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

Pinch, Benika and McGregor, Lynn and Dovala, Dustin and Thoma, Claudio and Sprague, Elizabeth and Gleim, Scott and Forrester, William and Brittain, Scott and Buckley, Dennis and D'Alessandro, Pier Luca and Tandeske, Laura and Edward, Harvey and Zachary, Hauseman (2020) A Strategy to Assess the Cellular Activity of E3 Ligases against Neo-Substrates using Electrophilic Probes. BioRXiv.

Abstract

Targeted protein degradation promises to enable small molecule-mediated modulation of currently undrugged proteins. While the well-characterized E3 ligases CRBN and VHL have successfully promoted the degradation of many proteins of interest, there are approximately 600 additional E3 ligase family members that may offer improved activity, substrate selectivity, or tissue distribution; however, characterizing the ability of these many ligases to promote targeted protein degradation has proven challenging. Here, we report the development of a rapid method to evaluate the ability of recombinant E3 ligase components to support the degradation of neo-substrates. 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 using covalent E3-target binder complexes of VHL-JQ1 and VHL-dasatinib and show the degradation of Brd4 and Lyn kinase, respectively. Applying COFFEE to SPSB2, a SOCS box and SPRY-domain E3 ligase not previously shown to degrade neo-substrates, we demonstrated the ability of this method to rapidly validate an uncharacterized ligase for degradation of neo-substrates.

Item Type: Article
Keywords: E3 ligase; targeted protein degradation; covalent
Date Deposited: 27 Apr 2021 00:45
Last Modified: 27 Apr 2021 00:45
URI: https://oak.novartis.com/id/eprint/43163

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