Azapeptides are a class of peptide mimics (peptidomimetics), which have served as valuable tools for the development of peptide based therapeutic agents. The therapeutic promise of azapeptides has been correlated to its primary sequence modification which translates into bio-active secondary structures that improves the pharmacological properties of the native peptide sequence. More specifically, azapeptides contain a semicarbazide within the peptide backbone which restricts the peptide bond torsion angles (φ, ψ) into pre-organized b-turn secondary structures. Thus, azapeptides have been shown to stabilize bio-active b-turn secondary structures responsible for high affinity and selective binding to a target receptor or enzyme in order to modulate its activity for therapeutic purpose. Moreover, azapeptides have been found to be stable in biological media thereby improving their therapeutic indices and pharmacokinetic properties. For example, Goserelin, an aza-Gly peptide analog has received FDA approval in 1989 for the treatment of prostate and breast cancers. Therefore, the systematic substitution of aza amino acid residues within bio-active sequences (aza-amino acid scanning) has been shown to be useful in the conversion of native peptides into lead therapeutic peptidomimetics. The submonomer approach for azapeptides synthesis has been especially useful in aza-amino acid scanning and in the production of diverse azapeptides for structure-activity relationship studies with therapeutic targets. In this thesis, the submonomer approach for azapeptide synthesis is put into practical use for the development of azapeptide inhibitors of the Insulin Receptor Tyrosine Kinase (IRTK) domain.
Overexpression or unregulated signal transduction of the IRTK has been associated with increased levels of gene expression and cell proliferation that are hallmarks of tumor progression. Thus, the inhibition of un-regulated tyrosine kinase phosphorylation of the insulin receptor may prove to be an efficient method of cancer therapy. Towards this goal, the synthetic pentapeptide, Ac-DIYET-NH2 derived from the activation loop of the insulin receptor was found to inhibit the autophosphorylation of the IRTK to about 80% at 4 mM. Moreover, molecular docking simulation studies indicated that, Ac-DIYET-NH2, was bound within the active site of the IRTK with a folded peptide structure that was reminiscent of a turn geometry.
In order to identify the location and importance of a turn structure on the inhibitory activity of Ac-DIYET-NH2, aza-modifications within the IYE region were developed for structure activity relationship studies. Submonomer solid phase synthesis was used for the production of azapeptides, featuring the introduction of new aza-Ile and aza-DOPA residues. The azapeptides were analyzed and purified by reverse-phase LCMS in order to ascertain purity (\u3e95%) and identity prior to structure-activity relationship studies. Molecular modeling and docking simulation studies revealed that the Ac-DIazaYET-NH2 sequence, adopted a β-turn conformation that bound to the kinase domain of the IRTK. The azapeptide β-turn conformation was also proven by CD and NMR spectroscopy. The inhibitory activity of the peptides, Ac-DIYET-NH2 vs Ac-DIazaYET-NH2, was evaluated in a single dose experiment (400 mM), which indicated minimal inhibitory activity (2, whereas, Ac-DIazaYET-NH2 displayed 50% inhibition of the IRTK autophosphorylation. These results validate the importance of the peptide b-turn geometry on the inhibition of IRTK phosphorylation. This finding is not only important towards the development of potent azapeptide inhibitors of the IRTK for potential anti-cancer applications, but also in the design of novel probes for studying the mechanisms and kinetics associated with this important class of tyrosine kinases
