Sequences of C₂₀ peptide and its peptomeric analogues.

<p>The peptoid-tolerant residues identified from this study are shown in bold and underlined.</p

A Readily Applicable Strategy to Convert Peptides to Peptoid-based Therapeutics

<div><p>Incorporation of unnatural amino acids and peptidomimetic residues into therapeutic peptides is highly efficacious and commonly employed, but generally requires laborious trial-and-error approaches. Previously, we demonstrated that C₂₀ peptide has the potential to be a potential antiviral agent. Herein we report our attempt to improve the biological properties of this peptide by introducing peptidomimetics. Through combined alanine, proline, and sarcosine scans coupled with a competitive fluorescence polarization assay developed for identifying antiviral peptides, we enabled to pinpoint peptoid-tolerant peptide residues within C₂₀ peptide. The synergistic benefits of combining these (and other) commonly employed methods could lead to a easily applicable strategy for designing and refining therapeutically-attractive peptidomimetics.</p> </div

Structures of peptoid residues corresponding to structurally identical peptides used for C₂₀ peptomeric analogues synthesis and clickable peptoid residues used for clicked C₂₀ analogues.

<p>Structures of peptoid residues corresponding to structurally identical peptides used for C₂₀ peptomeric analogues synthesis and clickable peptoid residues used for clicked C₂₀ analogues.</p

Structural comparison of alanine, proline and sarcosine.

<p>Structural comparison of alanine, proline and sarcosine.</p

Helical wheel of C₂₀ peptide and relative % inhibition of singly substituted peptomers.

<p>(A) Peptide residues identified as tolerating positions for peptoid substitution are shown in shaded circle. The heptad position is based on hRSV F fusion protein sequence. (B) Based on the results shown in panel (A), peptomers with single peptoid substitution were prepared and their relative % inhibition to C₂₀ peptide at 100 µM were examined using a 5HB-based competitive FP assay.</p

Relative % inhibition and ratio of molar ellipticities at 222 and 208 nm.

<p>Terminally and internally clicked (C1C₂₀, C2C₂₀) and unclicked (UC1C₂₀, UC2C₂₀) peptomers and a stapled peptide analog (SC₂₀) as a positive control were tested. % Inhibition of structurally constrained C₂₀ analogues relative to C₂₀ peptide were measured at 100 µM shown in bars. The ratio of molar ellipticities (unit: deg·cm²·dmole^-1) at 222 and 208 nm is used to evaluate the helical content in each peptomer and is shown in red line (left y-axis).</p

Crystallographic data and refinement statistics.

<p>Crystallographic data and refinement statistics.</p

The inhibitory C-terminal extension forms a well-defined structure in crystals of the Ulster NA domain.

<p>(A) Crystal structure of the Ulster HN NA domain dimer, shows a dimer-of-dimers tetramer. The C-terminal extension is highlighted in dark blue and red in each pair of dimers. (B) The Ulster HN dimer is shown, with the C-terminal extension in dark blue. The C-terminal extension begins at the base of the β-propeller domain, extends along the outside of the dimer interface, and then rises above the active site before inserting the C-terminus into the receptor-binding site.</p

The C-terminal extension inhibits the NA domain active site.

<p>(A) Top view of a single HN NA domain subunit, showing superpositions with the NDV Kansas low-pH and DANA-bound crystal structures. The additional residues of the C-terminal extension are highlighted in blue and overlap the ligand bound in the active site. The D198 loop is indicated, which changes conformation in the Ulster HN structure, contributing to blocking the active site. (B,C) Detailed comparison of contacts made by the Ulster HN C-terminus and DANA sialic acid within the active site. Dotted lines represent hydrogen bonds and polar contacts identified by Pymol.</p

Electron density maps for key regions of the C-terminal extension.

<p>(A) Omit map electron density for C-terminal residues 611–615, which insert into the NA domain active site. (B) Omit map electron density for residues surrounding the interchain disulfide bond at C596. Residues associated with different chains of the observed NA domain dimers are indicated with an A: or B: prefix. (C) Omit map electron density C-terminal extension residues 583–588, which engage a second sialic acid binding site. The omit maps was calculated using the SFCHECK <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002855#ppat.1002855-Vaguine1" target="_blank">[46]</a> program and contoured at 1.57 sigma.</p