Previous studies documented up‐regulation of peptidase neurolysin (Nln) after brain ischemia, however, the significance of Nln function in the post‐stroke brain remained unknown. The aim of this study was to assess the functional role of Nln in the brain after ischemic stroke. Administration of a specific Nln inhibitor Agaricoglyceride A (AgaA) to mice after stroke in a middle cerebral artery occlusion model, dose‐dependently aggravated injury measured by increased infarct and edema volumes, blood–brain barrier disruption, increased levels of interleukin 6 and monocyte chemoattractant protein‐1, neurological and motor deficit 24 h after stroke. In this setting, AgaA resulted in inhibition of Nln in the ischemic hemisphere leading to increased levels of Nln substrates bradykinin, neurotensin, and substance P. AgaA lacked effects on several physiological parameters and appeared non‐toxic to mice. In a reverse approach, we developed an adeno‐associated viral vector (AAV2/5‐CAG‐Nln) to overexpress Nln in the mouse brain. Applicability of AAV2/5‐CAG‐Nln to transduce catalytically active Nln was confirmed in primary neurons and in vivo. Over‐expression of Nln in the mouse brain was also accompanied by decreased levels of its substrates. Two weeks after in vivo transduction of Nln using the AAV vector, mice were subjected to middle cerebral artery occlusion and the same outcome measures were evaluated 72 h later. These experiments revealed that abundance of Nln in the brain protects animals from stroke. This study is the first to document functional significance of Nln in pathophysiology of stroke and provide evidence that Nln is an endogenous mechanism functioning to preserve the brain from ischemic injury.

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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.

The importance of the polo-like kinase 1 (PLK1) gene is increasing substantially both as a biomarker and as a target for highly specific cancer therapy. This is due to its involvement in multiple points of cell progression and carcinogenesis. PLK1 inhibitors' efficacy in treating human cancers has been limited due to the lack of a specific targeting strategy. Here, we describe a method of targeted downregulation of PLK1 in cancer cells and the concomitant rapid detection of surface lipidomic perturbations using desorption electrospray ionization mass spectrometry (DESI MS). The efficient delivery of siRNA targeting PLK1 gene selectively to the cancer cells is achieved by targeting overexpressed cell surface epithelial cell adhesion molecule (EpCAM) by the EpDT3 aptamer. The chimeric aptamer (EpDT3-siPLK1) showed the knockdown of PLK1 gene expression and PLK1 protein levels by quantitative PCR and western blotting, respectively. The abundant surface lipids, phosphatidylcholines (PCs), such as PC(32:1) (m/z 754.6), PC(34:1) (m/z 782.6), and PC(36:2) (m/z 808.6), were highly expressed in MCF-7 and WERI-RB1 cancer cells compared to normal MIO-M1 cells and they were observed using DESI MS. These overexpressed cell surface lipids in the cancer cells were downregulated upon the treatment of EpDT3-siPLK1 chimera indicating a novel role of PLK1 to regulate surface lipid expression in addition to the efficient selective cancer targeting ability. Our results indicate that DESI MS has a potential ability to rapidly monitor aptamer-mediated cancer therapy and accelerate the drug discovery process. Graphical abstract Binding of aptamer chimera to the cells and changes in lipid profile.

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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.