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Harnessing Covalent Chemical Probes: From Inhibition to Induced Proximity Modalities


Covalent drugs have garnered intense interest due to the unique ways in which they can modulate protein function. Over the past decade, several covalent drugs have been approved that target EGFR, BTK, KRASG12C, and the SARS-CoV-2 Mpro for oncology and antiviral indications. The success of these drugs has driven scientists to design strategies to discover and characterize electrophilic molecules, including specialized screening methods and selectivity-profiling techniques. These technologies have enabled the rapid discovery of covalent chemical probes that selectively modulate protein activity to facilitate study of protein function in different biological contexts. These covalent tool compounds have frequently been further optimized to yield enzymatic inhibitors that provide clinical benefit to patients. Electrophilic compounds have also proven exceptionally useful for inducing proximity between proteins using bifunctional molecules, specifically for targeted protein degradation (TPD). Bifunctional degraders, or proteolysis targeting chimeras (PROTACs), consist of an E3 ligase recruiter linked to a ligand for a target protein. While many degraders recruit the E3s CRBN and VHL to degrade target proteins, more than 600 E3 ligases exist in human cells which could be used for this purpose. To expand the scope of TPD, I discovered a novel covalent ligand for the E3 ligase FEM1B. This ligand, EN106, can be incorporated into bifunctional PROTACs that degrade BRD4 and BCR-ABL, demonstrating that FEM1B can be used to induce degradation of target proteins. While protein degradation is often desirable, there are many cases where protein stabilization could have a therapeutic benefit instead. Here, I describe a strategy for targeted protein stabilization enabled by bifunctional deubiquitinase targeting chimeras (DUBTACs) that induce proximity between a deubiquitinase (DUB) and a target protein. Discovery of a covalent, allosteric ligand for the DUB OTUB1 led to the discovery of DUBTACs that stabilize mutant cystic fibrosis transmembrane conductance regulator (CFTR), whose degradation is central to cystic fibrosis pathology, as well as the tumor suppressor kinase Wee1. This DUBTAC platform allows the modular design of molecules that induce stabilization, unlocking proteins whose gain-of-function would be therapeutically desirable as potential drug targets. Finally, I share the discovery and optimization of covalent inhibitors of the SARS-CoV-2 main protease (Mpro), as part of an effort to discover pan-coronavirus antiviral compounds. This thesis describes the discovery of covalent chemical probes and how they can be harnessed to modulate protein function in ways that highlight potential opportunities for future drug discovery.

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