Proteomic Ligandability Maps of Spirocycle Acrylamide Stereoprobes Identify Covalent ERCC3 Degraders

Abstract

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile way to discover ligands for proteins in native biological systems. Here, we describe a set of stereo-and regiochemically defined spirocycle acrylamides and the analysis of these electrophilic "stereoprobes" in human cancer cells by cysteine-directed ABPP. Despite showing attenuated reactivity compared to structurally related azetidine acrylamide stereoprobes, the spirocycle acrylamides preferentially liganded specific cysteines on diverse protein classes. One compound termed ZL-12A promoted the degradation of the TFIIH helicase ERCC3. Interestingly, ZL-12A reacts with the same cysteine (C342) in ERCC3 as the natural product triptolide, which did not lead to ERCC3 degradation but instead causes collateral loss of RNA polymerases. ZL-12A and triptolide cross-antagonized one another's protein degradation profiles. Finally, we provide evidence that the antihypertension drug spironolactone�previously found to promote ERCC3 degradation through an enigmatic mechanism�also reacts with ERCC3_C342. Our findings thus describe monofunctional degraders of ERCC3 and highlight how covalent ligands targeting the same cysteine can produce strikingly different functional outcomes.

Covalent Degrader of the Oncogenic Transcription Factor β-Catenin

“…A striking example of such functional modulation is a recent discovery by Erb, Cravatt, et al of a covalent ligand that stereoselectively targeted a pioneer transcription factor FOXA1 to rewire its transcriptional programming . Other examples of covalent ligand-assisted degradation include the TFIIH helicase ERCC3 and immune kinases such as ITK. , While challenging to prove, we conjecture that these intrinsically disordered regions within transcription factors may adopt a transient binding pocket that can be molecularly recognized by a covalent ligand to form an irreversible bond. Given that these disordered transcription factors may already be thermodynamically unstable, we hypothesize that it may not require significant binding energy to further destabilize these dynamic regions, leading to their unfolding and ultimately ubiquitination and degradation of these targets.…”