Human respiratory syncytial virus (hRSV) typically affects newborns and young children. Even though it can cause severe and, in some cases, lifelong respiratory infections, there are currently no FDA-approved therapeutics that control this virus. The hRSV F protein facilitates viral fusion, a critical extracellular event that can be targeted for therapeutic intervention by disrupting the assembly of a post-fusion 6-Helix bundle (6HB) within the hRSV F protein. Here we report the development of a fluorescence polarization assay using an engineered hRSV F protein 5-Helix Bundle (5HB). We generated the 5HB and validated its ability to form a 6HB in a fluorescence polarization assay. To test the potential of 5HB as a screening tool, we then investigated a series of truncated peptides derived from the “missing” sixth helix. Using this FP-based 5HB system, we have successfully demonstrated that short peptides can prevent 6HB formation and serve as potential hRSV fusion inhibitors. We anticipate this new 5HB system will provide an effective tool to identify and study potential antivirals to control hRSV infection.
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.
We have evaluated “NMEGylation”—the covalent attachment of a oligo-N-methoxyethylglycine (NMEG) chain—as a new form of peptide/protein modification to enhance the bioavailability of short peptides. OligoNMEGs are hydrophilic PEG-like molecules made by solid-phase synthesis, typically up to 40 monomers in length. They have been studied as non-fouling surface coatings, and as monodisperse mobility modifiers for free-solution conjugate capillary electrophoresis. However, polyNMEGs have not been demonstrated prior to this work as modifiers of therapeutic proteins. In prior published work, we identified a short peptide, “C20”, as a potential extracellular inhibitor of the fusion of human respiratory syncytial virus (hRSV) with mammalian cells. The present study was aimed at improving the C20 peptide's stability and solubility. To this end we synthesized and studied a series of NMEGylated C20 peptide-peptoid bioconjugates comprising different numbers of NMEGs at either the N- or C- terminus of C20. NMEGylation was found to greatly improve this peptide's solubility and serum stability; however, longer polyNMEGs (n > 3) deleteriously affected peptide binding to the target protein. By incorporating just one NMEG monomer, along with a glycine monomer as a flexible spacer, at C20's N-terminus (NMEG-Gly-C20), we increased both solubility and serum stability greatly, while recovering a binding affinity comparable to that of unmodified C20 peptide. Our results suggest that NMEGylation with an optimized number of NMEG monomers and a proper linker could be useful, more broadly, as a novel modification to enhance bioavailability and efficacy of therapeutic peptides.
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