Discovery of a pyridine-piperazine-based small molecule that enhances the activity of peptidase neurolysin

Abstract

Neurolysin (Nln) is a peptidase recognized for its cerebroprotective function in acute ischemic stroke. This study aimed to identify small molecule activators of Nln as research tools to further explore the role of this enzyme in stroke and other neurological disorders. Building on our previous computational screen of ∼140,000 compounds from the National Cancer Institute Developmental Therapeutics Program database, we extended experimental testing to the top 100 candidates using an Nln enzymatic assay. A pyridine-piperazine derivative (Py-Pip) was identified as a hit molecule and was characterized in detail. Py-Pip concentration-dependently enhanced the hydrolysis of both synthetic and natural substrates (neurotensin, angiotensin I, and bradykinin) by rat Nln, and displayed comparable activating effects on human and mouse orthologs. Importantly, Py-Pip exhibited a favorable selectivity profile, showing no potentiation of homologous metallopeptidases or unrelated enzymes. Kinetic analysis revealed that Py-Pip increases the catalytic efficiency (V_max/K_m) of Nln via a nonessential activation mechanism, whereas competition assays with inhibitor dynorphin A(1-13) confirmed that Py-Pip acts at a distinct, nonoverlapping site. Direct binding was further validated by orthogonal biophysical techniques, including differential scanning fluorimetry, microscale thermophoresis, and biolayer interferometry, whereas circular dichroism spectroscopy indicated activator-induced secondary structural changes. These findings validate that Nln activity can be enhanced by small molecules and establish Py-Pip as a novel, nonpeptide scaffold for developing potent, "drug-like" activators to investigate Nln biology and therapeutic potential. SIGNIFICANCE STATEMENT: This study reports the discovery of a novel nonpeptide small molecule that selectively enhances the activity of neurolysin (Nln), a peptidase implicated in cerebroprotection. Unlike previous peptide-based activators, this molecule provides a stable, "drug-like" scaffold and a structural foundation for the development of potent Nln activators to probe Nln biology and therapeutic potential in ischemic stroke.

Keywords: Allosteric modulation; Cerebroprotection; Enzyme activator; Enzyme kinetics; Metallopeptidase; Nonpeptide scaffold.