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.