2784
C.-Q. Wei et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2781–2784
(6) for the facile synthesis of SH2 domain ligands, which
may exhibit high affinity in both extracellular and whole
cell systems. As such, compound 6 may represent a
useful tool for the preparation of signal transduction
antagonists and potentially, may find value in the
development of anticancer therapeutics.
11. Burke, T. R.; Liu, D. G.; Gao, Y. J. Org. Chem. 2000, 65,
6288.
12. Evans, D. A.; Britton, T. C.; Ellman, J. A. Tetrahedron
Lett. 1987, 28, 6141.
13. Konig, W.; Geiger, R. Chem. Ber. 1970, 103, 788.
14. Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
15. Liu, D.-G.; Yao, Z.-j.; Gao, Y.; Burke, T. R., Jr. Org.
Prep. Proc. Int. 2000, 32, 197.
16. A biotinated phosphopeptide encompassing a Grb2 SH2
domain-binding sequence derived from SHC protein, was
bound at 25 ng/mL to 96-well plates coated with streptavidin
(Roche Diagnostics GmbH #1645692) overnight. Nonspecific
interactions were inhibited by 5% bovine serum albumin con-
taining TBS. Samples of recombinant purified Grb2 SH2-GST
fusion protein were pre-incubated with serial dilutions of inhi-
bitors, then added into each well. After extensive washing with
0.1% bovine serum albumin in TBS, bound Grb2 SH2 domain
was detected using anti-GST antibodies and goat anti-mouse
antibody conjugated to alkaline phosphatase. Quantitation of
bound alkaline phosphatase was achieved by a colorimetric
reaction employing p-nitrophenylphosphate as substrate.
17. ErbB-2 over-expressing breast cancer cells, MDA-MB-
453, were treated with inhibitors (30, 10, 3, 1, 0 mM) for 4 h in
serum-free IMEM medium. Cells were washed twice with PBS
to remove inhibitor, then cell lysates were prepared using 1%
Triton X-100 in PBS containing 0.2 mM NaVO4. Grb2 and
associated Grb2-binding proteins were immunoprecipitated
from each lysate (250 mg) with anti-Grb2 antibodies and col-
lected using protein G Agarose (Roches Diagnostics GmbH
#1243233). Immunoprecipitated proteins were separated by
SDS-PAGE on 4–20% Tris–glycine gradient gels (Invitrogen/
Novex), and pTyr-containing proteins were detected by wes-
tern blotting using anti-phosphotyrosine antibodies (Santa
Cruz #sc-7020) and visualized with ECL (Amersham). Mem-
branes were subsequently stripped and Grb2 proteins were re-
probed with an antibody recognizing the total Grb2 protein
(Santa Cruz #sc-8034) as an internal immunoprecipitation
control. A major tyrosine phosphorylated protein in these cells
is the p185 erbB-2.
Acknowledgements
Appreciation is expressed to Dr. James Kelley of the
LMC for mass spectral analysis of synthetic materials.
This work was supported in part by the Susan G.
Komen Breast Cancer Foundation.
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