A Strategy to Assess the Cellular Activity of E3 Ligases against Neo-Substrates using Electrophilic Probes

Abstract

Targeted protein degradation is a rapidly developing therapeutic modality that promises lower dosing and enhanced selectivity as compared to traditional occupancy-driven inhibitors, and the potential to modulate historically intractable targets. While the well-characterized E3 ligases CRBN and VHL have been successfully redirected to degrade numerous proteins, there are approximately 600 predicted additional E3 family members that may offer improved activity, substrate selectivity, and/or tissue distribution; however, characterizing the potential applications of these many ligases for targeted protein degradation has proven challenging. Here, we report the development of an approach to evaluate the ability of recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for hit finding to identify specific E3 ligase binders, this approach makes use of simple chemistry for Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE). We demonstrate this method by electroporating recombinant VHL, covalently functionalized with JQ1 or dasatinib, to induce degradation of BRD4 or kinase targets, respectively. Furthermore, by applying COFFEE to SPSB2, a SOCS box and SPRY-domain E3 ligase that has not previously been redirected for targeted protein degradation, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligases against neo-substrates.

From Conception to Development: Investigating PROTACs Features for Improved Cell Permeability and Successful Protein Degradation

“…However, how to evaluate the ability of E3 ligase components to hijack neosubstrate degradation remains an important challenge. In this regard, Pinch et al conveyed a method so-called Covalent Functionalization Followed by E3 Electroporation into live cells (COFFEE) that bypasses the need for hit finding to identify specific E3 ligase binders (Pinch et al, 2020). In this work, they covalently linked a BRD4 ligand, JQ1, and the multikinase inhibitor, dasatinib, to VHL and other E3 ligases via their solvent-exposed cysteines and they further electroporated the recombinant E3 ligases into live cells to form functional E3 ubiquitin ligase complexes capable of TOI degradation.…”