Novel Peptidomimetics for Inhibition of HER2:HER3 Heterodimerization in HER2-Positive Breast Cancer

Abstract

The current approach to treating HER2-overexpressed breast cancer is the use of monoclonal antibodies or a combination of antibodies with traditional chemotherapeutic agents or kinase inhibitors. Our approach is to target clinically validated HER2 domain IV with peptidomimetics and inhibit the protein-protein interactions (PPI) of HERs. Unlike antibodies, peptidomimetics have advantages in terms of stability, modification, and molecular size. We have designed peptidomimetics (compounds 5 and 9) that bind to HER2 domain IV, inhibit protein-protein interactions, and decrease cell viability in breast cancer cells with HER2 overexpression. We have shown, using enzyme fragment complementation and proximity ligation assays, that peptidomimetics inhibit the PPI of HER2:HER3. Compounds 5 and 9 suppressed the tumor growth in a xenograft mouse model. Furthermore, we have shown that these compounds inhibit PPI of HER2:HER3 and phosphorylation of HER2 as compared to control in tissue samples derived from in vivo studies. The stability of the compounds was also investigated in mouse serum, and the compounds exhibited stability with a half-life of up to 3 h. These results suggest that the novel peptidomimetics we have developed target the extracellular domain of HER2 protein and inhibit HER2:HER3 interaction, providing a novel method to treat HER2-positive cancer.

Keywords: HER2; HER3; breast cancer; peptidomimetic; protein-protein interaction.

Figure 1

Proposed mechanism of inhibition of EGFR heterodimerization by peptidomimetic compounds 5/9.

Figure 2

Structures of compounds 9, 5, 8 and control (CP).

Figure 3

A) Inhibition of heterodimerization of HER2:HER3 in HER2, HER3 transfected U2OS cells by compound 9 at different concentrations using enzyme fragment complementation assay (DiscoveRx). Dose-response curve for inhibition of HER2:HER3 heterodimerization by 9 in the presence of 0.3 μM NRG1 (triangles). Control compound (CP) in the presence of 0.3 μM NRG-1 (filled squares). B) HER2:HER3 heterodimerization and its inhibition by compound 9 observed by PLA. Concentration-dependent inhibition of HER2:HER3 heterodimers was observed. SKBR-3 cells showing only HER2:HER3 heterodimerization as red spots. A control compound (CP) did not inhibit HER2:HER3 heterodimerization. At a sub-optimum dose of compound 9 (0.4 μM), HER2:HER3 heterodimerization was inhibited to a lesser extent. At optimum dose of compound 9 (0.8 μM), HER2:HER3 heterodimerization was significantly inhibited. C) Effect of compound 9 on phosphorylation of HER2 kinase domain. SKBR-3 cells that overexpress HER2 protein upon treatment with compound 9 (1 μM). Compound 5 and lapatinib (L) (0.07 μM) and control compound (CP) are shown for comparison. The visualization of GAPDH was used to ensure equal sample loading in each lane. Phosphorylation was detected using p-HER2 antibody. Total HER2 protein is also shown.

Figure 4

A) Effect of compounds on HER2 expression studied by flow cytometry. BT-474 cells without any FITC-labeled antibody to HER2, BT474 cells with FITC-antiHER2, cells treated with lapatinib and labeled with FITC-anti-HER2, cells treated with compound 9 with FITC-anti-HER2. B) Cell treated with compound 5.

Figure 5

Antitumorigenic activity of compounds 9 (diamonds) and 5 (crosses). Compounds 5 and 9 (4 mg/Kg) delayed the tumor growth in athymic nude mice significantly compared to the control group (filled triangles) without any treatment (standard error plotted). For comparison, tumor suppression by lapatinib is also shown (squares). It is very clear that there is a significant difference between the control and treatment groups.

Figure 6

H and E staining of tumor sections of mouse with and without compound treatments. A) Tumor tissue without any compound treatment, B) compound 5 treatment, C) compound 9 treatment. Sections from treated tumors (B, C) showed areas of necrosis that presented as eosinophilic debris with no cellular structures and nuclei.

Figure 7

Western blot analysis of the HER2 phosphorylation level of treatment and control groups. Compounds 5 and 9 significantly inhibited HER2 phosphorylation compared to the untreated control group.

Figure 8

Confocal images of PLA on breast tumor sections. A) PLA on control mouse without any treatment. Red color fluorescence indicate HER2:HER3 heterodimerization. PLA on mouse treated with 4 mg/kg of B) compounds 9 and C) 5. Compounds specifically inhibited HER2:HER3 heterodimerization significantly compared to the control without any treatment as indicated by the small number of red dots.

Figure 9

HER2 expression in tumor sections of mice evaluated by fluorescently labeled anti-HER2 antibody (green fluorescence). Tissue section without any antibody treatment and labeled with nuclear stain DAPI. Tissue section without any compound treatment, compounds 9, 5, and lapatinib treatment are shown. Notice the HER2 expression in all the tumors suggesting that compounds do not affect the expression of HER2 protein.

Figure 10

Stability of compounds 9 and 5 incubated in mouse serum and analyzed at different intervals of time. The amount of remaining intact peptide was detected by mass spectrometry using freshly prepared compound 8 an analog of compounds 9 and 5 as internal standard. A) compound 9, B) compound 5. Compound 5 was more stable compared to compound 9 in serum as seen from the graph.