Effects of Peptidic Antagonists of Grb2-SH2 on Human Breast Cancer Cells

Abstract

The growth factor receptor-bound protein Src homology 2 (Grb2-SH2) plays an important role in the oncogenic Ras signaling pathway, which involves in cell proliferation and differentiation. Therefore, the antagonist of Grb2-SH2 has become a potential target for developing anticancer agents. Recently, we discovered a peptide 1 (Fmoc-Glu-Tyr-Aib-Asn-NH(2)) with high affinity for the Grb2-SH2 domain by using surface plasmon resonance (SPR)-biosensor technology. Herein, we report the further design of the lead peptide 1 by addition of an Arg-Gly-Asp sequence to 1 to enhance binding to Grb2-SH2 and inducing apoptosis in cancer cells. Both the linear and cyclic analogs of the newly designed compound were prepared along with an analog in which the N(alpha)-Fmoc group was removed. These peptide analogs were assayed for their affinity for the Grb2-SH2, their antiproliferative effect on human breast cancer cells, their specificity for cancer cells, and their effects on cytotoxicity and the cell cycle. MCF-7 and MDA-MB-453 breast cancer cells were treated with various concentrations of each peptide. The cell viability and cytotoxicity of peptide-treated cells were determined by using the cell proliferation kit (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-tetrazolium bromide, MTT) and cytotoxicity kit (lactate dehydrogenase, LDH), respectively. Effects of peptides on the cell cycle progression of cancer cells and apoptosis were analyzed by using flow cytometry. Results demonstrated that the peptide analog 2 (H-Arg-Gly-Asp-Glu-Tyr-Aib-Asn-Arg-Gly-Asp-NH(2)) had anti-proliferative effects on MCF-7 and MDA-MB-453 cells with an IC(50) of 45.7 microM and 47.4 microM, respectively. The cytotoxicity and percentage of sub-G1 in the cell cycle were increased in these cancer cells when cells were treated with higher concentration of the Arg-Gly-Asp-containing peptide 2. These results provide important information for the development of anti-cancer agents.

Subcellular micropatterning for visual immunoprecipitation reveals differences in cytosolic protein complexes downstream the EGFR

“…For Gab1, copatterning was remarkably reduced by ~95% with a dissociation half-life of 2.6 min, whereas SOS1 copatterning was reduced by ~25% (dissociation half-life of 1.4 min), indicating a more pronounced affinity of actinomycin D for the C-terminal SH3 domain of Grb2, which mediates binding of Gab1. In recent years, high affinity Grb2-binding peptides have been developed to block Grb2 association to cell surface receptors (Chen et al, 2010; Noguchi et al, 2017) or binding to downstream molecules (Kardinal et al, 2001; Gril et al, 2007). We therefore further tested two known Grb2 inhibitors, the SH3 domain blocking peptide VPPPVPPRRR (Vidal et al, 1999) and the most recently described Grb2 SH2 domain inhibitor cyclo(YpVNFΦrpPRR) (Wen et al, 2020).…”

Oncogenic Role of Grb2 in Breast Cancer and Grb2 Antagonists as Therapeutic Drugs

“…Fmoc-Glu-Tyr-Aib-Asn-NH2, another novel peptide which has capability to target Grb2-SH2 domain in human breast cancer. The antagonistic activity of this peptide was investigated against MDA-MB-453 cells (cells with overexpressed erb-B2 expression driven through Grb2 signaling) and MCF-7 cells, concomitant data proved the anti-proliferative effects of peptide antagonist in breast cancer cells [49]. A fusion protein was constructed by joining PTD (an arginine rich peptide with ability to cross cell membrane) and Grb2-SH2 domain to target the EGFR and HER-2 via Grb2.…”

Molecular docking and dynamic studies of human growth factor receptorbound protein (Grb) 2 insights to identify novel inhibitors

“…5 Grb2 inhibitors were unclear in preclinical levels and some were under development . 5 The association mechanism of SH2 domain of Grb2 with SOS was crucial step for development of new anticancer agent s. 6 The present study was implemented wit h energy based (E) pharmacophore modeling, common pharmacophore hypothesis (CPH), multiple docking, G calculations and molecular dynamics (MD) simulations to identify novel lead molecules to inhibit over expression of Grb2 during angiogenesis.…”