Design, synthesis, and evaluation of fluorescent cell-penetrating peptidic antagonists of Grb2-SH2 for targeting MCF-7 breast cancer cells

The growth factor receptor-bound protein-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signaling pathway, which is involved in cell proliferation and differentiation. We focused on developing peptidic antagonists of the Grb2-SH2 domain as anti-proliferative agents, however, the bioavailability or effectiveness of peptidic antagonists may be reduced by the cell membrane, which constitutes a barrier for maintaining the integrity of the cell and thus prevents peptidic antagonists from entering the cell. The objective of this study is to enhance the cell permeability of synthetic peptides by conjugating the cell-penetrating peptide (CPP), Gly-Arg-Lys-Lys-Arg-Arg (TAT) or Tyr-Thr-Ala-Ile-Ala-Trp-Val-Lys-Ala-Phe-Ile-Arg-Lys-Leu-Arg-Lys (YTA), to our leading peptidic antagonist of Grb2-SH2, Fmoc-Glu-Tyr-Aib-Asn-NH 2 ( THUROC-1 ). Peptides were synthesized by solid-phase method, purified by RP-HPLC, and characterized by mass spectrometry. Effects of peptides on the viability of breast cancer cells MCF-7 were analyzed by MTT assay. Peptide 8 (Glu-Tyr-Aib-Asn-Tyr-Thr-Ala-Ile-Ala-Trp-Val-Lys-Ala-Phe-Ile-Arg-Lys-Leu-Arg-Lys-NH 2 ), which was designed by conjugating YTA to the C-terminus of de-Fmoc analog of THUROC-1 , exhibited greater anti-proliferative effect (IC 50 = 67.1 μM) than that of THUROC-1 . The cell permeability of peptide was evaluated by analyzing cells treated with peptide 10 using confocal microscope, and results suggested that the cell permeability of THUROC-1 was enhanced by conjugation with the CPP sequence, leading to the enhanced anti-proliferative effect on MCF-7 cells. In conclusion, we developed the CPP-conjugated peptide 8 with enhanced cell-permeability and increased anti-proliferative effects on MCF-7 cells.