Peptidase neurolysin (Nln) is an enzyme that functions to cleave various neuropeptides. Upregulation of Nln after stroke has identified the enzyme as a critical endogenous cerebroprotective mechanism and validated target for the treatment of ischemic stroke. Overexpression of Nln in a mouse model of stroke results in dramatic improvement of stroke outcomes, while pharmacological inhibition aggravates them. Activation of Nln has therefore emerged as an intriguing target for drug discovery efforts for ischemic stroke. Herein, we report the discovery and hit-to-lead optimization of first-in-class Nln activators based on histidinecontaining dipeptide hits identified from a virtual screen. Adopting a peptidomimetic approach provided lead compounds that retain the pharmacophoric histidine moiety and possess single-digit micromolar potency over 40-fold greater than the hit scaffolds. These compounds exhibit 5-fold increased brain penetration, significant selectivity over highly homologous peptidases, greater than 65-fold increase in mouse brain stability, and 'drug-like' fraction unbound in the brain.

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Neurolysin (Nln) is a recently recognized endogenous mechanism functioning to preserve the brain from ischemic injury. To further understand the pathophysiological function of this peptidase in stroke and other neurological disorders, the present study was designed to identify small molecule activators of Nln.Using a computational approach, the structure of Nln was explored, followed by docking and in silico screening of ~140,000 molecules from the National Cancer Institute Developmental Therapeutics Program database. Top ranking compounds were evaluated in a Nln enzymatic assay, and two hit histidine-dipeptides were further studied in detail. The identified dipeptides enhanced the rate of synthetic substrate hydrolysis by recombinant (human and rat) and mouse brain-purified Nln in a concentrationdependent manner (micromolar A 50 and A max ≥ 300%), but had negligible effect on activity of closely related peptidases. Both dipeptides also enhanced hydrolysis of Nln endogenous substrates neurotensin, angiotensin I and bradykinin, and increased efficiency of the synthetic substrate hydrolysis (V max /K m ratio) in a concentration-dependent manner. The dipeptides and competitive inhibitor dynorphin A(1-13) did not affect each other's affinity for Nln, suggesting differing nature of their respective binding sites.Lastly, drug affinity responsive target stability (DARTS) and differential scanning fluorimetry (DSF) assays confirmed concentration-dependent interaction of Nln with the activator molecule. This is the first study demonstrating that Nln activity can be enhanced by small molecules, although the peptidic nature and low potency of the activators limit their application. The identified dipeptides provide a chemical scaffold to develop high-potency, drug-like molecules as research tools and potential drug leads. Significance statementThis study describes discovery of two molecules that selectively enhance activity of peptidase neurolysin (Nln)a newly recognized cerebroprotective mechanism in the post-stroke brain. The identified molecules will serve as a chemical scaffold for development of drug-like molecules to further study Nln, This article has not been copyedited and formatted. The final version may differ from this version.