✍🏼 Melissa Lim, Thang Do Cong, Lauren M. Orr, Ethan S. Toriki, Andrew C. Kile, James W. Papatzimas, Elijah Lee, Daniel K. Nomura

🏠 Department of Chemistry, University of California, Berkeley, Berkeley, CA USA

📑 bioRxiv (2024)

Abstract
Targeted protein degradation with monovalent molecular glue degraders is a powerful therapeutic modality for eliminating disease causing proteins. However, rational design of molecular glue degraders remains challenging. In this study, we sought to identify a transplantable and linker-less covalent handle that could be appended onto the exit vector of various protein-targeting ligands to induce the degradation of their respective targets. Using the BET family inhibitor JQ1 as a testbed, we synthesized and screened a series of covalent JQ1 analogs and identified a vinylsulfonyl piperazine handle that led to the potent and selective degradation of BRD4 in cells. Through chemoproteomic profiling, we identified DCAF16 as the E3 ligase responsible for BRD4 degradation—an E3 ligase substrate receptor that has been previously covalently targeted for molecular glue-based degradation of BRD4. Interestingly, we demonstrated that this covalent handle can be transplanted across a diverse array of protein-targeting ligands spanning many different protein classes to induce the degradation of CDK4, the androgen receptor, BTK, SMARCA2/4, and BCR-ABL/c-ABL. Our study reveals a DCAF16-based covalent degradative and linker-less chemical handle that can be attached to protein-targeting ligands to induce the degradation of several different classes of protein targets.

How the WOLF was used in this study
The WOLF cell sorter was used to generate isogenic RNF126 knockout cell lines by enabling precise single-cell isolation following CRISPR–Cas9 genome editing. After HEK293T cells were nucleofected with Cas9 ribonucleoprotein complexes targeting the RNF126 locus and expanded as a mixed knockout pool, the WOLF cell sorter was employed to sort individual viable cells into 96-well plates. This single-cell sorting step allowed clonal expansion from individual edited cells, ensuring the derivation of genetically uniform knockout clones. Resulting colonies were subsequently screened by immunoblotting to confirm loss of RNF126 expression, enabling downstream mechanistic and proteomic analyses to be performed on well-defined, isogenic cell lines.