The mTOR protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR-dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of PI3K typical of cells lacking TSC2, a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology.

SUMMARY In an effort to find new pharmacological modalities to overcome resistance to ATP-site inhibitors of Bcr-Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr-Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry we demonstrate that GNF-2 binds to the myristate binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analog of GNF-2 having improved pharmacokinetic properties, when utilized in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I Bcr-Abl and displayed in vivo efficacy against the recalcitrant T315I Bcr-Abl mutant in a murine bone-marrow transplantation model. These results demonstrate that therapeutically relevant inhibition of Bcr-Abl activity can be achieved using inhibitors that bind to the myristate binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

The candidate tumor suppressor ING1 was identified in a genetic screen aimed at isolation of human genes whose expression is suppressed in cancer cells. It may function as a negative growth regulator in the p53 signal transduction pathway. However, its molecular mechanism is not clear. The ING1 locus encodes alternative transcripts of p47ING1a , p33 ING1b, and p24 ING1c . Here we report differential association of protein products of ING1 with the mSin3 transcriptional corepressor complex. p33ING1b associates with Sin3, SAP30, HDAC1, RbAp48, and other proteins, to form large protein complexes, whereas p24ING1c does not. The ING1 immune complexes are active in deacetylating core histones in vitro, and p33ING1b is functionally associated with HDAC1-mediated transcriptional repression in transfected cells. Our data provide basis for a p33ING1b -specific molecular mechanism for the function of the ING1 locus.

The Eph family of receptor tyrosine kinases has drawn growing attention due to their role in regulating diverse biological phenomena. However, pharmacological interrogation of Eph kinase function has been hampered by a lack of potent and selective Eph kinase inhibitors. Here we report the discovery of compounds 6 and 9 using a rationally designed kinase-directed library which potently inhibit Eph receptor tyrosine kinases, particularly EphB2 with cellular EC 50 s of 40 nM. Crystallographic data of EphA3 and EphA7 in complex with the inhibitors show that they bind to the "DFG-out" inactive kinase conformation and provide valuable information for structure-based design of second generation inhibitors.The Eph/ephrin family is the largest among tyrosine kinases and is unique in that the ligands and receptors are both membrane bound providing the possibility for bidirectional cell-cell signaling. Genome analysis reveals that there are 14 Eph receptors and eight ephrin ligands. Eph receptor signaling is responsible for arguably the most diverse set of biological phenomena of any tyrosine kinase family including organ development, tissue remodeling, neuronal signaling, insulin secretion, and bone metabolism. 1, 2 Not surprisingly, deregulation of ephrindependent signaling has been implicated in pathological conditions related to all of these systems. 3 The involvement of Eph/ephrin-signaling in tumorigenesis has been the most extensively investigated due to frequent upregulation of Eph receptor or ligand expression in numerous tumor types. [4][5][6] The emerging picture is complicated by the diversity of biological function that is associated with individual receptors and ligands, including oncogenic or tumor suppressor functions. The possibility of targeting Eph/ephrins therapeutically may be the most straightforward in the context of inhibiting Eph/ephrin-signaling in the vasculature as a means of preventing tumor angiogenesis. 2,6 Currently only a few small molecule Eph kinase © 2009 Elsevier Ltd. All rights reserved. * Corresponding Authors: Sirano Dhe-Paganon, Tel: 416-946-3876, E-mail: sirano.dhepaganon(@)utoronto.ca Nathanael Gray, Tel: 617-582-8590, E-mail: nathanael_gray(@)dfci.harvard.edu . † These authors contributed equally to this work Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ¶ Once inside PyMol, press the play button (at the bottom right) in order to start the 3D movie NIH Public Access To date the majority of investigation into functions of Eph receptor tyrosine kinases has been accomplished using genetic and biochemical methods. Pharmacological app...

Overproduction of the Her2 oncoprotein has been found in Ϸ30% of breast tumors, and patients who have Her2 excesses typically have more aggressive disease. Here we show that the expression of the Her2 gene can be decreased by inhibiting the interaction of the two cancer-linked proteins, DRIP130͞CRSP130͞Sur-2 (a Raslinked subunit of human mediator complexes) and ESX (an epithelial-restricted transcription factor). Disruption of the interaction by a short cell-permeable peptide reduced the expression of the Her2 gene and specifically impaired the growth and viability of Her2-overexpressing breast cancer cells. The association of ESX with DRIP130 is mediated by a small hydrophobic face of an 8-aa helix in ESX, suggesting a therapeutic approach to incapacitating the Her2 gene by small organic molecules. O verexpression of the oncogene Her2 (neu͞ErbB2) has been found in Ϸ30% of breast tumors (1, 2). It is unclear why overexpression of this transmembrane receptor occurs in breast cancers, but patients who have Her2 excesses typically have more aggressive disease with enhanced metastasis and increased resistance to chemotherapy (1-6). Monoclonal antibodies against the Her2 protein have been successful in treating these Her2-positive patients (7-10). A humanized antibody called Herceptin has demonstrated tumor-inhibitory and chemosensitizing effects in clinical studies and is the only drug that the Food and Drug Administration has approved for treatment of Her2-overexpressing breast tumors (9, 10). The clinical success of the Her2-antibody therapy was an excellent example of the translation of basic cancer biology into clinical cancer treatment. However, the antibody therapy alone may not be ideal for therapeutic intervention of Her2-overexpressing breast cancers. In theory, down-regulation of Her2 may be accomplished efficiently by inhibiting the expression of the Her2 gene rather than targeting elevated levels of the Her2 proteins that are already overexpressed. By analogy with treatments for AIDS, cocktails of drugs, each with different mechanisms of action, might also be more effective at achieving complete remission of breast tumors. Thus, discovering a means to provide external control over Her2 expression, particularly through small organic molecules, remains appealing.One of the critical transcription factors that activate the Her2 gene in breast cancers is ESX (ESE-1͞ELF3͞ERT͞Jen), an Ets factor that is expressed specifically in epithelial cells including mammary glands (11-13). ESX binds and strongly activates the Her2 promoter (11), and the ESX-binding site in the Her2 promoter is absolutely required for the high-level expression of Her2 in breast cancer cells (14). The expression of ESX is boosted in Her2-overexpressing breast cancers (11); a simple correlation seems to exist between the expression levels of ESX and Her2 in breast cancer cells. Here we isolate DRIP130͞ CRSP130͞Sur-2, a Ras-linked metazoan-specific subunit of human mediator complexes, as a nuclear cofactor that binds specifically to the trans...

Inhibition of Bcr-Abl kinase activity by imatinib for the treatment of chronic myeloid leukemia (CML) currently serves as the paradigm for targeting dominant oncogenes with small molecules. We recently reported the discovery of GNF-2 (1) and GNF-5 (2) as selective non-ATP competitive inhibitors of cellular Bcr-Abl kinase activity that target the myristate binding site. Here, we used cell-based structure−activity relationships to guide the optimization and diversification of ligands that are capable of binding to the myristate binding site and rationalize the findings based upon an Abl−compound 1 cocrystal. We elucidate the structure−activity relationships required to obtain potent antiproliferative activity against Bcr-Abl transformed cells and report the discovery of new compounds (5g, 5h, 6a, 14d, and 21j-I) that display improved potency or pharmacological properties. This work demonstrates that a variety of structures can effectively target the Bcr-Abl myristate binding site and provides new leads for developing drugs that can target this binding site.

[M-H] -. The concentrations were determined by measuring UV absorbance at pH 7.5 (50 mM NH 4 HCO 3 buffer); ε = 67000 Lmol -1 cm -1 at λ max near 500 nm was used. The samples were aliquoted and stored at -20 ˚C.

Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors. Here we explore the activity of a previously reported allosteric inhibitor of BCR-Abl kinase, GNF-2, against two cellular isoforms of Abl tyrosine kinase: one that carries a myristate in the N terminus and the other that is deficient in N-myristoylation. Our results show that GNF-2 inhibits the kinase activity of non-myristoylated c-Abl more potently than that of myristoylated c-Abl by binding to the myristate-binding pocket in the C-lobe of the kinase domain. Unexpectedly, indirect immunofluorescence reveals a translocation of myristoylated c-Abl to the endoplasmic reticulum in GNF-2-treated cells, whereas GNF-2 has no detectable effect on the localization of non-myristoylated c-Abl. These results indicate that GNF-2 competes with the NH 2 -terminal myristate for binding to the c-Abl kinase myristate-binding pocket and that the exposed myristoyl group accounts for the localization to the endoplasmic reticulum. We also demonstrate that GNF-2 can inhibit enzymatic and cellular kinase activity of Arg, a kinase highly homologous to c-Abl, which is also likely to be regulated through intramolecular binding of an NH 2 -terminal myristate lipid. These results suggest that non-ATP-competitive inhibitors, such as GNF-2, can serve as chemical tools that can discriminate between c-Abl isoform-specific behaviors.The catalytic activity of a protein kinase can be modulated by binding of a ligand to a site distant from the active site, also referred to as the allosteric site (1). The ligand is referred to as an allosteric kinase inhibitor and induces a protein conformation that is not compatible with kinase activity. Allosteric inhibitors can potentially be exploited to elucidate kinase functions not discovered using ATP-competitive inhibitors, because they can exploit binding sites and regulatory mechanisms that are unique to a particular kinase.The c-Abl and Arg (Abl-related gene) proteins comprise the Abl family of non-receptor tyrosine kinases. Each family member has two isoforms: one that is myristoylated in the N terminus (1b or IV) and the other that is deficient in N-myristoylation due to an alternative splicing of the first exon (1a or I) (Fig. 1A). N-Myristoylation often serves as a mechanism for targeting proteins to cellular membranes. However, Abl family members localize to multiple subcellular compartments; whereas Arg is mostly found in the cytoplasm, c-Abl shuttles between the nucleus and the cytoplasm, where it localizes to the cytosol, endoplasmic reticulum, and mitochondria (2).The Abl family members share a high degree of sequence identity (ϳ90%) in the NH 2 -terminal half residues, including the SH3, 2 SH2, and kinase domains (3). The kinase domain is followed by proline-rich motifs that serve as binding sites for SH3 domains. A range of proteins are reported to bind directly or indirectly to the SH3, SH2, and proline-rich domains of c-Abl and are impl...