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

Pinch, Benika, Buckley, Dennis, Gleim, Scott, Brittain, Scott, Tandeske, Laura, D'Alessandro, Pier Luca, Hauseman, Zachary , Lipps, Jennifer, Xu, Lei, Harvey, Edward , Schirle, Markus, Sprague, Elizabeth, Forrester, William, Dovala, Dustin, McGregor, Lynn and Thoma, Claudio (2021) A Strategy to Assess the Cellular Activity of E3 Ligase Components against Neo-Substrates using Electrophilic Probes. Cell chemical biology. ISSN 24519456


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 well-characterized E3 ligases such as 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. 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 maleimide-thiol 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 tyrosine kinase targets, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F-box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only ligase receptors but also adaptors can be directly hi-jacked for neo-substrate degradation.

Item Type: Article
Date Deposited: 28 Sep 2021 00:45
Last Modified: 28 Sep 2021 00:45


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