Investigation of E3 ligase ligands for targeted protein degradation

Abstract

Targeted protein degradation (TPD) is a therapeutic strategy that harnesses the cell’s protein quality control machinery to selectively remove disease-relevant proteins. Central to many TPD approaches are E3 ubiquitin ligases, which can be recruited and rewired by small molecules to non-native substrates, leading to ubiquitination and subsequent proteasomal degradation of those substrates. One of the most widely studied E3 ligases in this context is cullin RING ligase 4 (CRL4) substrate receptor cereblon (CRBN), which has been successfully exploited by molecular glues and bifunctional degraders to modulate protein homeostasis. Here, I employ chemical biology and proteomics approaches to investigate ligands for CRBN and other E3 ligases and their utility in TPD. In Chapter 1, I provide an overview of the ubiquitin-proteasome system (UPS) and its role in regulating protein stability through E3 ligase-mediated ubiquitination. I survey several endogenous degrons – short motifs that direct substrates to E3 ligases – and discuss how these motifs can be harnessed for both small molecule degrader design and new E3 ligase recruitment. I introduce two major small molecule strategies for TPD: molecular glue degraders and heterobifunctional PROTACs. I describe key advances that have enabled the maturation of the TPD modality, including serendipitous discovery and structure-guided design of E3 ligase ligands, as well as approaches to identify molecules that recruit additional E3 ligases. I also highlight the mechanistic principles that govern degrader efficacy, including ternary complex formation and cooperativity, as well as the techniques employed to interrogate these principles. Finally, I briefly summarize additional induced proximity approaches for targeted degradation. In Chapter 2, I explored the potential of various E3 ligases and their cognate degrons as modular tools for TPD. I began by focusing on the CTLH E3 ligase substrate receptor Gid4, which recognizes an N-terminal proline degron. Using biochemical assays like DSF and TR-FRET, I characterized Gid4-ligand interactions and developed peptidic PROTACs linking Gid4 ligands to JQ1 for BRD4 degradation. While these PROTACs promoted ternary complex formation both in vitro and in cells, BRD4 degradation was inconsistent. To complement these efforts, I employed a GFP-based Pro/N-end model substrate, which demonstrated proline- and Gid4- dependent degradation. I further expanded my investigation to CRL family E3 ligases with C-end degrons, revealing promising activity for CRL2 and CRL4 substrate receptors with cognate dipeptide C-end degrons -GG and -EE. Overall, this chapter highlights both the promise and challenge of harnessing endogenous degrons to recruit additional E3 ligases for TPD. In Chapter 3, I detail the development of a 75-member library of glutarimide N-alkylated “capped imide” ligands, inspired by our prior photoaffinity labelling probes for lenalidomide. Initial screening suggested that several capped imides deplete CK1ɑ in a CRBN-dependent manner, and a few capped imides sensitized cellular proliferation without depleting known neosubstrates. I employed chemical proteomics in an attempt to structurally characterize the binding site of capped imides as well as enrichment and global proteomics approaches to identify targets responsible for their activity. However, extensive follow-up revealed batch-to-batch variability in the CRBN-dependent activities, likely tied to trace impurities and not the intended compounds. Nonetheless, I identified reproducible CRBN-independent anti-proliferative effects in select cell lines. These findings underscore the importance of comprehensive compound validation in degrader research and reveal potential lead scaffolds for further mechanistic investigation. In Chapter 4, I investigate capped imides further for CRBN-independent activity, and demonstrate that glutarimide N-alkylation is a viable prodrug strategy. I first validate that capped imides ablate CRBN-binding in vitro and in cells. Despite loss of CRBN-engagement, these ligands retain immunomodulatory activity – specifically suppression of TNFɑ expression – that is likely CRBN-independent. I profile a panel of hematopoietic and solid tumor cell lines for cell viability effects, revealing that capped imides have a generally safe anti-proliferative profile, with a few notable exceptions in lung and ovarian lineages that may merit investigation into the mechanistic target. Using quantitative global proteomics, I identified that lenalidomide and select capped imides promote CRBN-independent depletion of small GTPase Rab28 via autophagy. Finally, I demonstrate a proof-of-concept prodrug that efficiently releases lenalidomide upon enzymatic activation.