Chemical proteomics and biochemical investigation of chemical degraders that engage cereblon

Abstract

Chemical degraders are a unique class of molecules that re-wire ubiquitination for targeted protein degradation. These compounds typically function by inducing physical proximity between an E3 ligase and the target protein to promote ubiquitination and subsequent proteasomal degradation. Recently, various molecules that induce ternary complex formation exogenously to regulate protein functions have emerged. The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide and pomalidomide, for example, bind to the E3 ligase substrate adaptor cereblon (CRBN) to induce engagement of target proteins such as IKZF1 and IKZF3 that are subsequently ubiquitinated and degraded. Bifunctional chemical degraders, such as proteolysis-targeting chimeras (PROTACs) harnessed the concept of induced proximity for targeted protein degradation, where an E3 ligase such as CRBN is recruited to the protein target for subsequent ubiquitination and degradation. However, to fully characterize the biological targets of these compounds, a systematic method to reveal a holistic map of interactions mediated by chemical degraders is needed. In my PhD studies, I used chemical proteomics and biochemical approaches to study the interactome of chemical degraders in cells. In particular, I developed and characterized a photoaffinity labeling (PAL) probe named photolenalidomide and a covalent probe for lenalidomide. By using these probes and chemical proteomics, I discovered a novel target of lenalidomide, eukaryotic initiation factor 3 subunit i (eIF3i), which forms a ternary complex with CRBN and lenalidomide. With structural information of the binding surface revealed, I further associated eIF3i sequestration to the antiangiogenetic properties of lenalidomide. Finally, I investigated the development of new chemical modalities with enhanced cell-type and target selectivity via enzymatic activation in cells. In Chapter 1, I discuss the current technical progress in strategies and methods to study and characterize chemical degraders in vitro and in live cells. I highlight the methods that have been adapted and applied to the field of chemical degraders, including ubiquitin profiling, global proteomics, affinity-purification mass spectrometry and enzymatic strategies. Advantages and disadvantages of each method are described and illustrated with each use case. In particular, chemical proteomics strategy, including activity-based protein profiling and photoaffinity labeling (PAL)-assisted chemical proteomics, serves as a unique strategy that translates a transient interaction into a detectable modification. Both target identification and binding site identification can be achieved without cellular engineering. In Chapter 2, I describe the application of PAL-assisted chemical proteomics for cellular target identification with the development and characterization of a probe for lenalidomide, termed photolenalidomide. Photolenalidomide is a PAL probe of lenalidomide with a diazirine functional group and enrichment handle that preserves the substrate degradation profile, phenotypic anti-proliferative and immunomodulatory properties. By using photolenalidomide in chemical proteomics, direct targets from multiple myeloma MM.1S cells such as IKZF1 and CRBN, were identified with structural resolution. I also discuss my contributions in the collaborative efforts to elucidate the direct binding modes of lenalidomide enantiomers, phosphatidylethanol (PEth) and UM171 molecule. Taken together, I demonstrate that PAL-assisted chemical proteomics is a robust strategy for cellular target identification and enables unbiased profiling of the interactome of molecules and their binding modes. In Chapter 3, I discuss the discovery of eIF3i as a novel target of lenalidomide. In this study, eIF3i was identified by chemical proteomics as a direct interacting protein that forms a ternary complex with CRBN in the presence of lenalidomide. Upon binding with CRBN and lenalidomide, eIF3i is sequestered from the eIF3 complex, leading to the interruption of eIF3 and eIF3i functions. I then describe my efforts in connecting the sequestration of eIF3i to the antiangiogenetic properties of lenalidomide in HUVEC cells. eIF3i was also observed to interact with CRBN and lenalidomide in MM.1S cells after the degradation of other targets. In sum, I demonstrated through my work the structural information of eIF3i interaction in the presence of lenalidomide as well as the mechanistic link of the eIF3i sequestration to lenalidomide phenotypic functions. Serving as a potentially prevalent loss-of-function mechanism, targeted protein sequestration may serve as a new direction of studies on chemical degraders and protein regulation in live cells. In Chapter 4, I discuss my progress towards the development of a sugar-coated PROTAC as a strategy for enzymatically activated targeted protein degradation. Specifically, I show that bifunctional degraders functionalized by O-linked N-acetylglucosamine (O-GlcNAc) require the enzymatic activity of O-GlcNAcase (OGA) to activate the compound activity for targeted protein degradation. I demonstrate a proof of concept with the assessment of sugar-coated PROTACs in cellular degradation assays and CRBN engagement NanoBRET assays, followed by initial efforts towards in vitro characterization of sugar-coated PROTACs and cell-specific cytotoxicity. These data demonstrate that sugar-coated PROTAC can be used to control targeted protein degradation and cellular toxicity by the regulation of OGA, which will open intriguing directions for future exploration of engineered targeted protein degradation via other sugars or post-translational modifications.