Discovery of a Tunable Heterocyclic Electrophile 4‑Chloro-pyrazolopyridine That Defines a Unique Subset of Ligandable Cysteines

Electrophilic small molecules with novel reactivity are
powerful
tools that enable activity-based protein profiling and covalent inhibitor
discovery. Here, we report a reactive heterocyclic scaffold, 4-chloro-pyrazolopyridine
(CPzP) for selective modification of proteins via a nucleophilic aromatic
substitution (SNAr) mechanism. Chemoproteomic profiling
reveals that CPzPs engage cysteines within functionally diverse protein
sites including ribosomal protein S5 (RPS5), inosine monophosphate
dehydrogenase 2 (IMPDH2), and heat shock protein 60 (HSP60). Through
the optimization of appended recognition elements, we demonstrate
the utility of CPzP for covalent inhibition of prolyl endopeptidase
(PREP) by targeting a noncatalytic active-site cysteine. This study
suggests that the proteome reactivity of CPzPs can be modulated by
both electronic and steric features of the ring system, providing
a new tunable electrophile for applications in chemoproteomics and
covalent inhibitor design.