Developing a protein-based assay for identifying HRSV entry inhibitors and knowledge-based approaches to design peptidomimetics [electronic resource]

Biotherapeutics have regained their reputation as drug candidates in the drug market due to their remarkable specificity; however, the broader clinical use of biotherapeutics is often challenged by poor pharmacological properties. Therefore, there is enormous interest in developing peptidomimetics as alternative therapeutic options. Inspired by the first successful example of peptide-based antivirals, Fuzeon®, similar strategies to develop antivirals have been applied to many other viruses that share class I fusion protein-mediated viral fusion. Using human Respiratory Syncytial Virus (hRSV) as a model system, the second chapter of this dissertation demonstrates our successful effort to develop a protein-based assay using a 5-Helix Bundle (5HB) fluorescence polarization (FP) as a screening platform for hRSV fusion inhibitors. The remaining chapters in this thesis all utilize the 5HB-based FP assay to evaluate the potential of short peptides and their peptoid-based peptidomimetics as antivirals to control hRSV infections. The third chapter proposes that NMEGylation, an alternative to PEGylation that uses a covalent attachment of an oligo-N-methoxyethylglycine (NMEG) chain, may enhance the bioavailability of short therapeutic peptides. The incorporation of optimized numbers of NMEG monomers along with a glycine linker increases the solubility and serum stability greatly, suggesting that NMEGylation may open a new opportunity to use peptoids as modifiers of therapeutically attractive peptides and proteins. The fourth chapter demonstrates how our novel approach combining alanine, proline, and sarcosine scans can be useful to determine peptoid-replaceable peptide residues and further proves the usefulness of the combined scan strategy using the C20 peptide as a parent peptide. Furthermore, two different methods to promote the [Alpha]-helical conformation of C20 analogues by structurally constraining the parent C20 peptide using "hydrocarbon-stapling" or "clicking" are described. We report a constrained C20 analogue with improved binding affinity, and discuss our structural investigations of the constrained C20 analogues using CD spectroscopy. The studies in the fifth chapter show that phage display can be used for identifying novel hRSV entry inhibitors that are not derived from the original sequence of the hRSV fusion protein F like the peptides described in chapters 2, 3, and 4. We report two 12-mer peptides with a low micromolar binding affinity to the 5HB, and ongoing efforts are aimed towards better understanding the interaction of the 12-mer peptides with the 5HB by co-crystallizing the peptides and 5HB. Finally, the dissertation concludes with a sixth chapter that summarizes the current status of each research chapter. In addition, future prospects, new directions, and potential applications of the findings in this dissertation are presented in this chapter.

Keywords: Chemical and Systems Biology.
Advisor: Barron Annelise E. primary advisor. Thesis advisor
Jardetzky Theodore primary advisor. Thesis advisor
Chen James Kenneth advisor. Thesis advisor
Cochran Jennifer R. advisor. Thesis advisor
Wandless Thomas advisor. Thesis advisor