Identification and Characterization of Two Structurally Related Dipeptides that Enhance Catalytic Efficiency of Neurolysin

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 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.

Is Diminazene an Angiotensin-Converting Enzyme 2 (ACE2) Activator? Experimental Evidence and Implications

“…with synthetic substrates may be misleading in identification of enzyme modulators (Jayaraman et al, 2021), in this set of experiments the concentration-dependent effect of DIZE on hydrolysis of Ang II by ACE2 was studied using mass spectrometry and no substantial differences were documented (Fig. 4).…”

“…For ACE2 we used fluorogenic substrate Mca-Ala-Pro-Lys (Dnp) (Kulemina and Ostrov, 2011), for ACE and NEP, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(Dnp)-OH (Johnson and Ahn, 2000;Wösten-van Asperen et al, 2008) and for Nln and TOP, Mca-Pro-Leu-Gly-Pro-D-Lys(Dnp)-OH (Jayaraman et al, 2021). As detailed in results section, the first set of experiments were carried out in 75 mM Tris-HCl 0.1 M NaCl, 0.5 µM ZnSO 4 and 0.01% Triton-X (pH 7.4) which is the same assay buffer used in the original study reporting discovery of DIZE as an ACE2 activator (Kulemina and Ostrov, 2011).…”

“…The reaction was stopped by addition of 1 µl of 1.0 N LCMS grade HCl and storage at −80°C freezer. Quantification of Ang II and its hydrolysis product Ang-(1-7) was carried out by LC-MS/MS using a SCIEX Triple Quad™ 5500+ LC-MS/MS System following a protocol modified from our recent studies (Jayaraman et al, 2021;Rahman et al, 2021). In brief, 2 μl of sample was injected onto a 50 × 2.1 mm Kinetex EVO C18 column (Phenomenex).…”

The evolution of small molecule enzyme activators

“…Detailed SAR studies identified analogues 25–27 with increased activities over the original hit (AC 50 of 6.5 μM, 4.2 μM and 7.0 μM for compounds 25 , 26 and 27 respectively). 207–210 In vitro metabolic stability studies reported that each of the three analogues had significantly increased half-lives in mouse plasma compared to the original hit, which reported a half-life of 34.19 minutes, in comparison to compounds 25 and 26 , which presented the best half-lives of >1000 minutes. 205 The permeabilities of each of the compounds were obtained within the MDR1-MDCA cell line, in which 26 and 27 presented the best permeability.…”

Imidazole Bioisostere Activators of Endopeptidase Neurolysin with Enhanced Potency and Metabolic Stability

“…The development of small-molecule enzyme activator compounds is rapidly becoming a viable therapeutic discovery strategy for many diseases. , In our efforts to identify Nln activator compounds for stroke, we previously disclosed a peptido­mimetic scaffold represented by Nln-10c and Nln-11a , optimized from a dipeptide hit ( HY ) obtained from a high-throughput screen (Figure ). Both compounds possessed “drug-like” pharmaco­kinetic parameters, including brain penetration. Additionally, compound Nln-11a was determined to preferentially accumulate in the ischemic hemisphere of the IS middle cerebral artery occlusion (MCAO) mouse model .…”