Nonphosphorylated Peptide Ligands for the Grb2 Src Homology 2 Domain

Abstract

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...

Specificity and affinity motifs for Grb2 SH2-ligand interactions

“…Approximately 40-50% of breast tumors display increased expression levels of members of the erbB family of receptor tyrosine kinases, and suppression of Grb2 function in these cells inhibits cell proliferation (11,12). Because of the clear potential of Grb2 inhibitors as therapeutic agents, significant interest has grown in the development of inhibitors of the Grb2 SH2 domain (5,13,14). We show here that the conventional affinity-based library selections for Grb2 SH2 ligands result in phosphopeptides that display cross-reactivity toward related SH2 domains and we define ligands that express a desirable specificity profile.…”

“…The binding preferences of SH2 domains have been studied extensively through the use of peptide libraries, and predictions for the optimal binding motifs for a large number of SH2 domains have been obtained in this manner (3,4). In addition, such binding motifs have been used extensively as lead structures for the design of selective small-molecular SH2 inhibitors (5,6). Importantly, in traditional library screening strategies used to define SH2 ligand motifs the selection of ligands is exclusively based on the strength of the SH2-phospho-peptide interaction.…”

Identification of Small Ligands Targeting Breast Cancer by In Vivo Screening of Peptide Libraries in Breast Cancer Patients

“…Fu^lre/tbe excrtmg work of Ruoslahti et al [2,60,61,65] Peptides and peptidomimetics are very promising targeting agents because they can potentially bind targets with the same exquisite specificity as antibodies, and are likely to have far more favorable pharmacokinetics. Peptides can have direct agonist or inhibitory activity on therapeutic targets.…”

“…Phage-displayed RPLs have been used by our lab and others to isolate small ligands, some with nanomolar and even picomolar affinity, to a large variety of targets including several potential tumor targets and other clinically important targets [2,18,60,61,65,90]. One example oftiie use of small peptides (8 and 12 mer) in targeting tumors has been reported by Renschier et al [66,67] 7/6/01 Version Breast Protocol for IRB Page 4 of 46 who used phage displayed RPLs to identify peptides that bind to the antigen binding recepte> of.BLphoma eels and induce apoptosis in vitro.…”

Identification of Small Ligands Targeting Breast Cancer by In Vivo Screening of Peptide Libraries in Breast Cancer Patients

“…Peptides that are conformationally constrained often possess higher affinity for a target than their linear counterpart [1]. In previous work in our laboratory, we identified a peptide that needed to be in a cyclic conformation in order to bind to the SH2 domain of Grb2 [2]. The libraries were designed so X = any amino acid encoded by NNK, where N -G, A, T, C and K= G or T. The NNK cloning scheme, a method commonly used in phage display, eliminates the potential for two stop codons and still encodes all twenty amino acids.…”