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Nigus D Ambaye,
Stephanie C Pero,
Menachem J Gunzburg,
MinYin Yap, Daniel J Clayton,
Mark P Del Borgo,
Patrick Perlmutter,
Marie-Isabel Aguilar,
Girja S Shukla,
Elena Peletskaya,
Michelle M Cookson,
David N Krag,
Matthew C J Wilce,
Jacqueline A Wilce
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ABSTRACT: Growth-receptor-bound protein (Grb)7 is an adapter protein aberrantly overexpressed, along with the erbB-2 receptor in breast cancer and in other cancers. Normally recruited to focal adhesions with a role in cell migration, it is associated with erbB-2 in cancer cells and is found to exacerbate cancer progression via stimulation of cell migration and proliferation. The G7-18NATE peptide (sequence: WFEGYDNTFPC cyclized via a thioether bond) is a nonphosphorylated peptide that was developed for the specific inhibition of Grb7 by blocking its SH2 domain. Cell-permeable versions of G7-18NATE are effective in the reduction of migration and proliferation in Grb7-overexpressing cells. It thus represents a promising starting point for the development of a therapeutic against Grb7. Here, we report the crystal structure of the G7-18NATE peptide in complex with the Grb7-SH2 domain, revealing the structural basis for its interaction. We also report further rounds of phage display that have identified G7-18NATE analogues with micromolar affinity for Grb7-SH2. These peptides retained amino acids F2, G4, and F9, as well as the YDN motif that the structural biology study showed to be the main residues in contact with the Grb7-SH2 domain. Isothermal titration calorimetry measurements reveal similar and better binding affinity of these peptides compared with G7-18NATE. Together, this study facilitates the optimization of second-generation inhibitors of Grb7.
Journal of Molecular Biology 07/2011; 412(3):397-411. · 4.00 Impact Factor
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Nigus D Ambaye,
Reece C C Lim, Daniel J Clayton,
Menachem J Gunzburg,
John T Price,
Stephanie C Pero,
David N Krag,
Matthew C J Wilce,
Marie-Isabel Aguilar,
Patrick Perlmutter,
Jacqueline A Wilce
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ABSTRACT: Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to Penetratin, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of Penetratin (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and colocalizes with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (K(d) = 14.4 microM), similar to the affinity of G7-18NATE (K(d) = 35.4 microM). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the antiproliferative and antimigratory potential of this inhibitor.
Biopolymers 01/2011; 96(2):181-8. · 2.87 Impact Factor
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Nigus D. Ambaye,
Reece C. C. Lim, Daniel J. Clayton,
Menachem J. Gunzburg,
John T. Price,
Stephanie C. Pero,
David N. Krag,
Matthew C. J. Wilce,
Marie-Isabel Aguilar,
Patrick Perlmutter,
Jacqueline A. Wilce
[show abstract]
[hide abstract]
ABSTRACT: Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to Penetratin©, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of Penetratin© (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and colocalizes with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (Kd = 14.4 μM), similar to the affinity of G7-18NATE (Kd = 35.4 μM). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the antiproliferative and antimigratory potential of this inhibitor. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 96: 181–188, 2011.
Biopolymers 12/2010; 96(2):181 - 188. · 2.87 Impact Factor
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ABSTRACT: Grb7 is an adapter protein that is involved in signalling pathways that mediate eukaryotic cell proliferation and migration. Its overexpression in several cancer types has implicated it in cancer progression and led to the development of the G7-18NATE cyclic peptide inhibitor. Here, the preparation of crystals of G7-18NATE in complex with its Grb7 SH2 domain target is reported. Crystals of the complex were grown by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant at room temperature. X-ray diffraction data were collected from crystals to 2.4 Å resolution using synchrotron X-ray radiation at 100 K. The diffraction was consistent with space group P2(1), with unit-cell parameters a=52.7, b=79.1, c=54.7 Å, α=γ=90.0, β=104.4°. The structure of the G7-18NATE peptide in complex with its target will facilitate the rational development of Grb7-targeted cancer therapeutics.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2010; 66(Pt 12):1640-3. · 0.51 Impact Factor
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Ylva E Bergman,
Mark P Del Borgo,
Romila D Gopalan,
Sania Jalal,
Sharon E Unabia,
Marisa Ciampini, Daniel J Clayton,
Jordan M Fletcher,
Roger J Mulder,
Jacqueline A Wilce,
Marie-Isabel Aguilar,
Patrick Perlmutter
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ABSTRACT: The first synthesis of carbon-stapled beta(3)-peptides is reported. The precursor beta(3)-peptides, with O-allyl beta-serines located in an i/i+3 relationship, were prepared on solid phase. We show that efficient ring-closing metathesis (RCM) of these new beta(3)-peptides proceeds smoothly either in solution or on an appropriate solid support. All products were generated with high selectivity for the E-isomer.
Organic Letters 09/2009; 11(19):4438-40. · 5.86 Impact Factor
