Grb2 SH2 Domain Research Articles

Potentiating effect of distant sites in non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain

Without the presence of a phosphotyrosyl group, a phage library derived non-phosphorylated cyclic peptide ligand of Grb2-SH2 domain attributed its high affinity and specificity to well-defined and highly favored interactions of its structural elements with the binding pocket of the protein. We have disclosed a significant compensatory role of the Glu 2− sidechain for the absence of the phosphate functionality on Tyr 0 in the peptide ligand, cyclo(CH 2CO- Glu 2−-Leu- Tyr 0-Glu-Asn-Val-Gly- Met 5+-Tyr-Cys)-amide (termed G1TE). In this study, we report the importance of hydrophobic residue at the Tyr + 5 site in G1TE. Both acidic and basic amino acid substitutes are disfavored at this position, and replacement of Met with β- tert-butyl-Ala was found to improve the antagonist properties. Besides, the polarity of the cyclization linkage was implicated as important in stabilizing the favored binding conformation. Oxidation of the thioether linkage into sulfoxide facilitated the binding to Grb2-SH2 markedly. Simultaneous modification of the three distant sites within G1TE provided the best agent with an IC 50 of 220 nM, which is among the most potent non-phosphorous- and non-phosphotyrosine-mimic containing Grb2-SH2 domain inhibitors yet reported. This potent peptidomimetic provides a novel template for the development of chemotherapeutic agents for the treatment of erbB2-related cancer. Biological assays on G1TE( Gla 2− ) in which the original residue of Glu 2− was substituted by γ-carboxyglutamic acid (Gla) indicated that it could inhibit the interaction between activated GF receptor and Grb2 protein in cell homogenates of MDA-MB-453 breast cancer cells at the 2 μM level. More significantly, both G1TE( Gla 2− ) alone and the conjugate of G1TE( Gla 2− ) with a peptide carrier can effectively inhibit intracellular association of erbB2 and Grb2 in the same cell lines with IC 50 of 50 and 2 μM, respectively.

A novel macrocyclic tetrapeptide mimetic that exhibits low-picomolar Grb2 SH2 domain-binding affinity

The growth factor receptor-bound protein 2 (Grb2) is an SH2 domain-containing docking module that participates in the signaling of numerous oncogenic growth factor receptor protein-tyrosine kinases (PTKs). Presented herein is a 5-methylindolyl-containing macrocyclic tetrapeptide mimetic ( 5) that binds to Grb2 SH2 domain protein with K d=75 pM. This represents the highest affinity yet reported for a synthetic inhibitor against any SH2 domain. In whole cell assays this novel analogue is able to effectively block the association of Grb2 to cognate cytoplasmic erbB-2 at IC 50<10 nM without prodrug derivatization or the addition of carrier peptide motifs. Anti-mitogenic effects against erbB-2-dependent breast cancers are achieved at non-cytotoxic concentrations ( IC 50=0.6 μ M). Macrocycle 5 may be representative of a new class of therapeutically relevant Grb2 SH2 domain-directed agents.

A Ras activation pathway dependent on Syk phosphorylation of protein kinase C.

Protein kinase C (PKC) and Syk protein tyrosine kinase play critical roles in immune cell activation including that through the high-affinity IgE receptor, FcepsilonRI. Mechanisms by which PKC activation leads to the activation of Ras, a family of GTPases essential for immune cell activation, have been elusive. We present evidence that Tyr-662 and Tyr-658 of PKCbetaI and PKCalpha, respectively, are phosphorylated by Syk in the membrane compartment of FcepsilonRI-stimulated mast cells. These phosphorylations require prior PKC autophosphorylation of the adjacent serine residues (Ser-661 and Ser-657, respectively) and generate a binding site for the SH2 domain of the adaptor protein Grb-2. By recruiting the Grb-2/Sos complex to the plasma membrane, these conventional PKC isoforms contribute to the full activation of the Ras/extracellular signal-regulated kinase signaling pathway in FcepsilonRI-stimulated mast cells.

Global optimization of conformational constraint on non-phosphorylated cyclic peptide antagonists of the Grb2-SH2 domain

Following our earlier work on a phage library derived non-phosphorylated thioether-cyclized peptide inhibitor of Grb2 SH2 domain, a series of small peptide analogues with various cyclization linkage or various ring size were designed and synthesized and evaluated to investigate the optimal conformational constraint for this novel Grb2-SH2 blocker. Our previous SAR studies have indicated that constrained conformation as well as all amino acids except Leu 2 and Gly 7 in this lead peptide, cyclo(CH 2CO-Glu 1-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr-Cys 10)-amide (termed G1TE), was necessary for sustenance of the biological activity. In this study, in an effort to derive potent and bioavailable Grb2-SH2 inhibitor with minimal sequence, we undertook a systematic conformational study on this non-phosphorylated cyclic ligand by optimizing the ring linkage, ring configuration and ring size. The polarity and configuration of the cyclization linkage were implicated important in assuming the active conformation. Changing the flexible thioether linkage in G1TE into the relatively rigid sulfoxide linkage secured a 4-fold increase in potency ( 4, IC 50=6.5 μM). However, open chain, shortening or expanding the ring size led to a marked loss of inhibitory activity. Significantly, the introduction of ω-amino carboxylic acid linker in place of three C-terminal amino acids in G1TE can remarkably recover the apparently favorable conformation, which is otherwise lost because of the reduced ring size. This modification, combined with favorable substitutions of Gla for Glu 1 and Adi for Glu 4 in the resulting six-residue cyclic peptide, afforded peptide 19, with an almost equal potency ( 19, IC 50=23.3 μM) relative to G1TE. Moreover, the lipophilic chain in ω-amino carboxylic acid may confer better cell membrane permeability to 19. These newly developed G1TE analogues with smaller ring size and less peptide character but equal potency can serve as templates to derive potent and specific non-phosphorylated Grb2-SH2 antagonists.

Concise and enantioselective synthesis of Fmoc-Pmp(But)2-OH and design of potent Pmp-containing Grb2-SH2 domain antagonists.

[reaction: see text] L-Phosphonomethylphenylalanine (L-Pmp) is an important phosphatase-resistant pTyr analogue. A most concise and stereoselective approach to the synthesis of the suitably protected Fmoc-Pmp(Bu(t))(2)-OH was developed in order to incorporate the functionally significant L-Pmp residue into peptides and peptidomimetics efficiently using standard Fmoc protocol. With this key building block, we are able to efficiently synthesize a series of potent Pmp-containing Grb2-SH2 domain antagonists, which can be used as chemotherapeutic leads for the treatment of erbB2-overexpressed breast cancer.

Activation of Ras by Grb2-SOS: Demonstrating an Assembly Role of SH3 Domains

The animation shows a schematic representation of the assembly role of SH3 domain-containing proteins using Grb2 as an example. Growth factor stimulation leads to the activation of receptor tyrosine kinases and to the phosphorylation of the receptor tail and the stimulation of phosphorylation of related adaptor proteins (not shown). The resultant phosphotyrosines form docking sites for the adaptor protein Grb2 (through its SH2 domain). The Grb2 SH3 domains bind proline-rich motifs in SOS, the guanine nucleotide exchange factor for Ras, recruiting SOS to the membrane and colocalizing it with Ras. The resultant stimulation of Ras activates a mitogen-activated protein kinase cascade, leading to cell growth and differentiation. The animation could be useful in demonstrating how different protein domains allow dynamic regulation of protein activity, protein assembly, and protein recruitment, allowing cells to respond to signals from their extracellular environment. [ Resource Details ]

Structural basis for a non-phosphorus-containing cyclic peptide binding to Grb2-SH2 domain with high affinity

Blocking the interaction between phosphotyrosine (pTyr)-containing activated receptors and the Src homology 2 (SH2) domain of the growth factor receptor bound protein 2 (Grb2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferative agents due to its potential to shut down the Ras activation pathway. Generally, the pTyr-X-Asn minimal binding motif is required for a high-affinity ligand binding to the Grb2-SH2 domain. Using phage-display techniques, we discovered a non-pTyr-containing cyclic peptide G1 with moderate binding affinity from 10 7 different sequences. To understand the structural basis for the high-affinity binding of these novel non-phosphorus-containing inhibitors to the Grb2-SH2 domain, we extensively studied herein the unique functional requirements of the acidic side chain in Tyr-2 position due to the absence of the phosphate group in these non-phosphorylated peptides. A comprehensive SAR study was also carried out to develop potent Grb2-SH2 domain antagonists based upon this novel template. With both the peptidomimetic optimization of the amino acid side-chains and the constraint of the backbone conformation guided by molecular modeling, we developed several potent antagonists with low nanomolar range binding affinity, such as cyclic peptide 20 with an IC 50=0.026 μ M, which is one of the most potent non-phosphorus-containing Grb2-SH2 antagonists reported to date. Whole cell assays indicate that peptide 20 can penetrate the cell membranes and inhibit the association of Grb2 with p185 erbB2 in erbB2-overexpressing MDA-MA-453 cancer cells at low micromolar concentrations.

Identification of AMSH-LP containing a Jab1/MPN domain metalloenzyme motif

We have isolated a cDNA clone encoding a new AMSH ( a ssociated m olecule with the SH 3 domain of STAM) family protein, termed AMSH-like protein (AMSH-LP). AMSH-LP has similar characteristics to AMSH; both AMSH-LP and AMSH are expressed ubiquitously in various human tissues, contain a putative nuclear localization signal (NLS), an M pr/ P ad1/ N -terminal (MPN) domain, and a Jab1/MPN domain metalloenzyme (JAMM) motif in their structures, and are excluded from the nucleus when lacking either the NLS or MPN domain. Moreover, we observed an enhancement of interleukin 2 (IL-2)-mediated c- myc induction in AMSH-LP-transfected cells similar to that seen in AMSH-transfected cells, suggesting a functional similarity between AMSH-LP and AMSH. However, the present study demonstrated that AMSH-LP, unlike AMSH, fails to bind to the SH3 domains of STAM1 ( s ignal t ransducing a daptor m olecule 1) and Grb2. These results suggest that AMSH-LP and AMSH may have different functions.

Utilization of a beta-aminophosphotyrosyl mimetic in the design and synthesis of macrocyclic Grb2 SH2 domain-binding peptides.

Grb2 SH2 domains are protein-docking modules that exert important functions in both normal and pathogenic signal transduction processes. Development of synthetic Grb2 SH2 domain binding ligands is being pursued by several groups as potential new therapies for a variety of diseases, including certain cancers. In these efforts, macrocyclization has been successfully utilized to take advantage of preferential recognition by Grb2 SH2 domains of ligands in beta-bend conformations. Recent examples of this approach include olefin-metathesis-derived macrocycles that employ ring closure at the beta-position of key pTyr-mimicking residues. In the current study, a novel phosphatase-stable beta-amino-pTyr mimetic designated "Pmp(beta)" was utilized to prepare variants of previously reported olefin-metathesis-derived macrocycles. An initial set of simplified cyclic peptides lacking key naphthyl side chain functionality was first synthesized to determine optimum ring size, with results indicating that a four-unit ring-closing segment was appropriate. On the basis of these findings, macrolactamization was undertaken with a more highly functionalized, naphthyl-containing gamma-amino acid analogue. The resulting cyclic beta-amino peptide is the first of a new class of pTyr-mimetic-containing ligands that may have utility in the development of antagonists of both Grb2 SH2 domains and other pTyr-dependent signaling systems.

Potent Grb2-SH2 domain antagonists not relying on phosphotyrosine mimics.

Development of Grb2-SH2 domain antagonists is an effective approach to inhibit the growth of malignant cells by modulating Grb2-related Ras signaling. We report here potent Grb2-SH2 domain antagonists that do not rely on phosphotyrosine or its mimics. These non-phosphorylated antagonists were developed and further modified by constraining the backbone conformation and optimizing amino acid side chains of a phage library-derived peptide, G1TE. After extensive SAR studies and structural optimization, non-phosphorylated peptide 12 was discovered with an IC(50) of 75 nM. This potent peptidomimetic provides a novel template for the development of non-pTyr containing Grb2-SH2 domain antagonists and acts as a chemotherapeutic lead for the treatment of erbB2-related cancer.

Molecular dynamics, free energy, and SPR analyses of the interactions between the SH2 domain of Grb2 and ErbB phosphotyrosyl peptides.

We studied the interactions between the SH2 domain of growth factor receptor binding protein 2 (Grb2) and ErbB receptor-derived phosphotyrosyl peptides using molecular dynamics, free energy calculations, and surface plasmon resonance (SPR) analysis. Binding free energies for nine phosphotyrosyl peptides were calculated using the MM-PBSA continuum solvent method, and excellent qualitative agreement with the SPR experimental data, with a correlation coefficient of 0.92, was obtained. Consistent with previous experimental findings, phosphotyrosyl peptides with the consensus sequence pYXNX showed favorable binding affinity for the Grb2. Unexpectedly, phosphotyrosyl peptides with the consensus sequence pYQQD, which had not shown any specific binding affinity for the Grb2 in earlier studies, also showed favorable binding affinity for the Grb2 in our experimental and computational analyses. Component analysis of the calculated binding free energies revealed that van der Waals interaction between the Grb2 and the phosphotyrosyl peptide was the dominant factor for specificity and binding affinity. These results indicate that current methods of estimating binding free energies are efficient for obtaining important information about protein-protein interactions, which are essential for the transmission of signals in cellular signaling pathways.

Structure-based design of thioether-bridged cyclic phosphopeptides binding to Grb2-SH2 domain

A series of phosphotyrosine containing cyclic peptides was designed and synthesized based upon the phage library derived cyclopeptide, G1TE. Considering the type-I β-turn feature of peptidic ligand binding to Grb2 SH2 domain, we introduce α,α-disubstituted cyclic amino acid, Ach, into the 4th position of the cyclic peptide to induce a local right handed 3 10 helical conformation. In order to stabilize the favorable binding conformation, the bulky and hydrophobic amino acids, neopentylglycine (NPG) and phenylalanine, were introduced into the 8th and 2nd positions of the peptide ligand, respectively. To facilitate the sidechain of pTyr3 reaching into the phosphotyrosine binding pocket, a less bulky alanine was preferred in position 1. Based upon these global modifications, a highly potent peptide ligand 12 was discovered with an IC 50=1.68 nM, evaluated by ELISA binding essay. Ligand 12 is at least 10 5 more potent than the lead peptide, termed G1TE.

Design and synthesis of conformationally constrained, extended and reverse turn pseudopeptides as Grb2-SH2 domain antagonists

A series of conformationally constrained and flexible pseudopeptide derivatives of the tripeptide pYVN were prepared as potential antagonists of interactions of phosphotyrosine peptides with the Grb2-SH2 domain. The conformationally constrained compounds contained trans- and cis-cyclopropanes as replacements to enforce locally extended and reverse turn peptide conformations, respectively.

Manipulation of EphB2 Regulatory Motifs and SH2 Binding Sites Switches MAPK Signaling and Biological Activity

Signaling by the Eph family of receptor tyrosine kinases (RTKs) is complex, because they can interact with a variety of intracellular targets, and can potentially induce distinct responses in different cell types. In NG108 neuronal cells, activated EphB2 recruits p120RasGAP, in a fashion that is associated with down-regulation of the Ras-Erk mitogen-activated kinase (MAPK) pathway and neurite retraction. To pursue the role of the Ras-MAPK pathway in EphB2-mediated growth cone collapse, and to explore the biochemical and biological functions of Eph receptors, we sought to re-engineer the signaling properties of EphB2 by manipulating its regulatory motifs and SH2 binding sites. An EphB2 mutant that retained juxtamembrane (JM) RasGAP binding sites but incorporated a Grb2 binding motif at an alternate RasGAP binding site within the kinase domain had little effect on basal Erk MAPK activation. In contrast, elimination of all RasGAP binding sites, accompanied by the addition of a Grb2 binding site within the kinase domain, led to an increase in phospho-Erk levels in NG108 cells following ephrin-B1 stimulation. Functional assays indicated a correlation between neurite retraction and the ability of the EphB2 mutants to down-regulate Ras-Erk MAPK signaling. These data suggest that EphB2 can be designed to repress, stabilize, or activate the Ras-Erk MAPK pathway by the manipulation of RasGAP and Grb2 SH2 domain binding sites and support the notion that Erk MAPK regulation plays a significant role in axon guidance. The behavior of EphB2 variants with mutations in the JM region and kinase domains suggests an intricate pattern of regulation and target recognition by Eph receptors.

Analysis of protein tyrosine kinase inhibitors in recombinant yeast lacking the ERG6 gene.

Studies of small-molecule-protein interactions in yeast can be hindered by the limited permeability of yeast to small molecules. This diminished permeability is thought to be related to the unique sterol composition of fungal membranes, which are enriched in the steroid ergosterol. We report the construction of the novel Saccharomyces cerevisiae yeast strain DCY250, which is compatible with yeast two-hybrid-based systems and bears a targeted disruption of the ERG6 gene to ablate ergosterol biosynthesis and enhance permeability to small molecules. The small-molecule inhibitors of protein tyrosine kinases (PTKs) PP1, PP2, herbimycin A, and staurosporine were investigated with yeast tribrid systems that detect the activity of the PTKs v-Abl and v-Src. These tribrid systems function by expression of the PTK, a B42 activation domain fused to the phosphotyrosine-binding Grb2 SH2 domain, a DNA-bound LexA-GFP-(AAYANAA)(4) universal PTK substrate, and a lacZ reporter gene. Yeast genetic systems that lack functional ERG6 were found to be as much as 20-fold more sensitive to small-molecule inhibitors of PTKs than systems with ERG6, and these deficient systems may provide a useful platform for the discovery and analysis of small-molecule-protein interactions.

Cyclic phosphopeptides for interference with Grb2 SH2 domain signal transduction prepared by ring-closing metathesis and phosphorylation.

Cyclic phosphopeptides were prepared using ring-closing metathesis followed by phosphorylation. These cyclic phosphopeptides were designed to interact with the SH2 domain of Grb2, which is a signal transduction protein of importance as a target for antiproliferative drug development. Binding of these peptides to the Grb2 SH2 domain was evaluated by a surface plasmon resonance assay. High affinity binding to the Grb2 SH2 domain was maintained upon macrocyclization, thus indicating that this method can be used to assemble high affinity cyclic phosphopeptides that interfere with signal transduction cascades.

Editorial: “Current trends in peptide science”

The “Current Trends in Peptide Science” reviews in this issue feature papers on a variety of peptide approaches to combating human diseases, including peptide antimicrobials, inhibitors of enzymes, anticancer agents, and vaccines. In the first paper, Bryant, Lazarus, and their colleagues describe advances in opioid peptide research made through the introduction of the 2′6′-dimethyl-L-tyrosine (Dmt) residue into opioidmimetics, particularly in the context of Dmt-Tic compounds. The authors review the ability of Dmt—when substituted for Tyr in opioid peptides—to enhance the interaction and accompanying biological activities of the resulting compounds with respect to δ and μ-opioid receptors. Structural data on these materials are presented which have facilitated the development of pharmacophores for both δ-opioid receptor agonists and antagonists. Through studies of mixture-based peptide synthetic combinatorial libraries, Blondelle and her colleagues then describe their advances in the development of antimicrobial and antiviral peptides, and their application to the control of infectious diseases. Noting that immunization with peptides can induce humoral and cellular immune responses mediated by antibody and T lymphocytes, respectively, the authors further describe their efforts in evaluating synthetic peptides as vaccines, including applications to HIV-1 proteins. In the following paper, Cooperman reviews research into class I ribonucleotide reductases (RR's), which are established targets for cancer chemotherapeutic and antiviral agents. Oligopeptides are described which specifically inhibit the RR's from which they are derived; structural work on these peptides is presented which has led to a model compatible with binding data, from which detailed knowledge could lead to peptide inhibitors selective for tumor cells. Focusing on the key role of the growth factor receptor-bound protein 2 (Grb2) in the Ras signaling pathway, which renders Grb2 a potential target for the design of antitumor agents, Lung and Tsai review current approaches to the development of Grb2 SH2 domain peptide inhibitors intended to interrupt Grb2 recognition. The authors delineate the classes of inhibitors used in this context, which range from those containing nonhydrolyzable mimetics of phosphates to nonphosphorylated cyclic peptide ligands for the Grb2 SH2 domain. Then, Cachia and Hodges provide a comprehensive discussion of synthetic peptide vaccines which may have utility in the prevention and treatment of Pseudomonas aeruginosa—an opportunistic pathogen that causes urinary tract and respiratory system infections, and which becomes extremely difficult to treat in cystic fibrosis patients. These authors describe their progress in development of anti-adhesin peptides targeted at the prevention of host cell attachment, and toward the elucidation of rules in consensus sequence design that can facilitate synthetic peptide vaccine development. These reviews highlight and re-state the emerging importance of the diverse roles of peptides in potential treatment of human disorders ranging from cancer to HIV to cystic fibrosis. We hope that readers discover, and share, the optimism inherent in these reports.

Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.

Solution structure of the human Grb7-SH2 domain/erbB2 peptide complex and structural basis for Grb7 binding to ErbB2.

The solution structure of the hGrb7-SH2 domain in complex with a ten amino acid phosphorylated peptide ligand representative of the erbB2 receptor tyrosine kinase (pY1139) is presented as determined by nuclear magnetic resonance methods. The hGrb7-SH2 domain structure reveals the Src homology 2 domain topology consisting of a central beta-sheet capped at each end by an alpha-helix. The presence of a four residue insertion in the region between beta-strand E and the EF loop and resulting influences on the SH2 domain/peptide complex structure are discussed. The binding conformation of the erbB2 peptide is in a beta-turn similar to that found in phosphorylated tyrosine peptides bound to the Grb2-SH2 domain. To our knowledge this is only the second example of an SH2 domain binding its naturally occurring ligands in a turn, instead of extended, conformation. Close contacts between residues responsible for binding specificity in hGrb7-SH2 and the erbB2 peptide are characterized and the potential effect of mutation of these residues on the hGrb7-SH2 domain structure is discussed.

Macrocyclization in the design of Grb2 SH2 domain-binding ligands exhibiting high potency in whole-cell systems.

While most SH2 domains bind phosphotyrosyl (pTyr) containing peptides in extended fashion, the growth factor receptor-bound protein 2 (Grb2) SH2 domain preferentially binds ligands in bend conformations. Accordingly, incorporation of bend-inducing functionality into synthetic ligands could potentially enhance their affinity for this SH2 domain. A macrocyclic tripeptide mimetic that contains a simplified pTyr surrogate lacking an alpha-nitrogen has recently been shown to exhibit high Grb2 SH2 domain-binding affinity in extracellular ELISA-based assays. However, the same compound is largely ineffective in whole-cell assays. It is known that acidic functionality originating from the alpha-nitrogen of pTyr residues or from the alpha-position of P0 pTyr mimetics not only increases binding affinity of peptides to Grb2 SH2 domains in extracellular assays but also enhances potency in cell-based systems. Such functionality is absent from the previously reported macrocycle. Therefore, the current study was undertaken to examine the effects of introducing carboxylic functionality at the pTyr mimetic alpha-position of macrocyclic ligands. It was found that such a modification not only enhanced Grb2 SH2 domain binding in extracellular assays but also conferred high efficacy in whole-cell systems. The most potent compound of the current study exhibited an IC(50) value of 0.002 microM in an extracellular ELISA-based assay, and in MDA-MB-453 cells, it both inhibited the association of Grb2 with p185(erbB-2) and exhibited antimitogenic effects with submicromolar IC50 values.