Improving the stability of bioactive peptides using protein-based motifs
Walker, Jennifer Renee
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Peptide instability and poor delivery in humans hinders the development of effective peptide drugs. To search for novel protective motifs for stabilizing peptides, an in vivo screen of randomized inhibitor peptides was developed and two protective motifs were identified. The first motif was generated by the fusion of the inhibitory peptides to the small stable protein, Rop. The second motif was generated by the placement of one or more prolines at the terminal ends of peptides. Repeating the in vivo screen with random peptides fused to either Rop or encoding terminal proline residues revealed an increase in the frequency of identifiable inhibitor peptides. Next, an in vitro method was developed to further investigate the novel proline protective motif. Randomized synthetic peptides beginning and ending with one proline, two proline or an alanine and two proline residues were tested for increased half-life in various eukaryotic and prokaryotic cell extracts. Peptides protected at the amino terminus with an alanine and two proline residues (APP) exhibited the longest half-lives. Molecular modeling methods suggested that the APP motif exhibited two possible conformers with one possessing two right angles along the peptide backbone which may cause steric hindrance at the terminus. This increased steric strain within the APP motif may be responsible for increased resistance to peptidases. Then, APP was modified in search for more protective motifs. The alanine residue was replaced with amino acids having an increased tendency to form a cis bond, which has been proven to resist some peptidases. Five modified motifs were tested for increased peptide half-life in rat serum. The three proline residues (PPP) substituted motif displayed the longest half-life. This motif may provide a level of protection surpassing that of other known protective motifs. While extended stability is one of many factors in peptide efficacy, uptake by the target cell is also important. A discovery was made that indicated biotinylated peptides could be transported across the membranes of Gram-negative bacteria via the biotin uptake pathway. Biotinylation may thus provide an alternative pathway for antibiotic peptide uptake.