Proteomic Ligandability Maps of Spirocycle Acrylamide Stereoprobes Identify Covalent ERCC3 Degraders

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.