Synergistic effect of growth factor receptor-bound protein 2/epidermal growth factor receptor dual-targeting peptide inhibitor and salinomycin on osteosarcoma.

Context: The growth factor receptor-bound protein 2 (Grb2)-Sos1 interaction, mediated by modular domains, plays an essential role in the oncogenic MAPK signaling pathway in osteosarcoma (OS). Recently, a dual-targeting peptide that targets the epidermal growth factor receptor and Grb2-Src homology 3 domain in OS cells was designed and synthesized. Aims: We investigated the synergistic effects of the peptide and salinomycin (Sal), a chemotherapeutic drug with effective anti-OS properties in clinical therapy. Subjects and Methods: Flow cytometry was used to measure the targeting efficacy of the peptide. Migration and CCK-8 assays were used to explore whether Sal and the peptide could synergistically inhibit OS cell behavior. Western blotting was used to detect apoptosis. Statistical Analysis Used: Data were analyzed using the GraphPad Prism 5.01. Statistical analysis was performed using the Student's t-test for the direct comparisons and one-way analysis of variance for the comparisons among the multiple groups. Statistical significance was set at P < 0.05. Results: The peptide was shown to target OS cells. When applied together, Sal and the peptide synergistically inhibited OS cell migration, invasion, and proliferation through the inhibition of Grb2-Sos1. This synergistic treatment also promoted the apoptosis of OS cells and inhibited tumor volume in vivo. Conclusions: These data provide valuable insights into the molecular mechanisms of OS and may be beneficial in clinical therapy.

Synthesis and structural characterization of a monocarboxylic inhibitor for GRB2 SH2 domain.

A monocarboxylic inhibitor was designed and synthesized to disrupt the protein-protein interaction (PPI) between GRB2 and phosphotyrosine-containing proteins. Biochemical characterizations show compound 7 binds with the Src homology 2 (SH2) domain of GRB2 and is more potent than EGFR1068 phosphopeptide 14-mer. X-ray crystallographic studies demonstrate compound 7 occupies the GRB2 binding site for phosphotyrosine-containing sequences and reveal key structural features for GRB2-inhibitor binding. This compound with a -1 formal charge offers a new direction for structural optimization to generate cell-permeable inhibitors for this key protein target of the aberrant Ras-MAPK signaling cascade.

Rational design of cell-permeable cyclic peptides containing a d-Pro-l-Pro motif.

Cyclic peptides are capable of binding to challenging targets (e.g., proteins involved in protein-protein interactions) with high affinity and specificity, but generally cannot gain access to intracellular targets because of poor membrane permeability. In this work, we discovered a conformationally constrained cyclic cell-penetrating peptide (CPP) containing a d-Pro-l-Pro motif, cyclo(AFΦrpPRRFQ) (where Φ is l-naphthylalanine, r is d-arginine, and p is d-proline). The structural constraints provided by cyclization and the d-Pro-l-Pro motif permitted the rational design of cell-permeable cyclic peptides of large ring sizes (up to 16 amino acids). This strategy was applied to design a potent, cell-permeable, and biologically active cyclic peptidyl inhibitor, cyclo(YpVNFΦrpPRR) (where Yp is l-phosphotyrosine), against the Grb2 SH2 domain. Multidimensional NMR spectroscopic and circular dichroism analyses revealed that the cyclic CPP as well as the Grb2 SH2 inhibitor assume a predominantly random coil structure but have significant ß-hairpin character surrounding the d-Pro-l-Pro motif. These results demonstrate cyclo(AFΦrpPRRFQ) as an effective CPP for endocyclic (insertion of cargo into the CPP ring) or exocyclic delivery of biological cargos (attachment of cargo to the Gln side chain).

NHD2-15, a novel antagonist of Growth Factor Receptor-Bound Protein-2 (GRB2), inhibits leukemic proliferation.

The majority of chronic myeloid leukemia (CML) cases are caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1), creating the mutant fusion protein BCR-ABL1. Downstream of BCR-ABL1 is growth factor receptor-bound protein-2 (GRB2), an intracellular adapter protein that binds to BCR-ABL1 via its src-homology-2 (SH2) domain. This binding constitutively activates growth pathways, downregulates apoptosis, and leads to an over proliferation of immature and dysfunctional myeloid cells. Utilizing novel synthetic methods, we developed four furo-quinoxaline compounds as GRB2 SH2 domain antagonists with the goal of disrupting this leukemogenic signaling. One of the four antagonists, NHD2-15, showed a significant reduction in proliferation of K562 cells, a human BCR-ABL1+ leukemic cell line. To elucidate the mode of action of these compounds, various biophysical, in vitro, and in vivo assays were performed. Surface plasmon resonance (SPR) assays indicated that NHD2-15 antagonized GRB2, binding with a KD value of 119 ± 2 µM. Cellulose nitrate (CN) assays indicated that the compound selectively bound the SH2 domain of GRB2. Western blot assays suggested the antagonist downregulated proteins involved in leukemic transformation. Finally, NHD2-15 was nontoxic to primary cells and adult zebrafish, indicating that it may be an effective clinical treatment for CML.

Lymecycline reverses acquired EGFR-TKI resistance in non-small-cell lung cancer by targeting GRB2.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) were first-line treatments for NSCLC patients with EGFR-mutations. However, about 30 % of responders relapsed within six months because of acquired resistance. In this study, we used Connectivity Map (CMap) to discover a drug capable of reversing acquired EGFR-TKIs resistance. To investigate Lymecycline's ability to reverse acquired EGFR-TKIs resistance, two Icotinib resistant cell lines were constructed. Lymecycline's ability to suppress the proliferation of Icotinib resistant cells in vitro and in vivo was then evaluated. Molecular targets were predicted using network pharmacology and used to identify the molecular mechanism. Growth factor receptor-bound protein 2 (GRB2) is an EGFR-binding adaptor protein essential for EGFR phosphorylation and regulation of AKT/ERK/STAT3 signaling pathways. Lymecycline targeted GRB2 and inhibited the resistance of the cell cycle to EGFR-TKI, arresting disease progression and inducing apoptosis in cancer cells. Combined Lymecycline and Icotinib treatment produced a synergistic effect and induced apoptosis in HCC827R5 and PC9R10 cells. Cell proliferation in resistant cancer cells was significantly inhibited by the combined Lymecycline and Icotinib treatment in mouse models. Lymecycline inhibited the resistance of the cell cycle to EGFR-TKI and induced apoptosis in NSCLC by inhibiting EGFR phosphorylation and GRB2-mediated AKT/ERK/STAT3 signaling pathways. This provided strong support that Lymecycline when combined with EGFR targeting drugs, enhanced the efficacy of treatments for drug-resistant NSCLC.

Downregulation of long non-coding RNA ZXF1 restricts cell survival by targeting miR-634-GRB2 in lung adenocarcinoma.

Increasing evidence demonstrates that long non-coding RNAs (lncRNAs) play important regulatory roles in mediating initiation and progression of lung adenocarcinoma (LA), which is one of the most lethal in humans. A previous study reported that lncRNAZXF1 was dysregulated in LA and enhanced expression of ZXF1 promoted the invasion and metastasis in LA. However, the effect of ZXF1 on LA progression and its underlying mechanisms were not thoroughly investigated. In our in vitro experiments, qRT-PCR revealed that the expression level of ZXF1 in LA tissues and tumor cells were significantly higher than that in adjacent normal tissues and normal cells. Furthermore, bioinformatics analysis, luciferase reporter assay, western blot and RNA immunoprecipitation (RIP) assay showed that ZXF1 could directly interact with miR-634, which targets GRB2. Therefore, we propose that ZXF1 could function as an oncogene partly by sponging miR-634 and therefore regulating GRB2 expression in LA. Overall, this study demonstrated, for the first time, that the lncRNA ZXF1/miR-634/GRB2 axis plays crucial roles in modulating LA progression. Moreover, lncRNA ZXF1 might potentially improve LA prognosis and serve as a therapeutic target for the treatment of LA.

Insulin enhancement of the antitumor activity of chemotherapeutic agents in colorectal cancer is linked with downregulating PIK3CA and GRB2.

The present state of cancer chemotherapy is unsatisfactory. New anticancer drugs that marginally improve the survival of patients continue to be developed at an unsustainably high cost. The study aimed to elucidate the effects of insulin (INS), an inexpensive drug with a convincing safety profile, on the susceptibility of colon cancer to chemotherapeutic agents: 5-fluorouracil (FU), oxaliplatin (OXA), irinotecan (IRI), cyclophosphamide (CPA) and docetaxel (DOC). To examine the effects of insulin on cell viability and apoptosis, we performed an in vitro analysis on colon cancer cell lines Caco-2 and SW480. To verify the results, we performed in vivo analysis on mice bearing MC38 colon tumors. To assess the underlying mechanism of the therapy, we examined the mRNA expression of pathways related to the signaling downstream of insulin receptors (INSR). Moreover, we performed Western blotting to confirm expression patterns derived from the genetic analysis. For the quantification of circulating tumor cells in the peripheral blood, we used the maintrac method. The results of our study show that insulin-pretreated colon cancer cells are significantly more susceptible to commonly used chemotherapeutics. The apoptosis ratio was also enhanced when INS was administered complementary to the examined drugs. The in vivo study showed that the combination of INS and FU resulted in significant inhibition of tumor growth and reduction of the number of circulating tumor cells. This combination caused a significant downregulation of the key signaling substrates downstream of INSR. The results indicate that the downregulation of PIK3CA (phosphatidylinositol 3-kinase catalytic subunit alpha), which plays a critical role in cell signaling and GRB2 (growth factor receptor-bound protein 2), a regulator of cell proliferation and differentiation may be responsible for the sensitizing effect of INS. These findings were confirmed at protein levels by Western blotting. In conclusion, these results suggest that INS might be potentially applied to clinical use to enhance the therapeutic effectiveness of chemotherapeutic drugs. The findings may become a platform for the future development of new and inexpensive strategies for the clinical chemotherapy of tumors.

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.

Proteomic Analysis Reveals Grb2 as a Key Regulator of Periodic Mechanical Stress Transduction in Chondrocytes.

BACKGROUND/AIMS: Periodic mechanical stress could significantly promote chondrocyte proliferation and matrix synthesis. However, the mechanisms underlying the ability of chondrocyte detecting and responding to periodic mechanical stimuli have not been well delineated. METHODS: Quantitative proteomic analysis was performed to construct the differently expressed proteome profiles of chondrocyte under pressure. Then a combination of Western blot, quantitative real-time PCR, lentiviral vector and histological methods were used to confirm the proteomic results and investigate the mechanoseing mechanism. RESULTS: Growth factor receptor-bound protein 2 (Grb2), a component of integrin adhesome, was found a 1.49-fold increase in dynamic stress group. This process was mediated through integrin ß1, leading to increased phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (ERK1/2) respectively and then produce the corresponding biological effects. CONCLUSION: This was the first time to demonstrate Grb2 has such an important role in periodic mechanotransduction, and the proteomic data could facilitate the further investigation of chondrocytes mechanosensing.

SC1 Promotes MiR124-3p Expression to Maintain the Self-Renewal of Mouse Embryonic Stem Cells by Inhibiting the MEK/ERK Pathway.

BACKGROUND/AIMS: Self-renewal is one of the most important features of embryonic stem (ES) cells. SC1 is a small molecule modulator that effectively maintains the self-renewal of mouse ES cells in the absence of leukemia inhibitory factor (LIF), serum and feeder cells. However, the mechanism by which SC1 maintains the undifferentiated state of mouse ES cells remains unclear. METHODS: In this study, microarray and small RNA deep-sequencing experiments were performed on mouse ES cells treated with or without SC1 to identify the key genes and microRNAs that contributed to self-renewal. RESULTS: SC1 regulates the expressions of pluripotency and differentiation factors, and antagonizes the retinoic acid (RA)-induced differentiation in the presence or absence of LIF. SC1 inhibits the MEK/ERK pathway through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and pathway reporting experiments. Small RNA deep-sequencing revealed that SC1 significantly modulates the expression of multiple microRNAs with crucial functions in ES cells. The expression of miR124-3p is upregulated in SC1-treated ES cells, which significantly inhibits the MEK/ERK pathway by targeting Grb2, Sos2 and Egr1. CONCLUSION: SC1 enhances the self-renewal capacity of mouse ES cells by modulating the expression of key regulatory genes and pluripotency-associated microRNAs. SC1 significantly upregulates miR124-3p expression to further inhibit the MEK/ ERK pathway by targeting Grb2, Sos2 and Egr1.

Comparing pharmacophore models derived from crystallography and NMR ensembles.

NMR and X-ray crystallography are the two most widely used methods for determining protein structures. Our previous study examining NMR versus X-Ray sources of protein conformations showed improved performance with NMR structures when used in our Multiple Protein Structures (MPS) method for receptor-based pharmacophores (Damm, Carlson, J Am Chem Soc 129:8225-8235, 2007). However, that work was based on a single test case, HIV-1 protease, because of the rich data available for that system. New data for more systems are available now, which calls for further examination of the effect of different sources of protein conformations. The MPS technique was applied to Growth factor receptor bound protein 2 (Grb2), Src SH2 homology domain (Src-SH2), FK506-binding protein 1A (FKBP12), and Peroxisome proliferator-activated receptor-γ (PPAR-γ). Pharmacophore models from both crystal and NMR ensembles were able to discriminate between high-affinity, low-affinity, and decoy molecules. As we found in our original study, NMR models showed optimal performance when all elements were used. The crystal models had more pharmacophore elements compared to their NMR counterparts. The crystal-based models exhibited optimum performance only when pharmacophore elements were dropped. This supports our assertion that the higher flexibility in NMR ensembles helps focus the models on the most essential interactions with the protein. Our studies suggest that the "extra" pharmacophore elements seen at the periphery in X-ray models arise as a result of decreased protein flexibility and make very little contribution to model performance.

α-Lipoic acid inhibits human lung cancer cell proliferation through Grb2-mediated EGFR downregulation.

BACKGROUND: Alpha lipoic acid (α -LA) is a naturally occurring antioxidant and metabolic enzyme co-factor. Recently, α -LA has been reported to inhibit the growth of various cancer cells, but the precise signaling pathways that mediate the effects of α -LA on non-small cell lung cancer (NSCLC) development remain unclear. METHODS: The CCK-8 assay was used to assess cell proliferation in NSCLC cell lines after α -LA treatment. The expression of growth factor receptor-bound protein 2 (Grb2), cyclin-dependent kinase (CDK)-2, CDK4, CDK6, Cyclin D3, Cyclin E1, Ras, c-Raf, epidermal growth factor receptor (EGFR), ERK1/2 and activated EGFR and ERK1/2 was evaluated by western blotting. Grb2 levels were restored in α-LA-treated cells by transfection of a plasmid carrying Grb2 and were reduced in NSCLC cells via specific siRNA-mediated knockdown. RESULTS: α -LA dramatically decreased NSCLC cell proliferation by downregulating Grb2; in contrast, Grb2 overexpression significantly prevented α-LA-induced decrease in cell growth in vitro. Western blot analysis indicated that α-LA decreased the levels of phospho-EGFR, CDK2/4/6, Cyclins D3 and E1, which are associated with the inhibition of G1/S-phase transition. Additional experiments indicated that Grb2 inhibition partially abolished EGF-induced phospho-EGFR and phospho-ERK1/2 activity. In addition, α-LA exerted greater inhibitory effects than gefitinib on NSCLC cells by preventing EGF-induced EGFR activation. CONCLUSION: For the first time, these findings provide the first evidence that α-LA inhibits cell proliferation through Grb2 by suppressing EGFR phosphorylation and that MAPK/ERK is involved in this pathway.

MiR-200a modulates TGF-ß1-induced endothelial-to-mesenchymal shift via suppression of GRB2 in HAECs.

Endothelial-mesenchymal transition (EndMT) is closely associated with embryogenesis, injury restitution, tissue neogenesis, tumor progressions and viscera fibrosis. EndMT may occur in the proximal tubular endothelial cells, inducing fibroblasts to produce matrix and then accelerating the process of cardiac fibrosis. Transforming growth factor-ß1 (TGF-ß1), a profibrotic cytokine, was recently shown to be a crucial trigger of EndMT in tubular endothelial cells. Increasing evidence suggests that growth factor receptor-bound 2 (GRB2) dysfunction affects fibrocytes; thus, GRB2 may be a novel target for treating fibrosis. The miR-200 miRNA cluster (miR-429, miR-141, miR-200c, miR-200b and miR-200a) was reported to inhibit EndMT. However, the underlying mechanisms, specifically that of miR-200a, are unclear. To elucidate the vital role of miR-200a in EndMT, we established a cardiac interstitial fibrosis model with a widely used EndMT assay, TGF-ß1-induced EndMT in human aortic endothelial cells (HAECs). We found that overexpression of miR-200a blocked EndMT in HAECs by inhibiting fibroblast-specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA) expression and increasing platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial cadherin (VE-cadherin) expression, regardless of the presence of TGF-ß1. MiR-200a expression was suppressed during the EndMT process, in both time- and dose-dependent manners, following GRB2 upregulation. EndMT was promoted by ectopic expression of GRB2 via decreased CD31 and VE-cadherin. Furthermore, EndMT was partially inhibited by co-transfection of miR-200a with GRB2 ORF, likely by restoring CD31 and VE-cadherin expression. MiR-200a negatively regulated GRB2 protein levels via direct binding to the GRB2 3'UTR. Finally, these discoveries may provide novel insights into the functional mechanism of miR-200a in regulating fibrosis via the TGFß1/miR-200a/GRB2/EndMT pathway, and miR-200a may serve as a new target for treating fibrosis in the future.

Up-regulation of the active form of small GTPase Rab13 promotes macroautophagy in vascular endothelial cells.

The importance of macroautophagy (hereafter referred to as autophagy) in vascular endothelial cell (VEC) biology and dysfunction is increasingly recognized, but the molecular mechanisms of autophagy in VECs in the presence of serum are still poorly understood. Previously, we identified pterostilbene as a potent autophagy inducer of VECs in the presence of serum. In this study, we used pterostilbene as a tool to induce VEC autophagy and identified the differentially expressed genes using high-throughput DAN microarray. The small GTPase Ras-related protein in brain 13 (Rab13) was found to be the most significantly up-regulated gene in pterostilbene-treated human umbilical VECs (HUVECs). Knockdown of Rab13 blocked pterostilbene-induced mTOR inhibition and autophagy, whereas overexpression of the GTP-containing active form of Rab13 induced mTOR inhibition and autophagy in HUVECs. Using a combination of immunofluorescence and co-immunoprecipitation (co-IP) assays, we demonstrated that pterostilbene or up-regulation of the active form of Rab13 promoted the interaction between Rab13 and growth factor receptor-bound protein 2 (Grb2). Knockdown of Grb2 suppressed pterostilbene or up-regulation of the active form of Rab13-induced autophagy. Further mechanistic studies revealed that Rab13 activated the downstream AMP-activated protein kinase (AMPK) and blocked mammalian target of rapamycin (mTOR) signaling by its functional interaction with Grb2 to regulate autophagy in HUVECs. Our study firmly establishes Rab13 as a novel regulator of autophagy in VECs under nutrient-enriched conditions.

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.

Necdin controls EGFR signaling linked to astrocyte differentiation in primary cortical progenitor cells.

Cellular signaling mediated by the EGF receptor (EGFR) plays a key role in controlling proliferation and differentiation of cortical progenitor cells (CPCs). However, regulatory mechanisms of EGFR signaling in CPCs remain largely unknown. Here we demonstrate that necdin, a MAGE (melanoma antigen) family protein, interacts with EGFR in primary CPCs and represses its downstream signaling linked to astrocyte differentiation. EGFR was autophosphorylated and interacted with necdin in EGF-stimulated CPCs. Necdin bound to autophosphorylated EGFR via its tyrosine kinase domain. EGF-induced phosphorylation of ERK was enhanced in necdin-null CPCs, where the interaction between EGFR and the adaptor protein Grb2 was strengthened, suggesting that endogenous necdin suppresses the EGFR/ERK signaling pathway in CPCs. In necdin-null CPCs, astrocyte differentiation induced by the gliogenic cytokine cardiotrophin-1 was significantly accelerated in the presence of EGF, and inhibition of EGFR/ERK signaling abolished the acceleration. Furthermore, necdin strongly suppressed astrocyte differentiation induced by overexpression of EGFR or its ligand binding-defective mutant equivalent to a glioblastoma-associated EGFR variant. These results suggest that necdin acts as an intrinsic suppressor of the EGFR/ERK signaling pathway in EGF-responsive CPCs to restrain astroglial development in a cell-autonomous manner.

Grb2 depletion under non-stimulated conditions inhibits PTEN, promotes Akt-induced tumor formation and contributes to poor prognosis in ovarian cancer.

In the absence of extracellular stimulation the adaptor protein growth factor receptor-bound protein (Grb2) and the phospholipase Plcγ1 compete for the same binding site on fibroblast growth factor receptor 2 (FGFR2). Reducing cellular Grb2 results in upregulation of Plcγ1 and depletion of the phospholipid PI(4,5)P2. The functional consequences of this event on signaling pathways are unknown. We show that the decrease in PI(4,5)P2 level under non-stimulated conditions inhibits PTEN activity leading to the aberrant activation of the oncoprotein Akt. This results in excessive cell proliferation and tumor progression in a xenograft mouse model. As well as defining a novel mechanism of Akt phosphorylation with important therapeutic consequences, we also demonstrate that differential expression levels of FGFR2, Plcγ1 and Grb2 correlate with patient survival. Oncogenesis through fluctuation in the expression levels of these proteins negates extracellular stimulation or mutation and defines them as novel prognostic markers in ovarian cancer.

A Synthetic Lethality Screen Using a Focused siRNA Library to Identify Sensitizers to Dasatinib Therapy for the Treatment of Epithelial Ovarian Cancer.

Molecular targeted therapies have been the focus of recent clinical trials for the treatment of patients with recurrent epithelial ovarian cancer (EOC). The majority have not fared well as monotherapies for improving survival of these patients. Poor bioavailability, lack of predictive biomarkers, and the presence of multiple survival pathways can all diminish the success of a targeted agent. Dasatinib is a tyrosine kinase inhibitor of the Src-family kinases (SFK) and in preclinical studies shown to have substantial activity in EOC. However, when evaluated in a phase 2 clinical trial for patients with recurrent or persistent EOC, it was found to have minimal activity. We hypothesized that synthetic lethality screens performed using a cogently designed siRNA library would identify second-site molecular targets that could synergize with SFK inhibition and improve dasatinib efficacy. Using a systematic approach, we performed primary siRNA screening using a library focused on 638 genes corresponding to a network centered on EGFR, HER2, and the SFK-scaffolding proteins BCAR1, NEDD9, and EFS to screen EOC cells in combination with dasatinib. We followed up with validation studies including deconvolution screening, quantitative PCR to confirm effective gene silencing, correlation of gene expression with dasatinib sensitivity, and assessment of the clinical relevance of hits using TCGA ovarian cancer data. A refined list of five candidates (CSNK2A1, DAG1, GRB2, PRKCE, and VAV1) was identified as showing the greatest potential for improving sensitivity to dasatinib in EOC. Of these, CSNK2A1, which codes for the catalytic alpha subunit of protein kinase CK2, was selected for additional evaluation. Synergistic activity of the clinically relevant inhibitor of CK2, CX-4945, with dasatinib in reducing cell proliferation and increasing apoptosis was observed across multiple EOC cell lines. This overall approach to improving drug efficacy can be applied to other targeted agents that have similarly shown poor clinical activity.

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.

Protein-ligand interactions: probing the energetics of a putative cation-π interaction.

In order to probe the energetics associated with a putative cation-π interaction, thermodynamic parameters are determined for complex formation between the Grb2 SH2 domain and tripeptide derivatives of RCO-pTyr-Ac6c-Asn wherein the R group is varied to include different alkyl, cycloalkyl, and aryl groups. Although an indole ring is reputed to have the strongest interaction with a guanidinium ion, binding free energies, ΔG°, for derivatives of RCO-pTyr-Ac6c-Asn bearing cyclohexyl and phenyl groups were slightly more favorable than their indolyl analog. Crystallographic analysis of two complexes reveals that test ligands bind in similar poses with the notable exception of the relative orientation and proximity of the phenyl and indolyl rings relative to an arginine residue of the domain. These spatial orientations are consistent with those observed in other cation-π interactions, but there is no net energetic benefit to such an interaction in this biological system. Accordingly, although cation-π interactions are well documented as important noncovalent forces in molecular recognition, the energetics of such interactions may be mitigated by other nonbonded interactions and solvation effects in protein-ligand associations.