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Tankyrases (TNKS1 and TNKS2) are proteins in the poly ADP-ribose polymerase (PARP) family. They have been shown to directly bind to axin proteins, which negatively regulate the Wnt pathway by promoting β-catenin degradation. Inhibition of tankyrases may offer a novel approach to the treatment of APC-mutant colorectal cancer. Hit compound 8 was identified as an inhibitor of tankyrases through a combination of substructure searching of the Amgen compound collection based on a minimal binding pharmacophore hypothesis and high-throughput screening. Herein we report the structure- and property-based optimization of compound 8 leading to the identification of more potent and selective tankyrase inhibitors 22 and 49 with improved pharmacokinetic properties in rodents, which are well suited as tool compounds for further in vivo validation studies.

The methylation state of lysine residues within histone H3 is a major determinant of active and inactive regions of the genome. Enhancer of Zeste homolog 2 (EZH2) is a histone lysine methyltransferase that is part of the polycomb repressive complex 2 (PRC2). Elevated EZH2 expression levels have been linked to hypertrimethylation of histone H3 lysine 27 (H3K27), repression of tumor repressor genes, and the onset of several types of cancers. We used the AlphaLISA technology to develop a high-throughput assay for identifying small molecule inhibitors of EZH2. AlphaLISA Acceptor Beads coated with antibodies directed against methylated H3K27 provided a sensitive method of detecting EZH2 activity through measurement of K27 methylation of a biotinylated H3-based peptide substrate. Optimized assay conditions resulted in a robust assay (Z'0.7) which was successfully implemented in a high-throughput screening campaign. Small molecule inhibitors identified by this method may serve as powerful tools to further elucidate the potential importance of EZH2 in the development and treatment of cancer.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that, when genetically altered by mutation, amplification, chromosomal translocation or inversion, has been shown to play an oncogenic role in certain cancers. Small molecule inhibitors targeting the kinase activity of ALK have proven to be effective therapies in certain ALK-driven malignancies and one such inhibitor, crizotinib, is now approved for the treatment of EML4-ALK-driven, non-small cell lung cancer. In neuroblastoma, activating point mutations in the ALK kinase domain can drive disease progression, with the two most common mutations being F1174L and R1275Q. We report here crystal structures of the ALK kinase domain containing the F1174L and R1275Q mutations. Also included are crystal structures of ALK in complex with novel small molecule ALK inhibitors, including a classic type II inhibitor, that stabilize previously unobserved conformations of the ALK activation loop. Collectively, these structures illustrate a different series of activation loop conformations than has been observed in previous ALK crystal structures and provide insight into the activating nature of the R1275Q mutation. The novel active site topologies presented here may also aid the structure-based drug design of a new generation of ALK inhibitors.

Lysine demethylase 1 (LSD1) and Jumonji C domain-containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z' > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 µL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent K(m) of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.

The ubiquitin proteasome pathway (UPP) has been implicated in a number of pathogenic diseases: cancer, inflammation, metabolic disorders, and viral infection. The human genome contains well over 500 genes encoding proteins involved in the UPP. Ubiquitin ligases (E3s) comprise the largest subset of these genes, and together with an E2 partner, provide the substrate selectivity required for regulating cellular proteins through the covalent attachment of ubiquitin. Many ligases that have been identified in critical cellular pathways have no known substrates. Even those E3s with known substrates may have a yet unidentified role in the pathways on which they lie and as such may have additional substrates. It is critical to identify these substrates for discovery of selective small molecule inhibitors aimed at therapeutic intervention. Other methods, such as mass spectrometry, have been utilized for identifying ligase substrates, but these are labor-intensive and require a significant investment. In this study, we utilized protein microarrays for the identification of substrates of the HECT domain E3, Smurf1. Smurf1 is a critical regulator of TGF-beta and bone morphogenic protein signaling, and has been demonstrated to play a role in regulating cell polarity through the degradation of RhoA. We set out to identify novel Smurf1 substrates involved in the regulation of the aforementioned pathways. Proof-of-principle experiments with known Smurf1 substrates demonstrated efficient ubiquitination thereby validating this approach. Assaying a human protein microarray for ubiquitination with Smurf1 and the partner E2 ubiquitin ligase Ubch5 or Ubch7 identified 89 potential substrates of the Smurf1 E3 activity, which spanned a number of different biological pathways. Substrates identified utilizing protein microarray technology have been validated in vitro. Here we demonstrate the utility of this approach for identifying substrates of particular E2/E3 complexes.

The sections in this article are: 1 The Insulin Receptor 2 Insulin Receptor Substrate 1 3 Role of Insulin Receptor Substrate 1 Sequence Polymorphisms in Humans and the Pathophysiology of Diabetes 4 Growing Family of Insulin Receptor Substrates 5 Complementary and Alternative Pathways in Insulin Signaling 6 A Polygenic Model of Non-Insulin-Dependent Diabetes Mellitus 7 Differential Subcellular Localization of Insulin Receptor Substrates 1 and 2 8 Insulin-Stimulated Insulin Receptor Substrate Interactions 8.1 Src Homology-2 Domain-Mediated Interactions 8.2 Non-Src Homology-2 Domain-Mediated Interactions 9 Serine/Threonine Protein Kinases and the Final Biological Effects of Insulin 10 Differential Regulation of Insulin Receptor Substrates 1 and 2 and Phosphatidylinositol-3-Kinase 11 Linking Early Steps in Insulin Action to Late Postreceptor Events 11.1 Insulin Stimulation of Glucose Transport 11.2 Coupling of Insulin Action to the Nucleus of the Cell 12 Cross-Talk Between the Insulin-Signaling Network and Other Hormonal Response Pathways 13 Future Perspectives

The ability of the insulin receptor to phosphorylate multiple substrates and their subcellular localization are two of the determinants that contribute to diversity of signaling. We find that insulin receptor substrate (IRS)-1 is 2-fold more concentrated in the intracellular membrane (IM) compartment than in cytosol, whereas IRS-2 is 2-fold more concentrated in cytosol than in IM. Insulin stimulation induces rapid tyrosine phosphorylation of both IRS-1 and IRS-2. This occurs mainly in the IM compartment, even though IRS-2 is located predominantly in cytosol. Furthermore, after insulin stimulation, both IRS-1 and IRS-2 translocate from IM to cytosol with at½ of 3.5 min. Using an in vitro reconstitution assay, we have demonstrated an association between IRS-1 and internal membranes and have shown that the dissociation of IRS-1 from IM is dependent on serine/threonine phosphorylation of IM. By comparison, within 1 min after insulin stimulation, 40% of the total pool of the 85-kDa subunit of phosphatidylinositol 3-kinase (p85) is recruited from cytosol to IM, the greater part of which can be accounted for by binding to IRS-1 present in the IM. The p85 binding and phosphatidylinositol 3-kinase activity associated with IRS-2 rapidly decrease in both IM and cytosol, whereas those associated with IRS-1 stay at a relatively high level in IM and increase with time in cytosol despite a return of p85 to the cytosol and decreasing tyrosine phosphorylation of cytosolic IRS-1. These data indicate that IRS-1 and IRS-2 are differentially distributed in the cell and move from IM to cytosol following insulin stimulation. Insulin-stimulated IRS-1 and IRS-2 signaling occurs mainly in the IM and shows different kinetics; IRS-1-mediated signaling is more stable, whereas IRS-2-mediated signaling is more transient. These differences in substrate utilization and compartmentalization may contribute to the complexity and diversity of the insulin signaling network.

In the accompanying paper (Emkey, R., and Kahn, C. R. (1997) J. Biol. Chem. 272, 31172–31181), we demonstrated that phorbol 12-myristate 13-acetate (PMA) treatment of Fao cells induces tyrosine phosphorylation of several proteins including ErbB2 and ErbB3. In the present study we show that sphingomyelinase also results in the enhanced tyrosine phosphorylation of ErbB2 and ErbB3 in these cells. In contrast to activation by PMA, the sphingomyelinase-induced phosphorylation of these proteins is independent of protein kinase C. However, both agents stimulate tyrosine phosphorylation of the kinase Pyk2 suggesting that it may be involved in the PMA and sphingomyelinase activation of these ErbB proto-oncogenes. Insulin plays a negative regulatory role in the ligand and non-ligand-induced phosphorylation of the ErbB proto-oncogenes via two mechanisms. Prolonged insulin treatment resulted in decreased expression of both ErbB2 and ErbB3. Insulin also appears to negatively regulate the protein tyrosine kinase responsible for phosphorylating ErbB2 in PMA-stimulated cells. The former effect of insulin was relieved by treatment with inhibitors of phosphatidylinositol 3-kinase. The similarities in PMA and sphingomyelinase-induced effects and the negative regulatory role of insulin suggest a mechanism by which multiple ligands can synergize with or protect against the tumorigenic effects of phorbol esters.

Small molecule inhibitors of Janus kinase (JAK) family members (JAK1, JAK2, JAK3, and Tyk2) are currently being pursued as potential new modes of therapy for a variety of diseases, including the inhibition of JAK2 for the treatment of myeloproliferative disorders. Selective inhibition within the JAK family can be beneficial in avoiding undesirable side effects (e.g., immunosuppression) caused by parallel inhibition of other JAK members. In an effort to design an assay paradigm for the development of JAK2 selective inhibitors, we investigated whether compound selectivity differed between cellular and purified enzyme environments. A set of JAK2 inhibitors was tested in a high-throughput JAK family cell assay suite and in corresponding purified enzyme assays. The high-throughput JAK cell assay suite comprises Ba/F3 cells individually expressing translocated ETS leukemia (TEL) fusions of each JAK family member (TEL-JAK Ba/F3) and an AlphaScreen phosphorylated-STAT5 (pSTAT5) immunoassay. Compound potencies from the TEL-JAK Ba/F3 pSTAT5 assays were similar to those determined in downstream cell proliferation measurements and more physiologically relevant cytokine-induced pSTAT5 PBMC assays. However, compound selectivity data between cell and purified enzyme assays were discrepant because of different potency shifts between cell and purified enzyme values for each JAK family member. For any JAK small molecule development program, our results suggest that relying solely on enzyme potency and selectivity data may be misleading. Adopting the high-throughput TEL-JAK Ba/F3 pSTAT5 cell assay suite in lead development paradigms should provide a more meaningful understanding of selectivity and facilitate the development of more selective JAK inhibitors.

Developing Janus kinase 2 (Jak2) inhibitors has become a significant focus for small molecule drug discovery programs in recent years due to the identification of a Jak2 gain-of-function mutation in the majority of patients with myeloproliferative disorders (MPD). Here, we describe the discovery of a thienopyridine series of Jak2 inhibitors that culminates with compounds showing 100- to >500-fold selectivity over the related Jak family kinases in enzyme assays. Selectivity for Jak2 was also observed in TEL-Jak cellular assays, as well as in cytokine-stimulated peripheral blood mononuclear cell (PBMC) and whole blood assays. X-ray cocrystal structures of 8 and 19 bound to the Jak2 kinase domain aided structure–activity relationship efforts and, along with a previously reported small molecule X-ray cocrystal structure of the Jak1 kinase domain, provided structural rationale for the observed high levels of Jak2 selectivity.

Protein kinases are recognized as important drug targets due to the pivotal roles they play in human disease. Many kinase inhibitors are ATP competitive, leading to potential problems with poor selectivity and significant loss of potency in vivo due to cellular ATP concentrations being much higher than K(m). Consequently, there has been growing interest in the development of ATP-noncompetitive inhibitors to overcome these problems. There are challenges to identifying ATP-noncompetitive inhibitors from compound library screens because ATP-noncompetitive inhibitors are often weaker and commonly excluded by potency-based hit selection criteria in favor of abundant and highly potent ATP-competitive inhibitors in screening libraries. Here we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for protein kinase cyclin-dependent kinase 4 (CDK4) and the identification of ATP-noncompetitive inhibitors by high-throughput screening after employing a strategy to favor this type of inhibitors. We also present kinetic characterization that is consistent with the proposed mode of inhibition.

The family of signal transducers and activators of transcription (STATs) consists of seven transcription factors that respond to a variety of cytokines, hormones, and growth factors. STATs are activated by tyrosine phosphorylation, which results in their dimerization and translocation into the nucleus where they exert their effect on transcription of regulated target genes. The phosphorylation of STATs is mediated mainly by Janus kinases (JAKs). The JAK/STAT pathway plays a critical role in hematopoietic and immune cell function. Here we focus on one member of the STAT family, STAT5. STAT5 is phosphorylated by several JAKs, including Jak3, Jak2, and Tyk2, in response to interleukin-2, erythropoietin (EPO), and interleukin-22, respectively. Activation of STAT5 is essential to T cell development and has been associated with hematologic malignancies. Therefore, the ability to assess STAT5 phosphorylation is important for discovery efforts targeting these indications. The assay formats available to detect phosphorylated STAT5 (pSTAT5) are relatively low throughput and involve lengthy protocols. These formats include western blot analysis, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. The SureFire (Perkin Elmer, Waltham, MA) pSTAT5 assay is a homogeneous assay that utilizes AlphaScreen (Perkin Elmer) technology to detect pSTAT5 in cell lysates. We have used this assay format to evaluate EPO-induced STAT5 phosphorylation in HEL cells and successfully complete a small-scale screening campaign to identify inhibitors of this event. The results obtained in these studies demonstrate that the SureFire pSTAT5 assay is a robust, reliable assay format that is amenable to high-throughput screening (HTS) applications.

Abstract 5150 Essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) are myeloproliferative disorders (MPDs) characterized by a chronic over-production of cells of one or more blood cell lineages and/or bone marrow fibrosis which may, on occasion, progress to acute myeloid leukemia. The V617F gain of function mutation in the pseudokinase domain of Jak2, which results in constitutive activation of Jak2, is the most frequent mutation associated with MPD. Constitutively activated Jak2 can lead to dysregulated downstream signaling pathways (STAT, MAP kinase, and PI3 kinase) which in turn trigger abnormal growth, survival and differentiation of hematopoietic progenitors. Therefore, inhibition of constitutively activated Jak2 may offer therapeutic potential. Designing a Jak2V617F specific inhibitor encounters challenges due to the lack of enzymatic activity of the pseudokinase domain of Jak2. In lieu of a Jak2V617F mutant selective inhibitor, a highly selective inhibitor of Jak2 is likely an attainable goal. Jak2 is a member of the Jak family of kinases including Jak1, Jak3, and Tyk2. Highly selective Jak2 inhibitors may provide a better safety margin in chronic dosing settings in ET and PV patients since inhibiting other Jak family members could cause side-effects such as immunosuppression. Attaining the desired selectivity of Jak2 inhibition versus the other family members has been challenging and few compounds have been reported to date that have the desired Jak2 selectivity. This can be attributed to the high homology of the ATP binding pocket among Jak family members, but is also hampered by a lack of assays capable of distinguishing the Jak-selectivity profile in a physiologically relevant setting. We compared the potency and selectivity of compounds tested in a pSTAT5 AlphaScreen® assay panel consisting of isogenic Ba/F3 cell lines individually expressing translocated ETS leukemia (TEL) fusions of each Jak-family member (Ba/F3-TEL-Jak) with data from corresponding Jak enzyme assays. Here we report that the selectivity of inhibitor compounds illustrated in enzyme assays did not correlate with the selectivity profile in cell lines due to different shifts in potency for each family member between enzyme and cells (Figure 1). As a consequence the selectivity of compounds for Jak2 against Jak1 observed in enzyme assays may be reduced or reversed in cellular assays. On the other hand, Jak2 selectivity over Jak3 seen in the enzyme assays was conserved in the cellular assay. Thus, we propose that compounds that exhibit greater potency on Jak2 compared to Jak1 in the enzyme assays are needed and should be the main focus of medicinal chemistry efforts in order to attain Jak2 selectivity over Jak1 in a cellular context. We also compared the potency and selectivity of compounds in the isogenic Ba/F3-TEL-Jak cell lines with data obtained with cytokine stimulated peripheral blood mononuclear cells (PBMCs). The potency and selectivity of compounds in PBMCs are determined by measuring the inhibition of phosphorylation of STAT5 in TPO or GM-CSF stimulated platelets or monocytes (mediated by Jak2) and in IL-2 stimulated lymphocytes (mediated by Jak1 and Jak3). We found that potency correlated well between PBMCs and Ba/F3-TEL-Jak2 cells, and the rank order of compounds based on IC50 values obtained with Ba/F3-TEL-Jak cell lines were conserved well in PBMCs; the compound selectivity profiles derived from the Ba/F3-TEL-Jak cell assays were predictive of Jak2 selectivity profiles obtained in the PBMC assays. Therefore, inclusion of Ba/F3-TEL-Jak pSTAT5 cellular assays may be useful for Jak family inhibitor development. Our results also suggest that relying solely on enzyme potency and selectivity data can be misleading, and that evaluating cellular selectivity in a biologically relevant context may provide a more meaningful understanding of selectivity and lead to the development of more selective Jak2 compounds. Disclosures: Liu: Amgen, Inc: Employment. Yu:Amgen: Employment. Pistillo:Amgen: Employment. Lee:Amgen: Employment. Schenkel:Amgen: Employment. Geuns-Meyer:Amgen: Employment. Archibeque:Amgen: Employment. Sinclair:Amgen: Employment. Emkey:Amgen: Employment. Molineux:Amgen: Employment.

Essential thrombocythemia (ET), polythemia vera (PV) and myelofibrosis (MF) are myeloproliferative disorders (MPDs) characterized by a chronic excessive production of cells from one or more myeloid lineages and/or bone marrow fibrosis, and have the potential to progress to AML. Recently, gain-of-function mutations in intracellular tyrosine kinase Janus kinase 2 (Jak2) were identified to be associated with MPDs. Jak2 is a member of the Jak family of kinases including Jak1, Jak3, and Tyk2 and is the most proximal signaling component for a number of cytokine receptors. The most common Jak2 mutation identified in MPDs is a substitution of valine to phenylalanine at codon 617 (V617F) located in the pseudokinase domain, which results in the loss of its repressive function, and subsequently leads to the constitutive activation of Jak2 and downstream signaling pathways (STAT, MAP kinase, and PI3 kinase) which affect the survival, differentiation and proliferation of hematopoietic progenitors. Highly selective Jak2 inhibitors may provide a better therapeutic window in chronic dosing settings (ET and PV patients) than non-selective Jak inhibitors. Here we report the discovery of a potent and highly selective Jak2 inhibitor, AMG-Jak2-01. In enzyme assays, AMG-Jak2-01 was potent (3 nM) and selective over other Jak family kinases (> 2000-fold over Jak1, 10-fold over Jak3 and > 400-fold over Tyk2). In isogenic BaF3 cell lines expressing constitutively active Jak kinases (Tel-Jak fusion), AMG-Jak2-01 showed over 40-fold Jak2 selectivity against Jak1 or Tyk2 and 10-fold selectivity against Jak3 based on the inhibition of STAT5 phosphorylation (pSTAT5 – detected with AlphaScreen®). In cytokine-stimulated primary PBMC or whole blood assays, AMG-Jak2-01 demonstrated more than 25-fold Jak2 selectivity over Jak1and Jak3 based on inhibition of pSTAT5 (detected by flow cytometry). In vivo, oral administration of AMG-Jak2-01 at 100 mg/kg for 1 hr inhibited GM-CSF-induced pSTAT5 by approximately 70% (p In summary, we have discovered a potent Jak2 inhibitor, AMG-Jak2-01, which is selective over other Jak family members in both enzyme and cell-based assays and demonstrates inhibitory activity in GM-CSF-induced STAT5 phosphorylation and erythropoietin-induced reticulocytosis in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1975. doi:10.1158/1538-7445.AM2011-1975

The Ubiquitin Proteasome Pathway (UPP) has become a target rich pathway for therapeutic intervention as its role in pathogenic disease is better understood. In particular many E3 ligases within this pathway have been implicated in cancer, inflammation and metabolic diseases. It has been of great interest to develop biochemical assays to identify inhibitors of catalytic E3 ubiquitination activity as a means of abrogating the disease mechanism. Here we describe a homogeneous biochemical assay that utilizes native ubiquitin and Tandem-repeated Ubiquitin-Binding Entities (TUBEs) as a detection technology for ubiquitination activity. We developed a TUBEs based proximity AlphaLisa® assay for Mdm2, which is an E3 ligase that negatively regulates p53 tumor suppressor protein. We further demonstrate that this assay strategy or design can also be applied to the development of assays to detect autoubiquitination of other E3 ligases that are also of interest for therapeutic intervention. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.