Critical intracellular signals in normal and malignant cells are transmitted by the adaptor protein Grb2 by means of its Src homology 2 (SH2) domain, which binds to phosphotyrosyl (pTyr) residues generated by the activation of tyrosine kinases. To understand this important control point and to design inhibitors, previous investigations have focused on the molecular mechanisms by which the Grb2 SH2 domain selectively binds pTyr containing peptides. In the current study, we demonstrate that the Grb2 SH2 domain can also bind in a pTyr independent manner. Using phage display, an 11-amino acid cyclic peptide, G1, has been identified that binds to the Grb2 SH2 domain but not the src SH2 domain. Synthetic G1 peptide blocks Grb2 SH2 domain association (IC 50 10 -25 M) with a 9-amino acid pTyrcontaining peptide derived from the SHC protein (pTyr317). These data and amino acid substitution analysis indicate that G1 interacts in the phosphopeptide binding site. G1 peptide requires a YXN sequence similar to that found in natural pTyr-containing ligands, and phosphorylation of the tyrosine increases G1 inhibitory activity. G1 also requires an internal disulfide bond to maintain the active binding conformation. Since the G1 peptide does not contain pTyr, it defines a new type of SH2 domain binding motif that may advance the design of Grb2 antagonists.The binding characteristics of Src homology 2 (SH2) 1 domains determine their important role as regulators of intracellular signaling (1, 2). Signal flow requires a phosphotyrosyl (pTyr) residue in the target protein for binding by the SH2 domain (1, 3, 4). Interaction of SH2 domains with specific pTyr-containing proteins activates distinct signaling pathways. SH2 domains modulate the activities of c-src (5), alter the substrate specificity of c-abl proto-oncoproteins (6, 7), and transduce signals initiated at growth factor receptors (8) and cellular attachment systems (9). SH2 domains have been suggested as promising sites for therapeutic intervention (10). Consequently, there has been significant effort to understand the structural basis of SH2 domain binding to pTyr-containing targets (11-21).The Grb2 SH2 domain binds pTyr-containing motifs within several proteins including the adapter proteins SHC (22, 23), growth factor receptors such as members of the erbB family (23-27), morphology-determining proteins such as FAK (9), and cellular oncogenes such as 28). SH2 domain binding leads to activation of important downstream pathways by bringing the nucleotide exchange factor SOS1 to the membrane environment of p21 ras (29). Other pathways may be initiated through action of the Grb2 SH3 domain as well. These pathways are suggested by experiments showing that the SH3 domains of Grb2 can bind to other proteins including dynamin (30), Vav (31, 32), Cbl (33), and several as yet unidentified targets (34). A particularly important role for Grb2 in human cancer has been proposed for cells transformed by high levels of erbB2 (HER-2 or neu) expression (35,36). In these cells, the SH2 domain...

Grb7 is an adapter-type signaling protein, which is recruited via its SH2 domain to a variety of receptor tyrosine kinases (RTKs), including ErbB2 and ErbB3. It is overexpressed in breast, esophageal, and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide attractive targets for therapeutic intervention. We have utilized phage display random peptide libraries as a source of small peptide ligands to the SH2 domain of Grb7. Screening these libraries against purified Grb7 SH2 resulted in the identification of Grb7-binding peptide phage clones that contained a nonphosphorylated Tyr-X-Asn (YXN) motif. The tyrosinephosphorylated form of this motif is characteristic of Grb7 SH2 domain binding sites identified in RTKs and other signaling proteins such as Shc. Peptides that are non-phosphorylated have greater potential in the development of therapeutics because of the instability of a phosphate group in vivo. Using a biased library approach with this conserved YXN motif, we identified seven different peptide phage clones, which bind specifically to the SH2 domain of Grb7. These peptides did not bind to the SH2 domain of Grb2 (which also selects for Asn at pY ؉2 ) or Grb14, a closely related family member. The cyclic structure of the peptides was required to bind to the Grb7 SH2 domain. Importantly, the synthetic Grb7-binding peptide G7-18 in cell lysates was able to specifically inhibit the association of Grb7 with the ErbB family of RTKs, in particular ErbB3, in a dose-dependent manner. These peptides will be useful in the development of targeted molecular therapeutics for cancers overexpressing Grb7 and in the development of Grb7-specific inhibitors to gain a complete understanding of the physiological role of Grb7.

Serial administration of an RPL is well tolerated and serial panning in individual mice leading to consensus sequence motifs is possible. Based on these preclinical data the Food and Drug Administration has approved the implementation of human clinical trials with this technique.

The selective delivery of therapeutic agents to receptors overexpressed in cancer cells without harming the rest of the body is a major challenge in clinical oncology today. In this study, we report the design and synthesis of paclitaxel (PTX) conjugated with an erbB2-recognizing peptide (EC-1). The cyclic peptide EC-1 specifically binds to the extracellular domain of ErbB2 and selectively inhibits proliferation of breast cancer cells overexpressing ErbB2. PTX is a potent antitumor agent commonly used in the treatment of advanced metastatic breast cancer, yet patients have to suffer some side effects caused by its systemic toxicity. The aim of our conjugate is to specifically deliver antitumor agent PTX to breast cancer cells that overexpress oncogenic ErbB2 with the purpose to reduce toxicity and enhance selective killing of cancer cells. In this study, a concise and efficient synthetic route for the preparation of the PTX-EC-1 conjugate has been developed in 6% overall yield. This synthetic approach provides a general method for conjugating a highly functionalized and disulfide-bridge containing cyclopeptide to Taxol or other antitumor agents.