Exposing the Flexibility of Human Parainfluenza Virus Hemagglutinin-neuraminidase

Human parainfluenza virus type 3 (hPIV-3) is a clinically significant pathogen and is the causative agent of pneumonia and bronchiolitis in children. In this study the solution dynamics of human parainfluenza type 3 hemagglutinin-neuraminidase (HN) have been investigated. A flexible loop around Asp216 that adopts an open conformation in direct vicinity of the active site of the <i>apo</i>-form of the protein and closes upon inhibitor binding has been identified. To date, no available X-ray crystal structure has shown the molecular dynamics simulation-derived predominant loop-conformation states found in the present study. The outcomes of this study provide additional insight into the dynamical properties of hPIV-3 HN and may have important implications in defining HN glycan recognition events, receptor specificity, and antiparainfluenza virus drug discovery

Access to 3‑<i>O</i>‑Functionalized <i>N</i>‑Acetylneuraminic Acid Scaffolds

Direct access to 3-<i>O</i>-functionalized 2-α-<i>N</i>-acetylneuraminides and their corresponding 2,3-dehydro-2-deoxy-<i>N</i>-acetyl­neuraminic acid derivatives is described. Initially, a stereoselective ring-opening of the key intermediate <i>N</i>-acetylneuraminic acid (Neu5Ac) 2,3-β-epoxide with an alcohol provided the 3-hydroxy α-glycoside. <i>O</i>-Alkylation of the C3 hydroxyl group generated novel 3-<i>O</i>-functionalized Neu5Ac derivatives that provided the corresponding unsaturated derivatives upon elimination

Structural Insights into Human Parainfluenza Virus 3 Hemagglutinin–Neuraminidase Using Unsaturated 3‑<i>N</i>‑Substituted Sialic Acids as Probes

A novel approach to human parainfluenza virus 3 (hPIV-3) inhibitor design has been evaluated by targeting an unexplored pocket within the active site region of the hemagglutinin–neuraminidase (HN) of the virus that is normally occluded upon ligand engagement. To explore this opportunity, we developed a highly efficient route to introduce nitrogen-based functionalities at the naturally unsubstituted C-3 position on the neuraminidase inhibitor template <i>N</i>-acyl-2,3-dehydro-2-deoxy-neuraminic acid (<i>N</i>-acyl-Neu2en), via a regioselective 2,3-bromoazidation. Introduction of triazole substituents at C-3 on this template provided compounds with low micromolar inhibition of hPIV-3 HN neuraminidase activity, with the most potent having 48-fold improved potency over the corresponding C-3 unsubstituted analogue. However, the C-3-triazole <i>N</i>-acyl-Neu2en derivatives were significantly less active against the hemagglutinin function of the virus, with high micromolar IC₅₀ values determined, and showed insignificant <i>in vitro</i> antiviral activity. Given the different pH optima of the HN protein’s neuraminidase (acidic pH) and hemagglutinin (neutral pH) functions, the influence of pH on inhibitor binding was examined using X-ray crystallography and STD NMR spectroscopy, providing novel insights into the multifunctionality of hPIV-3 HN. While the 3-phenyltriazole-<i>N</i>-isobutyryl-Neu2en derivative could bind HN at pH 4.6, suitable for neuraminidase inhibition, at neutral pH binding of the inhibitor was substantially reduced. Importantly, this study clearly demonstrates for the first time that potent inhibition of HN neuraminidase activity is not necessarily directly correlated with a strong antiviral activity, and suggests that strong inhibition of the hemagglutinin function of hPIV HN is crucial for potent antiviral activity. This highlights the importance of designing hPIV inhibitors that primarily target the receptor-binding function of hPIV HN

Glycan structures present on array.

<p>Glycan structures present on array.</p

Binding of <i>C. jejuni</i> 11168 to Neu5Ac containing structures.

<p>Fluorescence intensities associated with <i>C. jejuni</i> 11168-GS (A) and <i>C. jejuni</i> 11168-O (B) binding to Neu5Ac containing glycans (25°C, black bar; 37°C, grey bar; and 42°C, white bar). For the structure of the individual glycans refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004927#pone-0004927-t002" target="_blank">Table 2</a>. * Significantly different to 25°C, P<0.05; # Significant difference between 37°C and 42°C, P<0.05.</p

Relative adherence of <i>C. jejuni</i> 11168 to sialidase treated Caco-2 cells.

<p>Relative adherence of 11168-GS (left) and 11168-O (right) to sialidase treated Caco-2 cells with respect to normal adherence levels (25°C, black bar; 37°C, grey bar; and 42°C, white bar).</p

Binding of <i>C. jejuni</i> 11168 to Man containing structures.

<p>Fluorescence intensities associated with <i>C. jejuni</i> 11168-GS (A) and <i>C. jejuni</i> 11168-O (B) binding to Man containing glycans (25°C, black bar; 37°C, grey bar; and 42°C, white bar). For the structure of the individual glycans refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004927#pone-0004927-t002" target="_blank">Table 2</a>. * Significantly different to 25°C, P<0.05; # Significant difference between 37°C and 42°C, P<0.05.</p

Relative binding of <i>C. jejuni</i> 11168 to Neu5Ac containing structures in the presence of sialyltransferase and sialidase inhibitors.

<p>Relative fluorescent levels of treated 11168-GS 42°C (A) and <i>C. jejuni</i> 11168-O 25°C (B) compared to normal <i>C. jejuni</i> binding to Neu5Ac containing glycans. (Neu5Ac2en, black bar; 4-guanidino-Neu5Ac2en, grey bar; and CMP, white bar). For the structure of the individual glycans refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004927#pone-0004927-t002" target="_blank">Table 2</a>.</p

Binding of <i>C. jejuni</i> 11168 to fucosylated structures.

<p>Fluorescence intensities associated with <i>C. jejuni</i> 11168-GS (A) and <i>C. jejuni</i> 11168-O (B) binding to fucosylated glycans (25°C, black bar; 37°C, grey bar; and 42°C, white bar). For the structure of the individual glycans refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004927#pone-0004927-t002" target="_blank">Table 2</a>. * Significantly different to 25°C, P<0.05; # Significant difference between 37°C and 42°C, P<0.05.</p

Lectin inhibition of <i>C. jejuni</i> adherence to intestinal cell line.

<p><i>C. jejuni</i> 11168-GS (A) and 11168-O (B) adherence to Caco-2 cells pre-treated with ECA, UEA-1, ConA, LFA, MAA and SNA (25°C, black bar; 37°C, grey bar; and 42°C, white bar). * Significant difference to non-lectin treated control, P<0.05; ND, Not determined.</p