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Identification and Characterization of Two Structurally Related Dipeptides that Enhance Catalytic Efficiency of
Srinidhi Jayaraman1, Joanna Kocot1, Shiva Hadi Esfahani1
1Department of Pharmaceutical Sciences (S.J., J.K., S.H.E., N.J.W., T.J.A., V.T.K.) and Center for Blood Brain Barrier Research (T.J.A., V.T.K.), School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas; Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan (A.U., A.D.); Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas (Y.M.); Department of Pharmaceutical Sciences, College of Pharmacy and Center for Drug Discovery, University of Nebraska Medical Center, Omaha, Nebraska (P.C.T.); Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota (H.A.); and Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, Florida (D.A.O.).
Abstract:
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. SIGNIFICANCE STATEMENT: This study describes discovery of two molecules that selectively enhance activity of peptidase Nln-a newly recognized cerebroprotective mechanism in the poststroke brain. The identified molecules will serve as a chemical scaffold for development of drug-like molecules to further study Nln and may become lead structures for a new class of drugs. In addition, our conceptual and methodological framework and research findings might be used for other peptidases and enzymes, the activation of which bears therapeutic potential.
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