Biology subjects

Orr, L. M.

Publications and source records attributed to Orr, L. M..

3 recordsLinked to original sources

bioRxiv2025

Development of Second-Generation Acyl Silane Photoaffinity Probes for Cellular Chemoproteomic Profiling

Deconvolution of the protein targets of hit compounds from phenotypic screens, often conducted in live cells, is critical for understanding mechanism of action and identifying potentially hazardous off-target interactions. While photoaffinity labeling and chemoproteomics are long-established approaches for discovering small-molecule-protein interactions in live cells, there are a relatively small number of photoaffinity labeling strategies that can be applied in intracellular settings. Recently, we reported a novel chemical framework for photoaffinity labeling based on the photo-Brook rearrangement of acyl silanes and demonstrated its ability, when appended to protein-targeting ligands, to label recombinant proteins. Here, we report the application of these probes to live cell photoaffinity workflows, demonstrate their complementarity to current state-of-the-art minimalist diazirine-based photoaffinity probes, and introduce a modular synthetic route to access acyl silane scaffolds with improved labeling properties.

biochemistry↗

bioRxiv2025

DCAF16-Based Covalent Degradative Handles for the Modular Design of Degraders

Targeted protein degradation (TPD) is a powerful strategy for targeting and eliminating disease-causing proteins. While heterobifunctional Proteolysis-Targeting Chimeras (PROTACs) are more modular, the rational design of monovalent or molecular glue degraders remains challenging. In this study, we generated a small library of BET-domain inhibitor JQ1 analogs bearing elaborated electrophilic handles to identify permissive covalent degradative handles and E3 ligase pairs. We identified an elaborated fumaramide handle that, when appended onto JQ1, led to the proteasome-dependent degradation of BRD4. Further characterization revealed that the E3 ubiquitin ligase CUL4DCAF16--a common E3 ligase target of electrophilic degraders--was responsible for BRD4 loss by covalently targeting C173 on DCAF16. While this original fumaramide handle, when appended onto other protein-targeting ligands, did not accommodate the degradation of other neo-substrates, a truncated version of this handle attached to JQ1 was still capable of degrading BRD4, now through targeting both C173 and C178. This truncated fumaramide handle, when appended on various protein targeting ligands, and was also more permissive in degrading other neo-substrates, including CDK4/6, SMARCA2/4, and the androgen receptor (AR). We further demonstrated that this optimized truncated fumaramide handle, when transplanted onto an AR DNA binding domain-targeting ligand, could degrade both AR and the undruggable truncation variant of AR, AR-V7, in androgen-independent prostate cancer cells in a DCAF16-dependent manner. Overall, we have identified a unique DCAF16-targeting covalent degradative handle that can be transplanted across several protein-targeting ligands to induce the degradation of their respective targets for the modular design of monovalent or bifunctional degraders.

biochemistry↗

bioRxiv2024

DCAF16-Based Covalent Handle for the Rational Design of Monovalent Degraders

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.

biochemistry↗