Structure-Guided Optimization of 4‑Chloro-Pyrazolopyridine Analogs for Covalent PREP Inhibition

Prolyl
endopeptidase (PREP) is a dynamic serine protease that cleaves
proline-containing peptides. PREP is also involved in numerous pathophysiological
processes through modulation of protein–protein interactions
and has been extensively studied in neurodegenerative diseases. In
this study, we report the structure-based design and synthesis of
covalent PREP inhibitors built on a 4-chloro-pyrazolopyridine (CPzP)
scaffold, previously identified through chemoproteomic screening to
target a noncatalytic cysteine residue within the active site. Guided
by crystallographic data and molecular docking studies, we optimized
initial hits to develop a potent inhibitor exhibiting nanomolar potency
in both biochemical and cellular assays, with high selectivity over
related serine proteases FAP and DPP4. Molecular dynamics simulations
indicated that modulation of the conformational flexibility of a dynamic
A-loop within PREP by CPzP analogs may contribute to inhibitory potency.
Collectively, this work introduces a new class of structurally distinct
inhibitors and provides tools to explore the diverse biological roles
of PREP.