteins, are those that cannot be targeted by a therapeutic that targets a well-defined binding pocket. Chemoproteomic strategies can expand the “druggable” proteome by mapping and profiling reactive, nucleophilic amino acids in the proteome that may interact with covalent small molecules. The work discussed in this dissertation highlights the utility of chemoproteomic strategies in drug discovery and drug development spaces. These strategies are first described in the context of targeted protein degradation, a powerful modality for eliminating disease-related proteins from cells. Targeted protein degradation therapeutics utilize proteolysis targeting chimeras (PROTACs) and molecular glue degraders to accomplish this. PROTACs and molecular glue degraders employ heterobifunctional or monovalent small molecules, respectively, to chemically induce the proximity of target proteins with E3 ubiquitin ligases to ubiquitinate and degrade target proteins via the proteasome. I review the E3 ligases that have been successfully exploited for targeted protein degradation, and discuss chemoproteomics-enabled covalent screening strategies for the discovery of new E3 ligase recruiters. Additionally, I provide a database of reactive cysteines across 97% of known E3 ligases, identified using mass-spectrometry based chemoproteomics. In one example highlighting the utility of chemoproteomic strategies, I employed chemoproteomic profiling to identify the targets of the electrophilic anti-cancer natural product dankastatin B, from the fungus Gymnascella dankaliensis. I discovered that dankastatin B covalently reacts with a cysteine on the voltage-dependent anion channel protein 3 (VDAC3) using activity-based chemoproteomic profiling. I demonstrate that the covalent modification of VDAC3 by dankastatin B leads to the initiation of intrinsic apoptosis in triple-negative breast cancer cells. Dankastatin B also covalently reacts with voltage-dependent anion channel 2 (VDAC2) and E3 ligase RNF114, which also contribute to the natural product’s anti-cancer activity, validated by knockdown studies. In another example, I present a robust strategy for the synthesis of photoaffinity labeling probes for covalent capture. Photoaffinity labeling probes are powerful tools for the identification of small molecule—protein interactions that work by inducing a covalent interaction between the drug-like molecule and proteins that it interacts with. I demonstrate that photoaffinity labeling probes containing an acyl silane moiety rather than the traditional diazirine moiety can be utilized in chemoproteomic activity-based profiling to identify potential off-targets of (+)-JQ1. I show that (+)-JQ1 linked to a photoreactive acyl silane alkyne probe binds to BRD4 and, through mass-spectrometry based chemoproteomic profiling, identify potential off-targets of (+)-JQ1 in leukemia cells. The data discussed provides evidence that acyl silanes may be a more tunable scaffold for photoaffinity labeling and covalent capture.