Ligandability of E3 Ligases for Targeted Protein Degradation Applications

Abstract

Targeted protein degradation (TPD) using proteolysis targeting chimeras (PROTACs) and molecular glue degraders has arisen as a powerful therapeutic modality for eliminating disease-causing proteins from cells. PROTACs and molecular glue degraders employ heterobifunctional or monovalent small molecules, respectively, to chemically induce the proximity of target proteins with E3 ubiquitin ligases to ubiquitinate and degrade specific proteins via the proteasome. Whereas TPD is an attractive therapeutic strategy for expanding the druggable proteome, only a relatively small number of E3 ligases out of the >600 E3 ligases encoded by the human genome have been exploited by small molecules for TPD applications. Here we review the existing E3 ligases that have thus far been successfully exploited for TPD and discuss chemoproteomics-enabled covalent screening strategies for discovering new E3 ligase recruiters. We also provide a chemoproteomic map of reactive cysteines within hundreds of E3 ligases that may represent potential ligandable sites that can be pharmacologically interrogated to uncover additional E3 ligase recruiters.

Figure 1.. Targeted protein degradation with heterobifunctional PROTACs.

PROTACs consist of a protein-targeting ligand linked to an E3 ubiquitin ligase recruiter via a linker to induce the proximity of an E3 ligase with a neo-substrate target protein to induce ubiquitination and proteasome-mediated degradation of the target protein.

Figure 2.

Existing E3 ligase recruiters for targeted protein degradation applications.

Figure 3.. Using chemoproteomic platforms to identify potential reactive and ligandable cysteines across the human E3 ligase family.

(A) ABPP-based chemoproteomic approaches using reactivity-based probes. Reactivity-based alkyne functionalized probes can be used to label reactive cysteines in complex proteomes, after which a TEV protease-cleavable biotin-azide tag can be appended to probe-labeled proteins using copper-catalyzed azide-alkyne cycloaddition (CuAAC). Probe-modified proteins can be avidinenriched and digested with trypsin, and probe-modified tryptic peptides can be eluted by TEV protease for quantitative proteomic analysis. (B) Aggregating our lab’s chemoproteomic datasets of all probe-modified cysteines from ABPP experiments, we have identified probe-modified cysteines across 97% of human E3 ligases. Data can be found in Supplemental Table 1. (C) Probe-modified tryptic peptides and structure of the reactive cysteines C188 and C287 in CRBN. Shown is the structure of CRBN-DDB1 with CRBN in blue, DDB1 in gray, and C188/C287 in magenta (PDB 4tz4). (D) Probe-modified tryptic peptides and structure of the reactive cysteine C77 in VHL. Shown is the structure of VHL-elongin B/C with VHL in blue, elongin B/C in gray, and C77 in magenta (PDB 1vcb).