Screening of a library of T7 phage-displayed peptides identifies alphaC helix in 14-3-3 protein as a CBP501-binding site

• Published in: Bioorganic & medicinal chemistry Vol. 19; no. 23; pp. 7049 - 7056
• Main Authors: Matsumoto, Yuki, Shindo, Yosuke, Takakusagi, Yoichi, Takakusagi, Kaori, Tsukuda, Senko, Kusayanagi, Tomoe, Sato, Hitoshi, Kawabe, Takumi, Sugawara, Fumio, Sakaguchi, Kengo
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ltd 01.12.2011; Elsevier
• Subjects: 14-3-3 Proteins - genetics; 14-3-3 Proteins - metabolism; 14-3-3ε; Amino Acid Sequence; Bacteriophage T7 - genetics; Bacteriophage T7 - metabolism; Binding Sites; Biological and medical sciences; CBP501; cdc25 Phosphatases - metabolism; Circular Dichroism; G2 checkpoint; Humans; Jurkat Cells; Medical sciences; Miscellaneous; Models, Molecular; Molecular Sequence Data; Peptide; Peptide Fragments - metabolism; Peptide Library; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Pharmacology. Drug treatments; Protein Structure, Secondary; T7 phage display; 14-3-3ε; G2 checkpoint; T7 phage display; Peptide; CBP501; D stereoisomer; Biotinylation; Oligopeptides; Peptides; Molecular structure; Binding site; Enzyme immunoassay; Secondary structure; Screening; Phage display; Chemical compound library; Circular dichroism spectrometry; Surface plasmon resonance; Helical structure; ELISA assay
• ISSN: 0968-0896; 1464-3391
• DOI: 10.1016/j.bmc.2011.10.004

CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix.