Functional evaluation of fluorescein-labeled derivatives of a peptide inhibitor of the EGF receptor dimerization.
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.
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan.
Kei
K
Toyama
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan.
Takaaki
T
Mizuguchi
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan.
Wataru
W
Nomura
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan. Electronic address: tamamura.mr@tmd.ac.jp.
Hirokazu
H
Tamamura
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D066246:ErbB Receptors / Q000378:metabolism*; D019793:Fluorescein / Q000737:chemistry*; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization
D010455:Peptides; C512478:EGFR protein, human; D066246:ErbB Receptors; D019793:Fluorescein
Cyclic peptide; Dimerization arm; EGF receptor; Fluorescein-labeled peptide; Intracellular delivery