Evaluation of rationally designed peptidomimetics for inhibition of protein-protein interactions of EGFRs in breast and lung malignancies

Protein-protein interactions (PPIs) propagate the extracellular and intracellular signals into the cells, which is required for the fundamental biological processes. Deregulation of PPIs results in many diseases such as cancers and autoimmune diseases. EGFR (Epidermal Growth Factor Receptor) receptor tyrosine kinase family is normally involved in signal transduction pathways leading to cell growth and differentiation. Overexpression of HER2, a member of EGFR family and deregulation of its signaling has implications in breast, ovarian and lung cancers. We have designed several peptidomimetics to block the HER2 mediated dimerization resulting in antiproliferative activity on cancer cells. Earlier studies in our lab have shown that a linear peptidomimetic compound 5, designed based on the crystal structure of HER-2 and trastuzumab has antiproliferative activity in nanomolar range. Linear peptides are enzymatically unstable due to the susceptibility to degradation by proteases or peptidases. The objective of this project was to design and investigate the structure-activity relationship of stable peptidomimetic analogs of linear compounds. Several strategies such as D-amino acid substitution, conformational constraining of the structure by cyclization and N- and C-termini modifications have been employed to impart metabolic stability to the structure. Among the compounds studied in this work, peptidomimetic compounds 18 and 21 in comparison with compound 5 exhibited better antiproliferative activity and selectivity for HER2 overexpressed breast, ovarian and lung cancer cell lines. The compounds were further evaluated for their ability to inhibit protein-protein interaction using enzyme complementation assay, proximity ligation assay and western blot analysis. Results suggested that compounds 18 and 21 are able to block HER2: HER3 interaction and inhibit phosphorylation of kinase domain of HER2. Besides, compounds have been evaluated for in vivo activity using athymic nude mice. Results suggested that HER2 heterodimerization is disrupted in the tumor tissues treated with compound 18. To evaluate the binding of compound to domain IV of HER2 protein, we have expressed and purified HER2 domain IV protein in S2 cell lines. Binding of compound 18 to HER2 protein was also studied by docking and SPR experiments. Peptidomimetic compound 18 showed a promising stability profile in vitro as well as in vivo. In conclusion, this work proves the significance of rationally designed peptidomimetics not only as potential therapeutic agents but also as valuable molecules to study the mechanism of PPIs.