Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications

Abstract

Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only a few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome-and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.

Targeted protein degradation: expanding the toolbox

“…These molecules are generally larger and more flexible than typical drug-like compounds, which can translate into poor membrane permeability and liability to efflux pumps 39,42,43 . While covalent binding to E3 ligases is acceptable 31,44 , PROTACs binding covalently to the protein target probably lose the substoichiometric nature of their mechanism 45 . Finally, what percentage of a target protein should be degraded to trigger a phenotypic response probably depends on the target and the readout, but needs to be systematically investigated.…”

Parthenolide Covalently Targets and Inhibits Focal Adhesion Kinase in Breast Cancer Cells

“…In previous works investigating the direct targets of parthenolide, multiple studies have revealed unique ligandable and functional cysteines within their respective proteins that could be targeted to influence cellular signaling and pathogenicity. Recent studies have shown that activity-based protein profiling (ABPP)-based chemoproteomic platforms can be utilized to uncover unique and functional druggable hotspots and modalities that can be accessed by covalently-acting small-molecules and natural products that may not be obvious using standard drug discovery paradigms (Backus et al, 2016;Bateman et al, 2017;Grossman et al, 2017;Hacker et al, 2017;Spradlin et al, 2018;Ward et al, 2018).…”

Small molecules that target the ubiquitin system

“…TRH 1-23 (2, Table 2) is a promising RNF4 inhibitor that was recently identified in an activity-based protein profiling (ABPP) screen. An analog of TRH 1-23, named CCW-16 3, was found to be a more a potent RNF4 inhibitor, exhibiting an IC 50 of 1.8 μM [63]. Von Hippel-Lindau (VHL) E3 Ubiquitin Ligase is one of the few E3 ligases, including cereblon, VHL, MDM2, and cIAP, that have been successfully exploited in targeted protein degradation strategies [71,72].…”