A Computational Approach to the Study of the Binding Mode of Dual ACE/NEP Inhibitors

Combined blockade of the renin−angiotensin−aldosterone system (RAAS) is an attractive therapeutic strategy for the treatment of cardiovascular diseases. Vasopeptidase inhibitors are a group of compounds capable of inhibiting more than one enzyme, which leads to potentiation of natriuretic peptide actions and suppression of the RAAS. In this study, molecular modeling has been used to elucidate key structural features that govern the binding and/or selectivity of a single compound toward the zinc catalytic sites of the N- and C-domains of the angiotensin-converting enzyme (ACE) and the neutral endopeptidase (NEP). Eleven dual inhibitors were categorized in three classes, according to their zinc binding groups. Analysis of their docked conformers revealed the molecular environment of the catalytic sites and the specific interactions between the inhibitors and amino acid residues that are important for selectivity and cooperativity. In addition, inhibitors were predicted to bind to the C-domain of the ACE with greater affinity than the N-domain, with an average difference in the free energy of binding ∼2−3 kcal mol−1. Residues that were identified to actively participate in the binding and stabilizating of the enzyme−inhibitor complexes were analyzed in a consensus way for both the ACE and the NEP. These atomic-level insights into enzyme−ligand binding can be used to drive new structure-based drug design processes in the quest for more selective and effective vasopeptidase inhibitors

Conformational Dynamics of the Anthrax Lethal Factor Catalytic Center

Anthrax lethal factor (LF) is a zinc-metalloprotease that together with the protective antigen constitutes anthrax lethal toxin, which is the most prominent virulence factor of the anthrax disease. The solution nuclear magnetic resonance and in silico conformational dynamics of the 105 C-terminal residues of the LF catalytic core domain in its apo form are described here. The polypeptide adopts a compact structure even in the absence of the Zn2+ cofactor, while the 40 N-terminal residues comprising the metal ligands and residues that participate in substrate and inhibitor recognition exhibit more flexibility than the C-terminal region

Simulated Interactions between Angiotensin-Converting Enzyme and Substrate Gonadotropin-Releasing Hormone: Novel Insights into Domain Selectivity^†

Human angiotensin-I converting enzyme (ACE) is a central component of the renin-angiotensin system and a major target for cardiovascular therapies. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. On the basis of the recently determined crystal structures of both ACE domains, we have studied their complexes with gonadotropin-releasing hormone (GnRH), which is cleaved releasing both the protected NH2- and COOH-terminal tripeptides. This is the first molecular modeling study of an ACE−peptide substrate complex that examines the structural basis of ACE's endopeptidase activity and offers novel insights into subsites that are distant from the obligatory binding site and were not identified in the crystal structures. Our data indicate that a bridging interaction between Arg500 of the N-domain and Arg8 of GnRH that involves a buried chloride ion may account for its role in the specificity of the N-domain for endoproteolytic cleavage of the substrate at the NH2-terminus in vitro. In support of this, the protected NH2-terminal dipeptide of GnRH exhibits stronger interactions than the protected COOH-terminal dipeptide with the N-domain of ACE. Further comparison of the models of ACE−substrate complexes promotes our understanding of how the two domains differ in their function and specificity and provides an extension of the pharmacophore model used for structure-based drug design up to the S7 subsite of the enzyme

Binding of svH1C to NKG2D.

<p>(A) The amount of bound peptide was measured after extensive washing with PBS containing 0.05% Tween-20. Fetuin (5 μM, red; 10 μM, yellow; 30 μM, green) and sialyllactose (12 μM, red; 20 μM, yellow; 40 μM, green) were included as inhibitors. Inhibition is shown by the average of single values from three separate assays. The average 100% value was 2.4 ng conjugate bound. (B) Graphical representation of inhibition of binding of svH1C to NKG2D by fetuin (circles) or sialyllactose (squares) as shown in (A).</p

A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D

<div><p>We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a K_D in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14⁺ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has characteristics of a checkpoint inhibitor.</p></div

Binding of svH1C to lectin-type receptors.

<p>The buffer in these assays was PBS containing 0.05% Tween-20 (see text for effects of different buffer compositions). The figure shows the amount of streptavidin-peroxidase bound to svH1c that was bound to the receptors. Siglec-1 and CLEC10a contained a C-terminal His tag and were assayed in separate experiments. The other receptors were Fc-chimeras and were included in the same assays. SEM was determined for six assays from four independent experiments. Inhibition by fetuin is shown by the average of single values in two assays in which the glycoprotein was added at 10 μM (red) or 30 μM (green).</p

svH1C bound to monocytes after digestion of PBMCs with neuraminidase.

<p>(A) Dot plots and histograms of cells in PBMC samples treated 30 min at 37°C with neuraminidase inactivated by heating 20 min in boiling water. (B) Histograms of PBMCs from the same donor as (A) after treatment with active neuraminidase. After enzyme digestion, cells were incubated 30 min on ice with 1 μM biotin-tagged svH1C, washed and incubated 30 min on ice with marker antibodies and streptavidin labeled with PerCP-Cy5.5. Binding to only the monocyte (mono) fraction was significantly increased by treatment with neuraminidase, as shown by the histogram. The upper dot plot in (B) represents the monocyte fraction from (A), whereas the lower dot plot represents the monocyte fraction from (B). The subset of monocytes to which svH1C bound is circled, which accounted for 35% of the total monocyte population. This experiment was performed three times with similar results.</p

Unravelling the Conformational Plasticity of the Extracellular Domain of a Prokaryotic nAChR Homologue in Solution by NMR

Pentameric ligand-gated ion channels (pLGICs) of the Cys loop family are transmembrane glycoproteins implicated in a variety of biological functions. Here, we present a solution NMR study of the extracellular domain of a prokaryotic pLGIC homologue from the bacterium Gloeobacter violaceus that is found to be a monomer in solution

¹H-¹H 2D TOCSY 400 MHz NMR spectrum of svH1C.

<p>The fingerprint of HN-Hα/aliph svH1C protons on a 16.0204 ppm spectrum was recorded in H2O/D2O (90%/10% v/v, at pH 7.0, T = 298K). The amino acid spin-patterns are indicated with dashed lines. Vertical bracket indicate the HN-Hα & ΗΝ-Ηβ (only for serine residues), while horizontal brackets denotes the amide HN and the lysine side-chain NH groups, which are involved in iso-peptide bonds.</p

Circular dichroism spectra of svH1C as a function of concentration.

<p>(A) 100 μM peptide in 50 mM borate, pH 9.0; (B) peptide solution in (A) diluted 1:3 with water; (C) peptide solution in (A) diluted 1:9 with water; (D) 100 μM peptide in 50 mM borate adjusted to pH 2.</p