Purpose: The Ras-Raf-mitogen-activated protein kinase kinase (MEK) pathway is overactive in many human cancers and is thus a target for novel therapeutics. We have developed a highly potent and selective inhibitor of MEK1/2. The purpose of these studies has been to show the biological efficacy of ARRY-142886 (AZD6244) in enzymatic, cellular, and animal models. Experimental Design: The ability of ARRY-142886 to inhibit purified MEK1 as well as other kinases was evaluated. Its effects on extracellular signal-regulated kinase (ERK) phosphorylation and proliferation in several cell lines were also determined. Finally, the inhibitor was tested in HT-29 (colorectal) and BxPC3 (pancreatic) xenograft tumor models. Results: The IC 50 of ARRY-142886 was determined to be 14 nmol/L against purified MEK1. This activity is not competitive with ATP, which is consistent with the high specificity of compound for MEK1/2. Basal and epidermal growth factor^induced ERK1/2 phosphorylation was inhibited in several cell lines as well as 12-O-tetradecanoylphorbol-13-acetate^induced ERK1/2 phosphorylation in isolated peripheral blood mononuclear cells.Treatment with ARRY-142886 resulted in the growth inhibition of several cell lines containing B-Raf and Ras mutations but had no effect on a normal fibroblast cell line.When dosed orally, ARRY-142886 was capable of inhibiting both ERK1/ 2 phosphorylation and growth of HT-29 xenograft tumors in nude mice. Tumor regressions were also seen in a BxPC3 xenograft model. In addition, tumors remained responsive to growth inhibition after a 7-day dosing holiday. Conclusions: ARRY-142886 is a potent and selective MEK1/2 inhibitor that is highly active in both in vitro and in vivo tumor models. This compound is currently being investigated in clinical studies.Excessive growth factor signaling leads to unregulated growth that can contribute to the pathogenesis of human cancer. The signaling cascade is initiated by the binding of peptide growth factors to their tyrosine kinase receptors at the plasma membrane. The receptor kinases are activated and through the recruitment of the growth factor receptor binding protein 2/son of sevenless complex to autophosphorylated sites on the receptors, the G protein Ras is induced to its active GTP-bound state. Ras recruits the serine/threonine kinase Raf to the plasma membrane, where it is then able to phosphorylate and activate mitogen-activated protein kinase kinases (MEK) 1 and 2, which are dual specificity protein kinases that phosphorylate serine/ threonine and tyrosine residues. The MEK kinases in turn phosphorylate and activate their only currently known substrates, extracellular signal-regulated kinases (ERK) 1 and 2. ERK1/2 proteins translocate to the nucleus where they phosphorylate and activate effector proteins and transcription factors, resulting in diverse cellular responses, including proliferation.The overexpression and/or mutation of epidermal growth factor (EGF) receptor (EGFR), erbB2, platelet-derived growth factor receptor, RET, and othe...
The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563-74. ©2016 AACR.
A monoclonal antibody has been produced which immunoprecipitates 58-and 53-kDa proteins which are rapidly tyrosine phosphorylated in insulin-treated cells. These proteins can also be tyrosine phosphorylated in vitro by the isolated human insulin receptor. Increased tyrosine phosphorylation of these proteins is also observed in cells expressing a transforming chicken c-Src (mutant Phe-527) and in cells with the activated tyrosine kinase domains of the Drosophila insulin receptor, human insulin-like growth factor I receptor, and human insulin receptor-related receptor. P58/53 did not appear to associate with either the GTPase activating protein of Ras (called GAP) or the phosphatidylinositol 3-kinase by either co-immunoprecipitation experiments or in Far Westerns with the SH2 domains of these two proteins. Since p58/53 did not appear, by immunoblotting, to be related to any previously described tyrosine kinase substrate such as the SH2 containing proteins SHC and the tyrosine phosphatase Syp, the protein was purified in sufficient amounts to obtain peptide sequence. This sequence was utilized to isolate a cDNA clone that encodes a previously uncharacterized 53-kDa protein which, when expressed in mammalian cells, is tyrosine phosphorylated by the insulin receptor.In the last few years, there has been extensive progress toward an understanding of the mechanism whereby tyrosine kinases such as growth factor receptors elicit subsequent biological responses (1, 2). The identification and isolation of endogenous substrates for these molecules have revealed that various enzymes such as phospholipase C␥ are tyrosine phosphorylated and activated by this modification (3). In addition, other proteins have a sequence which is homologous to a region of c-Src (called the SH2 domain) that allows these proteins to bind to tyrosine-phosphorylated proteins (4). This complex formation can itself stimulate the enzymatic activity of the SH2 containing protein (for example, the binding of the phosphatidylinositol 3-kinase to insulin receptor substrate-1) or redirect it to another cellular compartment where its enzymatic activity is required (for example, the translocation of the GTPase activating protein of Ras to the plasma membrane, the site of its substrate) (4). In the case of the insulin receptor (IR), 1 several cytosolic substrates have been described. These include the most extensively characterized substrate, called insulin receptor substrate-1 (IRS-1), which is tyrosine phosphorylated and subsequently bound by the phosphatidylinositol (PI) 3-kinase as well as several other SH2 containing proteins (5). A variety of experimental approaches have implicated this substrate as playing a role in mediating several biological responses (6 -9) although gene knockout mice which lack IRS-1 still exhibit most of their responsiveness to insulin (10, 11). In addition, recent studies have shown that growth hormone, interleukins 4 and 13, interferons-␣ and ␥, and leukemia inhibitory factor can all stimulate the tyrosine phosphorylation of .In...
Tyrosine kinases of the Janus kinase family initiate cellular responses through their association with receptors for ␣-helical cytokines. In addition to a tyrosine kinase domain, these enzymes possess a kinase-like (KL) domain, whose function remains elusive. To investigate the role of the KL domain of Tyk2 in interferon-␣͞ signaling, we transfected a library of Tyk2 cDNAs containing random point mutations in KL into Tyk2-negative cells and selected for loss-of-function Tyk2 mutants. Four such mutants, V584D, G596V, H669P, and R856G, were identified through this screen. Like the wild-type Tyk2, the mutant proteins were able to sustain the level of IFNAR1 receptor protein. However, all four mutants were incapable of restoring high-affinity interferon-␣ binding in Tyk2-negative cells and were also catalytically impaired, even when transiently overexpressed. Interferon-␣ induced phosphorylation, and gene expression could be detected in V584D-or G596V-expressing cells, but not in H669P-or R856G-expressing cells. Furthermore, H669P and R856G proteins were constitutively highly phosphorylated. All together, our findings demonstrate that an intact KL domain is essential for the intrinsic catalytic activity of Tyk2 and for the establishment of a high-affinity interferon-␣ receptor complex.I n mammals, the four Janus kinases (JAKs) (Tyk2, JAK1, JAK2, and JAK3) have been shown to participate in the early steps of signaling cascades originating from cell surface receptors engaged with ␣-helix-bundled cytokine ligands (1-3). One major pathway triggered by these cytokines involves the catalytic activation of the receptor-associated JAK proteins, tyrosine phosphorylation of the cytoplasmic regions of the receptors, recruitment of SH2-containing signal transducers and activators of transcription (STAT) proteins onto the phosphorylated motifs, and phosphorylation of these transcription factors. Activated STATs then translocate into the nucleus and induce the transcription of target genes (1,4,5). Little is known about the molecular mechanisms regulating the JAK enzymes that play a critical role in this signaling pathway.Sequence alignment of the JAKs reveals seven regions of homology, called JH1 to JH7. The amino-terminal region comprises JH3 through JH7 and has been implicated in receptor interaction and stability (3). JH1, located at the carboxyl terminus, is a tyrosine kinase (TK) domain that contains an activation loop, the phosphorylation of which is required for catalytic activation (6-9). The centrally located JH2 or kinase-like (KL) domain exhibits high sequence identity (up to 30%) with kinase domains but lacks intrinsic catalytic activity. Differences between the KL domain and canonical kinase domains are evident in conserved sequences defining the ATP-and substrate-binding cleft, including the glycine-rich loop; the HRDL motif, which is HGNV in KL; and the DFG motif, which is DPG (10). No tyrosine residues are present in the segment of KL corresponding to the activation loop of active kinase domains.Functional conse...
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