Evaluation of Cyclic Peptide Inhibitors of the Grb7 Breast Cancer Target: Small Change in Cargo Results in Large Change in Cellular Activity.

Grb7 is an adapter protein, overexpressed in HER2+ve breast and other cancers, and identified as a therapeutic target. Grb7 promotes both proliferative and migratory cellular pathways through interaction of its SH2 domain with upstream binding partners including HER2, SHC, and FAK. Here we present the evaluation of a series of monocyclic and bicyclic peptide inhibitors that have been developed to specifically and potently target the Grb7 SH2-domain. All peptides tested were found to inhibit signaling in both ERK and AKT pathways in SKBR-3 and MDA-MB-231 cell lines. Proliferation, migration, and invasion assays revealed, however, that the second-generation bicyclic peptides were not more bioactive than the first generation G7-18NATE peptide, despite their higher in vitro affinity for the target. This was found not to be due to steric hindrance by the cell-permeability tag, as ascertained by ITC, but to differences in the ability of the bicyclic peptides to interact with and penetrate cellular membranes, as determined using SPR and mass spectrometry. These studies reveal that just small differences to amino acid composition can greatly impact the effectiveness of peptide inhibitors to their intracellular target and demonstrate that G7-18NATE remains the most effective peptide inhibitor of Grb7 developed to date.

Discovery, Development, and Cellular Delivery of Potent and Selective Bicyclic Peptide Inhibitors of Grb7 Cancer Target.

Grb7 is a signaling protein with critical roles in tumor cell proliferation and migration and an established cancer therapeutic target. Here we explore chemical space to develop a new bicyclic peptide inhibitor, incorporating thioether and lactam linkers that binds with affinity (KD = 1.1 µM) and specificity to the Grb7-SH2 domain. Structural analysis of the Grb7-SH2/peptide complex revealed an unexpected binding orientation underlying the binding selectivity by this new scaffold. We further incorporated carboxymethylphenylalanine and carboxyphenylalanine phosphotyrosine mimetics and arrived at an optimized inhibitor that potently binds Grb7-SH2 (KD = 0.13 µM) under physiological conditions. X-ray crystal structures of these Grb7-SH2/peptide complexes reveal the structural basis for the most potent and specific inhibitors of Grb7 developed to date. Finally, we demonstrate that cell permeable versions of these peptides successfully block Grb7 mediated interactions in a breast cancer cell line, establishing the potential of these peptides in the development of novel therapeutics targeted to Grb7.

Unexpected involvement of staple leads to redesign of selective bicyclic peptide inhibitor of Grb7.

The design of potent and specific peptide inhibitors to therapeutic targets is of enormous utility for both proof-of-concept studies and for the development of potential new therapeutics. Grb7 is a key signaling molecule in the progression of HER2 positive and triple negative breast cancers. Here we report the crystal structure of a stapled bicyclic peptide inhibitor G7-B1 in complex with the Grb7-SH2 domain. This revealed an unexpected binding mode of the peptide, in which the staple forms an alternative contact with the surface of the target protein. Based on this structural information, we designed a new series of bicyclic G7 peptides that progressively constrain the starting peptide, to arrive at the G7-B4 peptide that binds with an approximately 2-fold enhanced affinity to the Grb7-SH2 domain (KD = 0.83 µM) compared to G7-B1 and shows low affinity binding to Grb2-, Grb10- and Grb14-SH2 domains (KD > 100 µM). Furthermore, we determined the structure of the G7-B4 bicyclic peptide in complex with the Grb7-SH2 domain, both before and after ring closing metathesis to show that the closed staple is essential to the target interaction. The G7-B4 peptide represents an advance in the development of Grb7 inhibitors and is a classical example of structure aided inhibitor development.

Cyclic Peptides Incorporating Phosphotyrosine Mimetics as Potent and Specific Inhibitors of the Grb7 Breast Cancer Target.

The Grb7 adaptor protein is a therapeutic target for both TNBC and HER2+ breast cancer. A nonphosphorylated cyclic peptide 1 (known as G7-18NATE) inhibits Grb7 via targeting the Grb7-SH2 domain, but requires the presence of phosphate ions for both affinity and specificity. Here we report the discovery of malonate bound in the phosphotyrosine binding pocket of the apo-Grb7-SH2 structure. Based on this, we carried out the rational design and synthesis of two analogues of peptide 1 that incorporate carboxymethylphenylalanine (cmF) and carboxyphenylalanine (cF) as mimics of phosphotyrosine (pY). Binding studies using SPR confirmed that affinity for Grb7-SH2 domain is improved up to 9-fold over peptide 1 under physiological phosphate conditions (KD = 2.1-5.7 µM) and that binding is specific for Grb7-SH2 over closely related domains (low or no detectable binding to Grb2-SH2 and Grb10-SH2). X-ray crystallographic structural analysis of the analogue bearing a cmF moiety in complex with Grb7-SH2 has identified the precise contacts conferred by the pY mimic that underpin this improved molecular interaction. Together this study identifies and characterizes the tightest specific inhibitor of Grb7 to date, representing a significant development toward a new Grb7-targeted therapeutic.

Targeting SH2 domains in breast cancer.

Breast cancer is among the most commonly diagnosed cancer types in women worldwide and is the second leading cause of cancer-related disease in the USA. SH2 domains recruit signaling proteins to phosphotyrosine residues on aberrantly activated growth factor and cytokine receptors and contribute to cancer cell cycling, metastasis, angiogenesis and so on. Herein we review phosphopeptide mimetic and small-molecule approaches targeting the SH2 domains of Grb2, Grb7 and STAT3 that inhibit their targets and reduce proliferation in in vitro breast cancer models. Only STAT3 inhibitors have been evaluated in in vivo models and have led to tumor reduction. Taken together, these studies suggest that targeting SH2 domains is an important approach to the treatment of breast cancer.

Progress towards the development of SH2 domain inhibitors.

Src homology 2 (SH2) domains are 100 amino acid modular units, which recognize and bind to tyrosyl-phosphorylated peptide sequences on their target proteins, and thereby mediate intracellular protein-protein interactions. This review summarizes the progress towards the development of synthetic agents that disrupt the function of the SH2 domains in different proteins as well as the clinical relevance of targeting a specific SH2 domain. Since 1986, SH2 domains have been identified in over 110 human proteins, including kinases, transcription factors, and adaptor proteins. A number of these proteins are over-activated in many diseases, including cancer, and their function is highly dependent on their SH2 domain. Thus, inhibition of a protein's function through disrupting that of its SH2 domain has emerged as a promising approach towards the development of novel therapeutic modalities. Although targeting the SH2 domain is a challenging task in molecular recognition, the progress reported here demonstrates the feasibility of such an approach.

The discovery of phenylbenzamide derivatives as Grb7-based antitumor agents.

Grb7 is a non-catalytic protein, the overexpression of which has been associated with the proliferative and migratory potentials of cancer cells. Virtual screening strategies involving a shape-based similarity search, molecular docking, and 2D-similarity searches complemented by experimental binding studies (Thermofluor and isothermal titration calorimetry) resulted in the identification of nine novel phenylbenzamide-based antagonists of the Grb7 SH2 domain. Moderate binding affinities were observed, ranging from K(d)=32.3 µM for lead phenylbenzamide NSC 104999 (1) to K(d)=1.1 µM for a structurally related compound, NSC 57148 (2). Deconvolution of the affinity data into its components revealed differences in lead binding, from being entropy based (lead 1) to enthalpically driven (NSC 100874 (3), NSC 55158 (4), and compound 2). Finally, the lead compound 1 was found to decrease the growth of MDA-MB-468 breast cancer cells, with an IC(50) value of 39.9 µM. It is expected that these structures will serve as novel leads in the development of Grb7-based anticancer therapeutics.

Interaction of the non-phosphorylated peptide G7-18NATE with Grb7-SH2 domain requires phosphate for enhanced affinity and specificity.

Src-homology (SH2) domains are an attractive target for the inhibition of specific signalling pathways but pose the challenge of developing a truly specific inhibitor. The G7-18NATE cyclic peptide is reported to specifically inhibit the growth factor receptor bound protein 7 (Grb7) adapter protein, implicated in the progression of several cancer types, via interactions with its SH2 domain. G7-18NATE effectively inhibits the interaction of Grb7 with ErbB3 and focal adhesion kinase in cell lysates and, with the addition of a cell permeability sequence, inhibits the growth and migration of a number of breast cancer cell lines. It is thus a promising lead in the development of therapeutics targeted to Grb7. Here we investigate the degree to which G7-18NATE is specific for the Grb7-SH2 domain compared with closely related SH2 domains including those of Grb10, Grb14, and Grb2 using surface plasmon resonance. We demonstrate that G7-18NATE binds with micromolar binding affinity to Grb7-SH2 domain (K(D) = 4-6 µm) compared with 50-200 times lower affinity for Grb10, Grb14, and Grb2 but that this specificity depends critically on the presence of phosphate in millimolar concentrations. Other differences in buffer composition, including use of Tris or 2-(N-Morpholino)ethanesulfonic acid or varying the pH, do not impact on the interaction. This suggests that under cellular conditions, G7-18NATE binds with highest affinity to Grb7. In addition, our findings demonstrate that the basis of specificity of G7-18NATE binding to the Grb7-SH2 domain is via other than intrinsic structural features of the protein, representing an unexpected mode of molecular recognition.

GRB7 is required for triple-negative breast cancer cell invasion and survival.

Triple-negative breast cancer (TNBC) is a heterogeneous disease that is usually associated with poor prognosis, and frequently associated with the basal-like breast cancer gene expression profile. There are no targeted therapeutic modalities for this disease, and no useful biomarkers. High GRB7 RNA expression levels are associated with an elevated risk of recurrence in patients with operable TNBC treated with standard adjuvant anthracycline and taxane therapy. To determine whether GRB7 is involved in the pathobiology of TNBC, we evaluated the biological effects of GRB7 inhibition in a panel of triple-negative cell lines-MDA-MB-468, MDA-MB-231, HCC70, and T4-2. We found GRB7 inhibition reduced cell motility and invasion of these cell lines and promoted cell death by apoptosis in 3D culture. These data suggest that GRB7 itself, or GRB7-dependent pathways, may prove to be important therapeutic targets in this disease.

Structural basis of binding by cyclic nonphosphorylated peptide antagonists of Grb7 implicated in breast cancer progression.

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.

Benzopyrazine derivatives: A novel class of growth factor receptor bound protein 7 antagonists.

Growth factor receptor bound protein 7 (Grb7) is an adapter protein that functions as a downstream effector of growth factor mediated signal transduction. Over-expression of Grb7 has been implicated in a variety of cancers such as breast, blood, pancreatic, esophageal, and gastric carcinomas. Inhibition of Grb7 has been shown to reduce the migratory and proliferative potential of these cancers, making it an attractive therapeutic target. Starting with a known peptide antagonist, the present work reports the application of a succession of computational ligand design tools comprising a ligand shape based similarity search, molecular docking and a 2D-similarity search to identify small molecular antagonists of the Grb7-SH2 domain from the NCI chemical database. Binding to the Grb7-SH2 domain was then experimentally tested using melting point shift assays and isothermal titration calorimetry. Overall, a total of 11 benzopyrazine based small molecular antagonists were identified with affinity for the Grb7-SH2 domain. Representative compounds tested using ITC were revealed to possess moderate binding affinity in the low micromolar range. Finally, the lead compound (NSC642056) was found to reduce the growth of a Grb7-expressing breast cancer cell line with an IC(50) of 86µM. It is expected that the identified antagonists will be useful additions to further explore the function of Grb7 and for the development of inhibitors with therapeutic potential.

Preparation and crystallization of the Grb7 SH2 domain in complex with the G7-18NATE nonphosphorylated cyclic inhibitor peptide.

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 Šresolution 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, ß=104.4°. The structure of the G7-18NATE peptide in complex with its target will facilitate the rational development of Grb7-targeted cancer therapeutics.

Grb7 SH2 domain structure and interactions with a cyclic peptide inhibitor of cancer cell migration and proliferation.

BACKGROUND: Human growth factor receptor bound protein 7 (Grb7) is an adapter protein that mediates the coupling of tyrosine kinases with their downstream signaling pathways. Grb7 is frequently overexpressed in invasive and metastatic human cancers and is implicated in cancer progression via its interaction with the ErbB2 receptor and focal adhesion kinase (FAK) that play critical roles in cell proliferation and migration. It is thus a prime target for the development of novel anti-cancer therapies. Recently, an inhibitory peptide (G7-18NATE) has been developed which binds specifically to the Grb7 SH2 domain and is able to attenuate cancer cell proliferation and migration in various cancer cell lines. RESULTS: As a first step towards understanding how Grb7 may be inhibited by G7-18NATE, we solved the crystal structure of the Grb7 SH2 domain to 2.1 A resolution. We describe the details of the peptide binding site underlying target specificity, as well as the dimer interface of Grb 7 SH2. Dimer formation of Grb7 was determined to be in the muM range using analytical ultracentrifugation for both full-length Grb7 and the SH2 domain alone, suggesting the SH2 domain forms the basis of a physiological dimer. ITC measurements of the interaction of the G7-18NATE peptide with the Grb7 SH2 domain revealed that it binds with a binding affinity of Kd = approximately 35.7 microM and NMR spectroscopy titration experiments revealed that peptide binding causes perturbations to both the ligand binding surface of the Grb7 SH2 domain as well as to the dimer interface, suggesting that dimerisation of Grb7 is impacted on by peptide binding. CONCLUSION: Together the data allow us to propose a model of the Grb7 SH2 domain/G7-18NATE interaction and to rationalize the basis for the observed binding specificity and affinity. We propose that the current study will assist with the development of second generation Grb7 SH2 domain inhibitors, potentially leading to novel inhibitors of cancer cell migration and invasion.

NMR analysis of G7-18NATE, a nonphosphorylated cyclic peptide inhibitor of the Grb7 adapter protein.

G7-18NATE is a nonphosphorylated, cyclic peptide that specifically inhibits the Grb7 adapter protein implicated in several pathways critical to cell proliferation and migration. It has been shown that G7-18NATE is able to compete with natural ligands for the Grb7 SH2 phosphotyrosine binding site, and to attenuate cell migration in a pancreatic cancer cell line. It is thus an important lead in the development of a selective inhibitor of Grb7 and potential novel anticancer therapeutics. The current study reports the solution properties of G7- 18NATE determined using NMR spectroscopy, in both water (pH 2-3) and phosphate buffer (pH 6.0), with 100 mM NaCl. The spectra reveal that G7-18NATE exists in two distinguishable conformational states on the NMR timescale, most likely due to cis-trans proline isomerization. In addition, the chemical shift data are consistent with a tendency of G7-18NATE to form a turn about the YDN motif, known to be important for binding, and suggest that this turn is stabilized in low salt and low pH conditions. Low NH temperature coefficients of Tyr-5 and Asn-7 amide protons may reflect their involvement in the formation of hydrogen bonds that stabilize such a turn. Overall, however, the peptide does not form a rigid structure, but exists in a highly flexible state in solution. Averaged 3JNH-H coupling constants and a lack of interresidue NOEs are characteristic of such peptide solution behavior. This suggests that there is scope for increasing the rigidity of the peptide that may enhance its binding affinity and specificity for Grb7.

Specific peptide ligand for Grb7 signal transduction protein and pancreatic cancer metastasis.

BACKGROUND: Pancreatic cancer is one of the most aggressive malignancies, with high rates of invasion and metastasis and with generally poor prognosis. We previously found that metastasis was strongly associated with the expression of growth factor receptor-bound protein 7 (Grb7), which contains a Src homology 2 (SH2) domain. In this study, we evaluated Grb7 protein as a molecular target of therapy for metastatic pancreatic cancer. METHODS: Grb7 protein expression was measured by immunohistochemistry in 36 human pancreatic cancer specimens and adjacent normal pancreatic tissue. We synthesized a nonphosphorylated peptide inhibitor that binds specifically to the SH2 domain of Grb7. Intracellular signaling was assessed by immunoprecipitation and immunoblot assays in cultured human pancreatic cancer cells. Cell migration was measured with a modified Boyden chamber method. Peritoneal metastasis of the pancreatic cancer cells was measured with a mouse model. All statistical tests were two-sided. RESULTS: We found that 22 (61%) of 36 pancreatic cancer specimens had higher levels of Grb7 protein than their corresponding normal pancreatic tissue specimens. Grb7 expression was statistically significantly different between specimens from patients without lymph node metastasis (stage N0; two of the 10 patients) and patients with lymph node metastasis (stages N1 + N2; 20 of the 26 patients) (P = .006). The Grb7 peptide inhibitor selectively blocked the interaction between Grb7 and focal adhesion kinase and blocked the phosphorylation of Grb7 protein. In vivo Grb7 peptide inhibitor statistically significantly attenuated cell migration (for control peptide, 87.5 cells migrated, 95% confidence interval [CI] = 82.6 to 92.4 cells; for Grb7 peptide, 5.7 cells migrated, 95% CI = 2.3 to 9.0 cells; P < .001) and peritoneal metastasis of the pancreatic cancer cells in a mouse model, as assessed by the number of nodules (control = 72.6 nodules, 95% CI = 55.8 to 89.4 nodules; and for Grb7 peptide = 3.2 nodules, 95% CI = 1.6 to 4.8 nodules; P < .001, t test) and their weight (control = 4.13 g, 95% CI = 3.40 to 4.86 g; Grb7 peptide = 0.19 g, 95% CI = 0.06 to 0.32 g; P < .001, t test). CONCLUSIONS: The Grb7 peptide inhibitor appears to be a promising molecularly targeted therapeutic agent against metastatic pancreatic cancer.

Grb7-based molecular therapeutics in cancer.

Traditional anti-cancer drugs preferentially kill rapidly growing tumour cells rather than normal cells. However, most of these drugs have no preferential selection towards cancer cells and are taken up by the whole body, resulting in significant adverse side effects. Therapeutic molecules that could specifically inhibit undesirable phenotypes are an attractive way of eliminating cancer cells. There is a widespread effort to develop inhibitors against signal transduction molecules that play a key role in the proliferative, migratory and invasive properties of a cancer cell. Grb7 is an adaptor-type signalling protein that is recruited via its Src-homology 2 (SH2) domain to a variety of tyrosine kinases. Grb7 is overexpressed in breast, oesophageal and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide attractive targets for therapeutic intervention.