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(2)CO-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 beta-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 gamma-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 microM 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 microM, respectively.     

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.     

Development of l-3-aminotyrosine suitably protected for the synthesis of a novel nonphosphorylated hexapeptide with low-nanomolar Grb2-SH2 domain-binding affinity

Synthesis of orthogonally protected (2S)-2-amino-3-(3-amino-4-hydroxy-phenyl)-propionic acid (10) suitable for solid phase peptide synthesis and its first use for the preparation of nonphosphorylated Grb2-SH2 domain antagonists (4a-c) are reported. The 3-aminotyrosine containing sulfoxide-cyclized hexapeptide (4b) exhibited potent Grb2-SH2 domain binding affinity with IC50 = 50 nM, which represents the highest affinity yet reported for a peptide inhibitor against Grb2-SH2 domain with only 6 residues free of phosphotyrosine or phosphotyrosine mimics. This potent small peptidomimetic 4b may be representative of a new class of therapeutically relevant Grb2-SH2 domain-directed agents, and acts as a chemotherapeutic lead for the treatment of erbB2-related cancers.     

Structural and energetic aspects of Grb2-SH2 domain-swapping

The SH2 domain of growth factor receptor-bound protein 2 (Grb2) has been the focus of numerous studies, primarily because of the important roles it plays in signal transduction. More recently, it has emerged as a useful protein to study the consequences of ligand preorganization upon energetics and structure in protein-ligand interactions. The Grb2-SH2 domain is known to form a domain-swapped dimer, and as part of our investigations toward correlating structure and energetics in biological systems, we examined the effects that domain-swapping dimerization of the Grb2-SH2 domain had upon ligand binding affinities. Isothermal titration calorimetry was performed using Grb2-SH2 in both its monomeric and domain-swapped dimeric forms and a phosphorylated tripeptide AcNH-pTyr-Val-Asn-NH(2) that is similar to the Shc sequence recognized by Grb2-SH2 in vivo. The two binding sites of domain-swapped dimer exhibited a 4- and a 13-fold reduction in ligand affinity compared to monomer. Crystal structures of peptide-bound and uncomplexed forms of Grb2-SH2 domain-swapped dimer were obtained and reveal that the orientation of residues V122, V123, and R142 may influence the conformation of W121, an amino acid that is believed to play an important role in Grb2-SH2 ligand sequence specificity. These findings suggest that domain-swapping of Grb2-SH2 not only results in a lower affinity for a Shc-derived ligand, but it may also affect ligand specificity.     

Binding affinity difference induced by the stereochemistry of the sulfoxide bridge of the cyclic peptide inhibitors of Grb2-SH2 domain: NMR studies for the structural origin

The SAR study on a phage library-derived non-phosphorylated cyclic peptide ligand of Grb2-SH2 domain indicates that the configuration of the cyclization linkage is crucial for assuming the active binding conformation. When the thioether linkage was oxidized to the two chiral sulfoxides, the R-configured sulfoxide-cyclized peptide displayed 10-30 times more potency than the corresponding S-configured one in binding affinity to the Grb2-SH2 domain. In this paper, the solution structures of such a pair of sulfoxide-bridged cyclic peptide diastereoisomers, i.e., cyclo[CH(2)CO-Gla(1)-L-Y-E-N-V-G-NPG-Y-(R/S)C(O)(10)]-amide, were determined by NMR and molecular dynamics simulation. Results indicate that the consensus sequence of Y(3)-E(4)-N(5)-V(6) in both diastereoisomers adopt a beta-turn conformation; however, the R-configured peptide forms an extended structure with a circular backbone conformation, while the S-configured isomer forms a compact structure with key residues buried inside the molecule. The average root-mean-square deviations were found to be 0.756 and 0.804 A, respectively. It is apparent that the chiral S-->O group played a key role in the solution structures of the sulfoxide-bridged cyclic peptides. The R-sulfoxide group forms an intramolecular hydrogen bond with the C-terminal amide, conferring a more rigid conformation with all residues protruding outside except for Leu2, in which the Gla1 and Tyr3 share an overlapping function as previous SAR studies proposed. Additionally, the extended structure endows a more hydrophilic binding surface of the R-configured peptide to facilitate its capture by its targeted protein. In comparison, the S-configured sulfoxide was embedded inside the ligand peptide leading to a compact structure, in which the essential residues of Gla1, Tyr3, and Asn5 form multiple intramolecular hydrogen bonds resulting in an unfavorable conformational change and a substantial loss of the interaction with the protein. The solution structures disclosed by our NMR and molecular dynamics simulation studies provide a molecular basis for understanding how the chirality of the cyclization linkage remarkably discriminates in terms of the binding affinity, thus advancing the rational design of potent non-phosphorylated inhibitors of Grb2-SH2 domain as antitumor agents.     

Discovery of thioether-bridged cyclic pentapeptides 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 (Grb 2) is considered to be an effective and non-cytotoxic strategy to develop new anti-proliferate agents due to its potential to shut down the Ras activation pathway. In this study, a series of phosphotyrosine containing cyclic pentapeptides were designed and synthesized based upon the phage library derived cyclopeptide, G1TE. 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 micromolar range binding affinity, such as cyclic peptide 15 with an K_d = 0.359 μM, which is providing a novel template for the development of Grb2-SH2 domain antagonists as potential therapeutics for certain cancers.     

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 IC₅₀ of 86 ??M. 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.     

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 beta-turn feature of peptidic ligand binding to Grb2 SH2 domain, we introduce alpha,alpha-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.     

Functional preference of the constituent amino acid residues in a phage-library-based nonphosphorylated inhibitor of the Grb2-SH2 domain.

A nonphosphorylated disulfide-bridged peptide, cyclo(Cys-Glu1-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr9-Cys)-amide (termed G1) has been identified, by phage library, that binds to the Grb2-SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2-SH2 domain association (IC50 of 15.5 microM) with natural phosphopeptide ligands. As a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve side-chains of amino acids Glu1, Tyr3, Glu4, Asn5, and Met8. Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2-SH2 domain in a beta-turn-like conformation. Replacement of Tyr3 or Asn5 with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y-X-N consensus sequence similar to that found in natural pTyr-containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu1, i.e. the amino acid N-terminal to Y3, remarkably reduces the binding affinity. The position of the Glu1 side-chain is confirmed to provide a complementary role for pTyr3, as demonstrated by the low micromolar inhibitory activity (IC50 = 1.02 microM) of the nonphosphorylated peptide 11, G1(Gla1), in which Glu1 was replaced by gamma-carboxy-glutamic acid (Gla).     

Structural requirements for Tyr in the consensus sequence Y-E-N of a novel nonphosphorylated inhibitor to the Grb2-SH2 domain.

The phage library derived, nonphosphorylated and thioether-cyclized peptide, termed G1TE, cyclo(CH(2)CO-Glu(1)-Leu-Tyr(3)-Glu-Asn-Val-Gly-Met-Tyr-Cys(10))-amid e, represents a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, with an IC(50) of 20 microM. The retention of binding affinity is very sensitive with respect to peptide ring-size alterations and Ala mutations. We demonstrated previously that the Glu(1) side chain and its closely related analogs partially compensate for the absence of the phosphate functionality on Tyr(3), and, based on molecular modeling, these acidic side-chains complex with the Arg67 and Arg86 side-chains of the protein in the binding cavity. In this study we judiciously altered and incorporated various natural and unnatural amino acids as Tyr replacements within the -YEN- motif, and we demonstrate the functional importance and structural requirement of Tyr(3) for effective binding of this novel non-phosphorylated ligand to the Grb2-SH2 domain. The phenyl side-chain moiety and a polar functional group with specific orientation in position Y(3) of the peptide are particularly required. Using SPR binding assays, a submicromolar inhibitor (IC(50) = 0.70 microM) was obtained when Glu(1) was replaced with alpha-aminoadipate and Tyr(3) was replaced with 4-carboxymethyl-Phe, providing peptide 14, G1TE(Adi(1), cmPhe(3)). Peptide 14 also inhibited Grb2/p185(erb)(B-2) protein association in cell homogenates of erbB-2-overexpressing MDA-MA-453 cancer cells at near one micromolar concentrations.     

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(2)CO-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 microM). However, open chain, shortening or expanding the ring size led to a marked loss of inhibitory activity. Significantly, the introduction of omega-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 microM) relative to G1TE. Moreover, the lipophilic chain in omega-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.     

用Gateway 系统构建携带Grb2-SH2 基因重组慢病毒载体

目的:构建携带Grb2-SH2基因重组慢病毒载体。方法:把Grb2-SH2基因从合成的工程载体PUC-57中导到入门载体pentr-3C中,再利用LR反应重组到目的载体plenti,通过酶切和测序分析对比验证Grb2-SH2基因后,将plenti-Grb2-SH2质粒利用慢病毒转染系统转染人胚胎肾上皮细胞株293T细胞获得携带Grb2-SH2慢病毒载体。结果:构建的重组质粒经酶切及测序和分析比对鉴定正确,目的基因片段大小约为500bp;该质粒与包装质粒共转染293T细胞获取的慢病毒,转染效率约为90%。结论:成功构建转载质粒plenti-Grb2-SH2及携带Grb2-SH2基因慢病毒载体。     

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. Copyright © 2011 John Wiley & Sons, Ltd.     

Affinity chromatography for purification of the modular protein growth factor receptor-bound protein 2 and development of a screening test for growth factor receptor-bound protein 2 Src homology 3 domain inhibitor using peroxidase-linked ligand

Growth factor receptor-bound protein 2 (Grb2) is an adapter protein involved in the Ras-dependent signaling pathway that plays an important role in human cancers initiated by oncogenic receptors. Grb2 is constituted by one Src homology 2 domain surrounded by two SH3 domains, and the inhibition of the interactions produced by these domains could provide an antitumor approach. In evaluating chemical libraries, to search for potential Grb2 inhibitors, it was necessary to elaborate a rapid test for their screening. We have developed, first, a batch method based on the use of an affinity column bearing a Grb2-SH3 peptide ligand to isolate highly purified Grb2. We subsequently describe a very rapid 96-well screening of inhibitors based on a simple competition between purified Grb2 and a peroxidase-coupled proline-rich peptide.     

Use of SPR to Study the Interaction of G7-18NATE Peptide with the Grb7-SH2 Domain

Surface plasmon resonance (SPR) is a useful biosensor technique for the study of biomolecular interactions, with the potential for high-throughput screening of ligand interactions with drug targets. The key to its successful use, however, is in the appropriate design of the experiment, including the mode of immobilization to the biosensor chip. We report an investigation of the use of SPR for measuring the affinity of the G7-18NATE peptide ligand for its Grb7-SH2 domain target involved in the migratory and proliferative potential of cancer cells. Previous studies have shown that the cyclic non-phosphorylated peptide, G7-18NATE, inhibits Grb7 interactions with upstream binding partners and is able to inhibit both cell migration and proliferation of cancer cells. We report the synthesis of a biotinylated G7-18NATE covalently attached to a linker (G7-18NATE-ASASASK-Biotin) and compare its interaction with the Grb7-SH2 domain by SPR using three different immobilization strategies; immobilisation of the peptide via streptavidin, immobilization of glutathione S-transferase (GST)-Grb7-SH2 domain via anti-GST antibody, and immobilization of biotinylated Grb7-SH2 domain via streptavidin. This revealed that sensorgrams free from non-specific binding and displaying simple kinetics were most readily achieved by immobilising the protein rather than the peptide, in spite of the lower response associated with this method. K _D values of ~300 μM were determined for both strategies at pH 7.4. This compared with a K _D value of 4.4 μM at pH 6 demonstrating the importance of pH on this interaction. Overall, the immobilised protein systems are most suitable for future comparative screening efforts using SPR.     

Small nonphosphorylated Grb2-SH2 domain antagonists evaluated by surface plasmon resonance technology

The growth factor receptor-binding protein-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signal transduction pathway, which involves cell proliferation and differentiation. Therefore, the Grb2-SH2 domain has been chosen as our target for development of potential antiproliferative agents. Herein, we report the study of the inhibitory effects of small nonphosphorylated peptide analogs interacting with the Grb2-SH2 domain protein by surface plasmon resonance (SPR) technology. A set of 8 related peptide analogs were synthesized, purified, and characterized. Their inhibitory effects on Grb2-SH2 were evaluated by the SPR technology developed with the BIACORE X instrument. The lead peptide, Fmoc-Glu-Tyr-Aib-Asn-NH2 (Fmoc-E-Y-Aib-N; Fmoc: 9-fluorenylmethyoxycarbonyl; Aib=alpha-amino isobutyric acid) inhibited Grb2-SH2 domain function with an IC50 value of 8.7 microM. A molecular modeling study of the lead peptide indicated that the glutamate in the Fmoc peptide is ideally positioned to form a strong salt bridge to Arg 67 in the Grb2-SH2 domain, using both its backbone carbonyl and its acidic group. Residue Glu 89 in Grb2-SH2 flips inward to fill the binding site and partially replace the phosphate group as a hydrogen-bond acceptor. Results of these studies provide important information for further development of potent nonphosphorylated peptide inhibitors of the Grb2-SH2 domain.     

Crystal structures of a high-affinity macrocyclic peptide mimetic in complex with the Grb2 SH2 domain

The high-affinity binding of the growth factor receptor-bound protein 2 (Grb2) SH2 domain to tyrosine-phosphorylated cytosolic domains of receptor tyrosine kinases (RTKs) is an attractive target for therapeutic intervention in many types of cancer. We report here two crystal forms of a complex between the Grb2 SH2 domain and a potent non-phosphorus-containing macrocyclic peptide mimetic that exhibits significant anti-proliferative effects against erbB-2-dependent breast cancers. This agent represents a "second generation" inhibitor with greatly improved binding affinity and bio-availability compared to its open-chain counterpart. The structures were determined at 2.0A and 1.8A with one and two domain-swapped dimers per asymmetric unit, respectively. The mode of binding and specific interactions between the protein and the inhibitor provide insight into the high potency of this class of macrocylic compounds and may aid in further optimization as part of the iterative rational drug design process.