Grb2 SH2 Domain Research Articles

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.

Fatty acids and cancer-amplified ZDHHC19 promote STAT3 activation through S-palmitoylation.

Signal transducer and activator of transcription 3 (STAT3) plays a critical role in regulating cell fate, inflammation and immunity1,2. Cytokines and growth factors activate STAT3 through kinase-mediated tyrosine phosphorylation and dimerization3,4. It remains unknown whether other factors could promote STAT3 activation through different mechanisms. Here we show that STAT3 is posttranslationally S-palmitoylated at the Src Homology 2 (SH2) domain, promoting its dimerization and transcriptional activation. Fatty acids could directly activate STAT3 by enhancing its palmitoylation, in synergy with cytokine stimulation. We further identified ZDHHC19 as a palmitoyl acyltransferase (PAT) regulating STAT3. Cytokine stimulation enhances STAT3 palmitoylation by promoting ZDHHC19–STAT3 association mediated by Grb2 SH3 domain. Silencing ZDHHC19 blocks STAT3 palmitoylation and dimerization, impairing cytokine and fatty acid-induced STAT3 activation. Importantly, ZDHHC19 is frequently amplified in multiple human cancers, including in 39% of lung squamous cell carcinomas (LSCCs). High ZDHHC19 levels correlate with high nuclear STAT3 in patient samples. In addition, ZDHHC19 knockout in LSCC cells significantly blocks STAT3 activity, and inhibits fatty acid-induced tumorsphere formation and high-fat diet (HFD)-induced tumorigenesis in vivo. Taken together, we reveal that fatty acid and ZDHHC19-mediated palmitoylation are additional signals regulating STAT3, linking deregulation of palmitoylation to inflammation and cancer.

Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways. Extrapolation to the full-length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine-phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated-tyrosine-mimic Y492E-FL-Grb7 protein (Y492E-FL-Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild-type FL Grb7(WT-FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization Kd of WT-FL-Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT-FL-Grb7 in comparison the tyrosine-phosphorylation mimic Y492E-FL-Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.

NMR assignment of free 1H, 15N and 13C-Grb2-SH2 domain.

Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein composed of three domains, an N-terminal SH3 (nSH3), SH2 and a C-terminal SH3 (cSH3) domains. This multi-domain protein has been reported to be a key factor in many signaling pathways related to controlling cell survival, differentiation, and growth. The Grb2-SH2 domain has been a focus for the study of the interaction with peptides and small molecules to act as inhibitors in uncontrolled cell growth, and consequently inhibit tumor proliferation. Here we describe the almost complete assignment of the free SH2 domain at pH 7. This work prepares the ground for further structural studies, backbone dynamics, mapping of interactions and drug screening and development. TalosN secondary structure prediction showed great similarity with the available structures in the PDB.

Toward Understanding the Functional State of the Grb7 Protein

Human growth factor receptor bound protein 7 (Grb7) proteins are the family of adaptor proteins that include Grb7, Grb10 and Grb14. These proteins have a conserved topology that includes a proline rich N‐terminal domain, an RA domain, a PH domain and a C‐terminal SH2 domain. Grb7 proteins bind with activated receptor tyrosine kinase (RTK) receptors through their SH2 domain to mediate downstream signal transduction events. Previous studies in our laboratory have shown the Grb7SH2 domain exists in a dimeric form and this may control the dimerization of the full‐length protein (FLGrb7). In our current studies, we show a FLGrb7 phosphorylation mimic, Y492E‐FLGrb7, results in a monomeric form of the protein, by in vitro biophysical measurements. Specifically, size exclusion and analytical ultracentrifugation results demonstrate Y492E‐FLGrb7 is essentially monomeric, while under the same conditions wild‐type Grb7 (WTFLGrb7) exists as a dimer. Further, the size and shape of these proteins were determined through dynamic light scattering measurements. These studies suggest the phosphorylation mimic Y492E‐FLGrb7 may exist in a more open, as opposed to compact globular, conformational form. We interpret the above results with a view towards understanding the functional state of Grb7 protein, and its role in controlling cell signaling pathways via interaction with its binding partners.Support or Funding InformationNIH5‐SC3‐6M116728This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Silico Screening to Identify Inhibitors of Growth Factor Receptor 2-Focal Adhesion Kinase Interaction for Therapeutic Treatment of Pathological Cardiac Hypertrophy.

The focal adhesion kinase-growth factor receptor 2 (FAK-Grb2) protein-protein interaction is implicated in pathogenesis of stress-induced cardiac hypertrophy. The focal adhesion targeting (FAT) domain of FAK unfolds to form a structural intermediate that interacts with a multibinding hot spot in the SH2 domain of Grb2. Disruption of the Grb2-FAT interaction is a therapeutic strategy for prevention of pathological cardiac hypertrophy. A pharmacophore was generated on the basis of structural and electrostatic properties of FAT bound to FAK using the Forge tool (Cresset). This pharmacophore was used as a query for Blaze server (Cresset) to screen a selectively enriched chemical library of 4,32,508 small molecules. The compounds selected were further filtered by hierarchical flexible docking approach using AutoDock v4. From the favorably docked compounds, five were selected on the basis of good adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties using SwissADME, MedChem Designer v.3, and MOLINSPIRATION. Stability of the binding mode of the inhibitors was further confirmed by molecular dynamic simulation study with AMBER v15 for a simulation time of 50 ns in aqueous environment. PM2307 was identified as the best inhibitor in terms of pharmacophoric features, dock score, and in silico ADMET analysis. The calculated binding affinity of PM2307 was better than that of the FAT-Grb2 complex as well as a previously reported small molecule inhibitor. PM2307 is also a quinolyl derivative sharing a similar scaffold with ofloxacin drugs, asserting its drug-like properties. Thus, it was proposed as a lead compound for development of drugs for pathological cardiac hypertrophy.

EGF Stimulates Rab35 Activation and Gastric Cancer Cell Migration by Regulating DENND1A-Grb2 Complex Formation

Aims: The aim of this study was to reveal the specific molecular mechanisms by which DENND1A accepts EGF signaling and activates Rab35 in gastric cancer.Methods: The expression of proteins related to DENND1A was examined by western blot analysis. Activation of Rab35 was assessed by GST-pulldown. The interaction of DENND1A and Grb2 was assessed by GST-pulldown and co-immunoprecipitation assays. The relationship between DENND1A and cell migration and invasion was detected using wound healing and transwell by gene overexpression and RNA interference.Results: EGF stimulation significantly promoted cell migration, whereas transfection with siRab35 partially inhibited EGF-promoted cell migration. DENND1A is also involved in these processes and active Rab35. Moreover, DENND1A binds to the N-terminal and C-terminal SH3 domains of Grb2 through PRD. Of special interest is the observation that EGFR can recruit Grb2-DENND1A complex under EGF stimulation. Further results reveal that the higher the expression of DENND1A, the shorter progression-free survival of gastric cancer patients.Conclusion: In summary, we confirmed that EGF-Grb2-DENND1A-Rab35 signaling pathway with the interaction of DENND1A and Grb2 as a regulatory center could regulate gastric cancer cell migration and invasion. Ultimately, the expression level of DENND1A predicts the survival status of gastric cancer patients and may become one of the important targets for the treatment of gastric cancer.

3056 - Analysis of Novel Inhibitors of the GRB2 SH2 Domain That Decrease Proliferation in Chronic Myeloid Leukemia

3056 - Analysis of Novel Inhibitors of the GRB2 SH2 Domain That Decrease Proliferation in Chronic Myeloid Leukemia

Protein-ligand binding enthalpies from near-millisecond simulations: Analysis of a preorganization paradox.

Calorimetric studies of protein-ligand binding sometimes yield thermodynamic data that are difficult to understand. Today, molecular simulations can be used to seek insight into such calorimetric puzzles, and, when simulations and experiments diverge, the results can usefully motivate further improvements in computational methods. Here, we apply near-millisecond duration simulations to estimate the relative binding enthalpies of four peptidic ligands with the Grb2 SH2 domain. The ligands fall into matched pairs, where one member of each pair has an added bond that preorganizes the ligand for binding and thus may be expected to favor binding entropically, due to a smaller loss in configurational entropy. Calorimetric studies have shown that the constrained ligands do in fact bind the SH2 domain more tightly than the flexible ones, but, paradoxically, the improvement in affinity for the constrained ligands is enthalpic, rather than entropic. The present enthalpy calculations yield the opposite trend, as they suggest that the flexible ligands bind more exothermically. Additionally, the small relative binding enthalpies are found to be balances of large differences in the energies of structural components such as ligand and the binding site residues. As a consequence, the deviations from experiment in the relative binding enthalpies represent small differences between these large numbers and hence may be particularly susceptible to error, due, for example, to approximations in the force field. We also computed first-order estimates of changes in configurational entropy on binding. These too are, arguably, paradoxical, as they tend to favor binding of the flexible ligands. The paradox is explained in part by the fact that the more rigid constrained ligands reduce the entropy of binding site residues more than their flexible analogs do, at least in the simulations. This result offers a rather general counterargument to the expectation that preorganized ligands should be associated with more favorable binding entropies, other things being equal.

Receptor Tyrosine Kinase Signalling in the Absence of Kinase Activity and Cancer of Non‐Genetic Origin

FGFR2‐expressing cancer cells with low concentrations of the adaptor protein Grb2 show high prevalence for metastatic outcome. In non‐stimulated cells the SH3 domain (and not the SH2 domain(s)) of Plcγ1 directly competes for a proline‐rich binding site at the very C‐terminus of FGFR2 with the C‐terminal SH3 domain of Grb2. Reduction of Grb2 concentration permits access of Plcγ1 to the receptor. Recruitment of Plcγ1 in this way is sufficient to up‐regulate phospholipase activity. This results in increased cell proliferation, motility and promotion of cell invasive behaviour in the absence of extracellular receptor stimulation. Therefore tumour growth and metastatic outcome can be dictated by the constitutive competition between Grb2 and Plcγ1 for the phosphorylation‐independent binding site on FGFR2. Since the majority of receptor tyrosine kinases have proline‐rich sequences in their C‐termini, the possibility of a second tier of signal transduction in the absence of growth factor stimulation, or kinase‐activating mutations emerges – leading to cancer of non‐genetic origin. Further examples of this second tier of signalling have been investigated and mechanistic detail of how protein concentration fluctuations in response to cellular stress can lead to pathogenesis has been established.Support or Funding InformationThis work is funded by CRUKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epidermal growth factor receptors containing a single tyrosine in their C-terminal tail bind different effector molecules and are signaling-competent

The EGF receptor is a classic receptor tyrosine kinase. It contains nine tyrosines in its C-terminal tail, many of which are phosphorylated and bind proteins containing SH2 or phosphotyrosine-binding (PTB) domains. To determine how many and which tyrosines are required to enable EGF receptor-mediated signaling, we generated a series of EGF receptors that contained only one tyrosine in their C-terminal tail. Assays of the signaling capabilities of these single-Tyr EGF receptors indicated that they can activate a range of downstream signaling pathways, including MAP kinase and Akt. The ability of the single-Tyr receptors to signal correlated with their ability to bind Gab1 (Grb2-associated binding protein 1). However, Tyr-992 appeared to be almost uniquely required to observe activation of phospholipase Cγ. These results demonstrate that multiply phosphorylated receptors are not required to support most EGF-stimulated signaling but identify Tyr-992 and its binding partners as a unique node within the network. We also studied the binding of the isolated SH2 domain of Grb2 (growth factor receptor-bound protein 2) and the isolated PTB domain of Shc (SHC adaptor protein) to the EGF receptor. Although these adapter proteins bound readily to wild-type EGF receptor, they bound poorly to the single-Tyr EGF receptors, even those that bound full-length Grb2 and Shc well. This suggests that in addition to pTyr-directed associations, secondary interactions between the tail and regions of the adapter proteins outside of the SH2/PTB domains are important for stabilizing the binding of Grb2 and Shc to the single-Tyr EGF receptors.

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.

Grb2 regulates the proliferation of hematopoietic stem and progenitors cells

Although Hematopoietic Stem and Progenitor Cell (HSPC) proliferation, survival and expansion have been shown to be supported by the cooperative action of different cytokines, little is known about the intracellular signaling pathways that are activated by cytokines upon binding to their receptors. Our study showed that Growth factor receptor-bound protein 2 (Grb2) mRNAs are preferentially expressed in HSC compared to progenitors and differentiated cells of the myeloid and erythroid lineages. Conditional deletion of Grb2 induced a rapid decline of erythroid and myeloid progenitors and a progressive decline of HSC numbers in steady state conditions. We showed that when transplanted, Grb2 deleted bone marrow cells could not reconstitute irradiated recipients. Strinkingly, Grb2 deletion did not modify HSPC quiescence, but impaired LT-HSC and progenitors ability to respond a proliferative signal induced by 5FU in vivo and by various cytokines in vitro. We showed finally that Grb2 links IL3 signaling to the ERK/MAPK proliferative pathway and that both SH2 and SH3 domains of Grb2 are crucial for IL3 signaling in progenitor cells. Our findings position Grb2 as a key adaptor that integrates various cytokines response in cycling HSPC.

Insight into the Selectivity of the G7-18NATE Inhibitor Peptide for the Grb7-SH2 Domain Target.

Growth factor receptor bound protein 7 (Grb7) is an adaptor protein with established roles in the progression of both breast and pancreatic cancers. Through its C-terminal SH2 domain, Grb7 binds to phosphorylated tyrosine kinases to promote proliferative and migratory signaling. Here, we investigated the molecular basis for the specificity of a Grb7 SH2-domain targeted peptide inhibitor. We identified that arginine 462 in the BC loop is unique to Grb7 compared to Grb2, another SH2 domain bearing protein that shares the same consensus binding motif as Grb7. Using surface plasmon resonance we demonstrated that Grb7-SH2 binding to G7-18NATE is reduced 3.3-fold when the arginine is mutated to the corresponding Grb2 amino acid. The reverse mutation in Grb2-SH2 (serine to arginine), however, was insufficient to restore binding of G7-18NATE to Grb2-SH2. Further, using a microarray, we confirmed that G7-18NATE is specific for Grb7 over a panel of 79 SH2 domains, and identified that leucine at the βD6 position may also be a requirement for Grb7-SH2 binding. This study provides insight into the specificity defining features of Grb7 for the inhibitor molecule G7-18NATE, that will assist in the development of improved Grb7 targeted inhibitors.

Grb2 carboxyl-terminal SH3 domain can bivalently associate with two ligands, in an SH3 dependent manner

Src homology domain containing leukocyte protein of 65 kDa (SLP65), the growth factor receptor binding protein 2 (Grb2), and the guanine nucleotide exchange factor for the Rho family GTPases (Vav), self associate in unstimulated B cells as components of the preformed B cell receptor transducer module, in an SH3-dependent manner. The complex enables the B cell to promptly respond to BCR aggregation, resulting in signal amplification. It also facilitates Vav translocation to the membrane rafts, for activation. Here we uncover the molecular mechanism by which the complex may be formed in the B cell. The C-terminal SH3 domain (SH3C) of Grb2 bivalently interacts with the atypical non-PxxP proline rich region of SLP65, and the N-terminal SH3 domain (SH3N) of Vav, both the interactions crucial for the proper functioning of the B cell. Most surprisingly, the two ligands bind the same ligand binding site on the surface of Grb2 SH3C. Addition of SLP65 peptide to the Grb2-Vav complex abrogates the interaction completely, displacing Vav. However, the addition of Vav SH3N to the SLP65-Grb2 binary complex, results in a trimeric complex. Extrapolating these results to the in vivo conditions, Grb2 should bind the SLP65 transducer module first, and then Vav should associate.

Exhaustive search of linear information encoding protein-peptide recognition.

High-throughput in vitro methods have been extensively applied to identify linear information that encodes peptide recognition. However, these methods are limited in number of peptides, sequence variation, and length of peptides that can be explored, and often produce solutions that are not found in the cell. Despite the large number of methods developed to attempt addressing these issues, the exhaustive search of linear information encoding protein-peptide recognition has been so far physically unfeasible. Here, we describe a strategy, called DALEL, for the exhaustive search of linear sequence information encoded in proteins that bind to a common partner. We applied DALEL to explore binding specificity of SH3 domains in the budding yeast Saccharomyces cerevisiae. Using only the polypeptide sequences of SH3 domain binding proteins, we succeeded in identifying the majority of known SH3 binding sites previously discovered either in vitro or in vivo. Moreover, we discovered a number of sites with both non-canonical sequences and distinct properties that may serve ancillary roles in peptide recognition. We compared DALEL to a variety of state-of-the-art algorithms in the blind identification of known binding sites of the human Grb2 SH3 domain. We also benchmarked DALEL on curated biological motifs derived from the ELM database to evaluate the effect of increasing/decreasing the enrichment of the motifs. Our strategy can be applied in conjunction with experimental data of proteins interacting with a common partner to identify binding sites among them. Yet, our strategy can also be applied to any group of proteins of interest to identify enriched linear motifs or to exhaustively explore the space of linear information encoded in a polypeptide sequence. Finally, we have developed a webserver located at http://michnick.bcm.umontreal.ca/dalel, offering user-friendly interface and providing different scenarios utilizing DALEL.

Grb7 protein RA domain oligomerization.

The growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is often coamplified with the erythroblastosis oncogene B 2 receptor in 20% to 30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The ras associating and pleckstrin homology domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half Lin11, Isl-1, Mec-3 (LIM) domains isoform 2 and filamin α. These interactions are believed to play a role in regulating Grb7-mediated cell migration function. The full-length Grb7 protein has been shown to dimerize, and the oligomeric state of the Grb7SH2 domain has been extensively studied; however, the oligomerization state of the ras associating and pleckstrin homology domains, and the importance of this oligomerization in Grb7 function, is yet to be fully known. In this study, we characterize the oligomeric state of the Grb7RA domain using size exclusion chromatography, nuclear magnetic resonance, nuclear relaxation studies, glutaraldehyde cross linking, and dynamic light scattering. We report the Grb7RA domain can exist in transient multimeric forms and, based upon modeling results, postulate the potential role of Grb7RA domain oligomerization in Grb7 function.

Blockade of Y177 and Nuclear Translocation of Bcr-Abl Inhibits Proliferation and Promotes Apoptosis in Chronic Myeloid Leukemia Cells.

The gradual emerging of resistance to imatinib urgently calls for the development of new therapy for chronic myeloid leukemia (CML). The fusion protein Bcr-Abl, which promotes the malignant transformation of CML cells, is mainly located in the cytoplasm, while the c-Abl protein which is expressed in the nucleus can induce apoptosis. Based on the hetero-dimerization of FKBP (the 12-kDa FK506- and rapamycin-binding protein) and FRB (the FKBP-rapamycin binding domain of the protein kinase, mTOR) mediated by AP21967, we constructed a nuclear transport system to induce cytoplasmic Bcr-Abl into nuclear. In this study, we reported the construction of the nuclear transport system, and we demonstrated that FN3R (three nuclear localization signals were fused to FRBT2098L with a FLAG tag), HF2S (two FKBP domains were in tandem and fused to the SH2 domain of Grb2 with an HA tag) and Bcr-Abl form a complexus upon AP21967. Bcr-Abl was imported into the nucleus successfully by the nuclear transport system. The nuclear transport system inhibited CML cell proliferation through mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 5 (STAT5) pathways mainly by HF2S. It was proven that nuclear located Bcr-Abl induced CML cell (including imatinib-resistant K562G01 cells) apoptosis by activation of p73 and its downstream molecules. In summary, our study provides a new targeted therapy for the CML patients even with Tyrosine Kinase Inhibitor (TKI)-resistance.

Targeted Covalent Inhibition of Grb2-Sos1 Interaction through Proximity-Induced Conjugation in Breast Cancer Cells.

Targeted covalent inhibitors of protein-protein interactions differ from reversible inhibitors in that the former bind and covalently bond the target protein at a specific site of the target. The site specificity is the result of the proximity of two reactive groups at the bound state, for example, one mild electrophile in the inhibitor and a natural cysteine in the target close to the ligand binding site. Only a few pharmaceutically relevant proteins have this structural feature. Grb2, a key adaptor protein in maintaining the ERK activity via binding Sos1 to activated RTKs, is one: the N-terminal SH3 domain of Grb2 (Grb2N-SH3) carries a unique solvent-accessible cysteine Cys32 close to its Sos1-binding site. Here we report the design of a peptide-based antagonist (a reactive peptide) that specifically binds to Grb2N-SH3 and subsequently undergoes a nucleophilic reaction with Cys32 to form a covalent bond thioether, to block Grb2-Sos1 interaction. Through rounds of optimization, we eventually obtained a dimeric reaction reactive peptide that can form a covalent adduct with endogenous Grb2 protein inside the cytosol of SK-BR-3 human breast cancer cells with pronounced inhibitory effect on cell mobility and viability. This work showcases a rational design of Grb2-targeted site-specific covalent inhibitor and its pronounced anticancer effect by targeting Grb2-Sos1 interaction.

Synthesis of Grb2 SH2 Domain Proteins for Mirror-Image Screening Systems.

Grb2 is an adaptor protein that mediates cellular signal transduction. Grb2 contains an SH2 domain that interacts with phosphotyrosine-containing sequences in EGFR and other signaling molecules, and it is a promising molecular target for anticancer agents. To identify novel inhibitors of the Grb2 SH2 domain from natural products and their mirror-image isomers, screening systems using both enantiomers of a synthetic Grb2 SH2 domain protein were established. A pair of synthetic procedures for the proteins were investigated: one employed a single native chemical ligation (NCL) of two segment peptides, and the other used the N-to-C-directed NCL of three segment peptides for easier preparation. Labeling at the N-terminus or the Ala115 residue of the Grb2 SH2 domain provided functional probes to detect binding to a phosphotyrosine-containing peptide. The resulting synthetic-protein-based probes were applied to bioassays, including chemical array analysis and enzyme-linked immunosorbent assays.