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 neurologic disorders, the present study was designed to identify small molecule activators of Nln. Using a computational approach, the structure of Nln was explored, which was 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 an 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 concentration-dependent manner (micromolar A50 and Amax ≥ 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 (Vmax/Km 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.
|Original language||English (US)|
|Number of pages||12|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - Nov 1 2021|
Bibliographical noteFunding Information:
This study was funded by National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant R01NS106879] with additional support from TTUHSC School of Pharmacy. Discovery of small molecule activators of Nln described herein is the subject of a published patent application: PCT Int. Appl. (2020) WO2020047185. No author has an actual or perceived conflict of interest with the contents of this article. 1S.J., J.K., and S.H.E. contributed equally to this work. https://doi.org/10.1124/jpet.121.000840. S This article has supplemental material available at jpet.aspetjournals.org.
This study was funded by National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant R01NS106879] with additional support from TTUHSC School of Pharmacy. Discovery of small molecule activators of Nln described herein is the subject of a published patent application: PCT Int. Appl. (2020) WO2020047185.
Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't