Functional evaluation of fluorescein-labeled derivatives of a peptide inhibitor of the EGF receptor dimerization.

Abstract A cyclic decapeptide ( 1 , ), which acts on the extracellular region of the EGF receptor, preventing it from dimerizing, has been developed. Peptide 2 , which was labeled with fluorescein at the N-terminus of peptide 1 , was synthesized based on structure–activity relationship studies. Peptide 2 essentially retained the inhibitory activity of peptide 1 against the receptor autophosphorylation. Confocal microscopy studies revealed that in carcinoma cells, the fluorescence of peptide 2 was localized inside some vesicles. Treatment of intact cells by peptide 1 in combination with peptide 2 decreased the fluorescence intensity significantly compared to treatment with only peptide 2 . These results indicate that peptide 2 competes with peptide 1 for binding to the cellular surface. Six derivatives of peptide 2 , in which constituent amino acids, with the exception of two cysteines and proline were randomized, were synthesized and used to treat the cells. Peptides 6 and 9 showed the highest fluorescence intensity in cells. From the results of the EGF receptor autophosphorylation assay, these two derivatives were proven to have higher inhibitory activity than peptide 2 , which would therefore be a useful delivery peptide and fluorescent probe to find new inhibitors against the EGF receptor. Peptides 6 and 9 are promising leads for EGF receptor inhibitors.