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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.

Pyridylmethylsulfonamide series were the first reported example of positive allosteric modulators (PAM) of the mGlu2 receptor. The hydroxyacetophenone scaffold is a second series of mGlu2 PAMs we have identified. This series of molecules are potent mGlu2 potentiators and possess significant CysLT1 (cysteinyl leukotriene receptor 1) antagonist activity, showing in vivo efficacy in a dural plasma protein extravasation (PPE) model of migraine. In this paper, we describe the dual SAR, pharmacokinetics and preclinical in vivo efficacy data for a tetrazole containing hydroxyacetophenone scaffold.

Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.

There has been significant interest in developing a transient receptor potential A1 (TRPA1) antagonist for the treatment of pain due to a wealth of data implicating its role in pain pathways. Despite this, identification of a potent small molecule tool possessing pharmacokinetic properties allowing for robust in vivo target coverage has been challenging. Here we describe the optimization of a potent, selective series of quinazolinone-based TRPA1 antagonists. High-throughput screening identified 4, which possessed promising potency and selectivity. A strategy focused on optimizing potency while increasing polarity in order to improve intrinsic clearance culminated with the discovery of purinone 27 (AM-0902), which is a potent, selective antagonist of TRPA1 with pharmacokinetic properties allowing for30-fold coverage of the rat TRPA1 IC50 in vivo. Compound 27 demonstrated dose-dependent inhibition of AITC-induced flinching in rats, validating its utility as a tool for interrogating the role of TRPA1 in in vivo pain models.

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.

A series of α-amidosulfones were found to be potent and selective agonists of CB2. The discovery, synthesis, and structure–activity relationships of this series of agonists are reported. In addition, the pharmacokinetic properties of the most promising compounds are profiled.

The CB2 receptor is an attractive therapeutic target for analgesic and anti-inflammatory agents. Herein we describe the discovery of a novel class of oxadiazole derivatives from which potent and selective CB2 agonist leads were developed. Initial hit 7 was identified from a cannabinoid target-biased library generated by virtual screening of sample collections using a pharmacophore model in combination with a series of physicochemical filters. 7 was demonstrated to be a selective CB2 agonist (CB2 EC50 = 93 nM, Emax = 98%, CB1 EC5010 microM). However, this compound exhibited poor solubility and relatively high clearance in rat, resulting in low oral bioavailability. In this paper, we report detailed SAR studies on 7 en route toward improving potency, physicochemical properties, and solubility. This effort resulted in identification of 63 that is a potent and selective agonist at CB2 (EC50 = 2 nM, Emax = 110%) with excellent pharmacokinetic properties.

Here we report the discovery, by high-throughput screening, of three novel (2-amino-5-keto)thiazole compounds that act as selective potentiators of nicotinic acetylcholine receptors. Compound selectivity was assessed at seven human nicotinic acetylcholine receptors (alpha1beta1gammadelta, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7) expressed in mammalian cells or Xenopus oocytes. At alpha2beta4, alpha4beta2, alpha4beta4, and alpha7, but not alpha1beta1gammadelta, alpha3beta2, or alpha3beta4, submaximal responses to nicotinic agonists were potentiated in a concentration-dependent manner by all compounds. At similar concentrations, no potentiation of 5-hydroxytryptamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, GABA(A), and N-methyl-d-aspartate receptors or voltage-gated Na(+) and Ca(2+) channels was observed. Furthermore, these compounds did not inhibit acetylcholine esterase. Further profiling revealed that these compounds enhanced the potency and maximal efficacy of a range of nicotinic agonists at alpha4beta2 nicotinic acetylcholine receptors, a profile typical of allosteric potentiators. At concentrations required for potentiation, the compounds did not displace [(3)H]epibatidine from the agonist-binding site, and potentiation was observed at all agonist concentrations, suggesting a noncompetitive mechanism of action. Blockade of common second messenger systems did not affect potentiation. At concentrations higher then required for potentiation the compounds also displayed intrinsic agonist activity, which was blocked by competitive and noncompetitive nicotinic acetylcholine receptor (nAChR) antagonists. These novel selective nicotinic receptor potentiators should help in clarifying the potential therapeutic utility of selective nAChR modulation for the treatment of central nervous system disorders.

The neurotransmitter serotonin (5HT) has been shown to modulate mobility, feeding, egg-laying, and defecation behaviors in the saprophytic nematode Caenorhabditis elegans. Although the effects of serotonin on these behaviors in parasitic nematodes is under study, little is known about the diversity, ontogeny, signaling, and pharmacology of serotonin receptors in these organisms. In an effort to increase our understanding of this system, we cloned and characterized a novel cDNA (5HT1Hc) from the parasitic nematode Haemonchus contortus that has high amino acid sequence homology with known G-protein coupled 5HT1-receptors from invertebrates and vertebrates. Transcript expression studies in four development stages (egg, L1/L2, L3, and adult) revealed the presence of the mRNA in the L1/L2, L3, and adult stages. Membranes from insect cells (Sf9) expressing the 5HT1Hc-receptor cDNA displayed nanomolar binding affinity to serotonin and a unique pharmacological profile distinct from known invertebrate and mammalian 5HT-receptors. Receptor signaling studies with mammalian AV12 cells expressing the 5HT1Hc-receptor and the promiscuous G-protein, Gα15, demonstrated dose-dependent intracellular signals with serotonin acting as an agonist. Together, these studies describe a novel invertebrate 5HT-receptor with high affinity for the indolealkylamine, serotonin, and pharmacological properties that do not conform to any known members of this superfamily of metabotropic receptors.

Osteoprotegerin (OPG), a secreted member of the tumor necrosis factor receptor superfamily, is a potent inhibitor of osteoclast formation and bone resorption. Because OPG functions physiologically as a locally generated (paracrine) factor, we used high-throughput screening to identify small molecules that enhance the activity of the promoter of the human OPG gene. We found three structurally unrelated compounds that selectively increased OPG gene transcription, OPG mRNA levels, and OPG protein production and release by osteoblastic cells. Structural analysis of one compound, a benzamide derivative, led to the identification of four related molecules, which are also OPG inducers. The most potent of these compounds, Cmpd 5 inhibited osteoclast formation and parathyroid hormone-induced calvarial bone resorption. In vivo, Cmpd 5 completely blocked resorptive activity (serum calcium, osteoclast number) in parathyroid hormone-treated rats. Furthermore, Cmpd 5 reduced the ability of a rat breast cancer to metastasize to bone. Finally, the compound also prevented bone loss in a rat adjuvant arthritis model. These results provide proof of the concept that low molecular weight compounds can enhance OPG production in ways that can result in effective therapies.

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.

A class of 2-acyliminobenzimidazoles has been developed as potent and selective inhibitors of anaplastic lymphoma kinase (ALK). Structure based design facilitated the rapid development of structure–activity relationships (SAR) and the optimization of kinase selectivity. Introduction of an optimally placed polar substituent was key to solving issues of metabolic stability and led to the development of potent, selective, orally bioavailable ALK inhibitors. Compound 49 achieved substantial tumor regression in an NPM-ALK driven murine tumor xenograft model when dosed qd. Compounds 36 and 49 show favorable potency and PK characteristics in preclinical species indicative of suitability for further development.

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.

Piperidine carboxamide 1 was identified as a novel inhibitor of anaplastic lymphoma kinase (ALK enzyme assay IC(50) = 0.174 μM) during high throughput screening, with selectivity over the related kinase insulin-like growth factor-1 (IGF1R). The X-ray cocrystal structure of 1 with the ALK kinase domain revealed an unusual DFG-shifted conformation, allowing access to an extended hydrophobic pocket. Structure-activity relationship (SAR) studies were focused on the rapid parallel optimization of both the right- and left-hand side of the molecule, culminating in molecules with improved potency and selectivity over IGF1R.

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.

Anaplastic lymphoma kinase (ALK) when expressed as a fusion protein with nucleophosmin (NPM) has been implicated as a driving oncogene in a subset of lymphomas. Recent reports of ALK expression in a number of other cancers have raised the possibility that an ALK inhibitor may benefit patients with these diseases as well. In a campaign to identify and develop a selective ALK inhibitor, 2 assays were devised to measure the phosphorylation of tyrosine residue 1604 of ALK (pY(1604) ALK). Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen(®)) and phosflow platforms were used to detect modulation of pY(1604) ALK to determine the relative potency of a set of small-molecule inhibitors. Prior to making use of these assays in diverse settings, the authors attempted to ensure their equivalence with a direct comparison of their performance. The pY(1604) ALK assays correlated well both with each other and with assays of ALK enzyme activity or ALK-dependent cell proliferation. The AlphaScreen(®) assay was amenable to automation and enabled rapid, high-throughput compound assessment in an NPM-ALK-driven cell line, whereas the phosflow assay enabled the authors to characterize the activity of compounds with respect to their impact on targeted enzymes and pathways. Results show that both AlphaScreen(®) and phosflow ALK assays exhibited diverse characteristics that made them desirable for different applications but were determined to be equally sensitive and robust in the detection of inhibition of pY(1604) ALK.

G protein-coupled receptors (GPCRs) are the target of approximately 40% of all approved drugs and continue to represent a significant portion of drug discovery portfolios across the pharmaceutical industry. As a result, GPCRs are the focus of many high-throughput screening (HTS) campaigns. Historically, ligand-binding assays were used to identify compounds that targeted GPCRs. Current GPCR drug discovery efforts have moved toward the utilization of functional cell-based assays for HTS. Many of these assays monitor the accumulation of a second messenger such as cAMP or calcium in response to GPCR activation. Calcium stores are released from the endoplasmic reticulum when Galphaq-coupled GPCRs are activated. Although Galphai- and Galphas-coupled receptors do not normally result in this mobilization of intracellular calcium, they can often be engineered to do so by expressing a promiscuous or a chimeric Galphaprotein, which couples to the calcium pathway. Thus calcium mobilization is a readout that can theoretically be used to assess activation of all GPCRs. The fluorometric imaging plate reader (FLIPR) has facilitated the ability to monitor calcium mobilization in the HTS setting. This assay format allows one to monitor activation and inhibition of a GPCR in a single assay and has been one of the most heavily utilized formats for screening GPCRs.

G protein-coupled receptors (GPCRs) are the target of approximately 40% of all approved drugs and continue to represent a significant portion of drug discovery portfolios across the pharmaceutical industry. As a result, GPCRs are the focus of many high-throughput screening (HTS) campaigns. Historically, ligand-binding assays were used to identify compounds that targeted GPCRs. Current GPCR drug discovery efforts have moved toward the utilization of functional cell-based assays for HTS. Many of these assays monitor the accumulation of a second messenger such as cAMP or calcium in response to GPCR activation. Calcium stores are released from the endoplasmic reticulum when Galphaq-coupled GPCRs are activated. Although Galphai- and Galphas-coupled receptors do not normally result in this mobilization of intracellular calcium, they can often be engineered to do so by expressing a promiscuous or a chimeric Galphaprotein, which couples to the calcium pathway. Thus calcium mobilization is a readout that can theoretically be used to assess activation of all GPCRs. The fluorometric imaging plate reader (FLIPR) has facilitated the ability to monitor calcium mobilization in the HTS setting. This assay format allows one to monitor activation and inhibition of a GPCR in a single assay and has been one of the most heavily utilized formats for screening GPCRs.

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.

A G-protein coupled receptor to niacin (nicotinic acid) was identified recently but the physiological/pharmacological role of the receptor remains poorly defined. We present our studies to demonstrate that HM74A, but not HM74, binds niacin at high affinities and effectively mediates Gi signaling events in human embryonic kidney HEK293 cells as well as in 3T3L1 adipocytes expressing HM74A. Furthermore, HM74A, but not HM74, expressed in differentiated 3T3L1 adipocytes effectively mediated inhibition of lipolysis by niacin. Our results provided direct evidence indicating that HM74A, but not HM74, was sufficient to mediate anti-lipolytic effect of niacin in adipose tissue.

To increase subtype selectivity and provide a novel means to alter receptor function, we discovered and characterization potentiators for the metabotropic glutamate 2 receptor (mGlu2). A class of 3-pyridylmethylsulfonamides (e.g., 3-MPPTS; 2,2,2-trifluoro-N-[3-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide) were found to be potent, subtype-selective potentiators of human and rat mGlu2. The sulfonamides increased agonist potency in functional assays but did not displace orthosteric radiolabeled antagonist or agonist binding to cloned mGlu2 receptors. Rather, the modulators increased the affinity of most of the orthosteric agonists including glutamate, DCG-IV (2S,2′R,3′R)-2-(2′,3′-dicarboxylcyclopropyl)glycine), and LY354740 (1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-bicaroxylate monohydrate). In striatal brain slices, LY354740 inhibited evoked excitatory postsynaptic potentials (EPSPs) equally well following either a low- (0.06 Hz) or high (4 Hz)-frequency stimulation of corticostriatal afferents. In contrast, the mGlu2 potentiator cyPPTS (2,2,2-trifluoro-N-[3-(cyclopentyloxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide) inhibited striatal EPSPs only at higher frequencies of stimulation (2 and 4 Hz). Several sulfonamides including 4-MPPTS, 4-APPES (N-[4-(4-carboxamidophenoxy)phenyl]-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride monohydrate) and/or CBiPES N-[4′-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride) were tested in mGlu2/3 agonist-sensitive rodent model(s) of anxiety and psychosis. As seen with LY354740, both 4-MPPTS and 4-APPES were efficacious in a rat fear-potentiated startle paradigm. Likewise in mice, CBiPES attenuated a stress-induced hyperthermia and PCP-induced hyperlocomotor activity. Furthermore, CBiPES mediated alteration in PCP-induced hyperlocomotor activity was sensitive to mGlu2/3 antagonist pretreatment. Taken together, the data indicate mGlu2 receptor potentiators have a unique use-dependent effect on presynaptic glutamate release, and show efficacy in several mGlu2/3-sensitive animal models of psychiatric disorders.

The neurotransmitter serotonin (5HT) has been shown to modulate mobility, feeding, egg-laying, and defecation behaviors in the saprophytic nematode Caenorhabditis elegans. Although the effects of serotonin on these behaviors in parasitic nematodes is under study, little is known about the diversity, ontogeny, signaling, and pharmacology of serotonin receptors in these organisms. In an effort to increase our understanding of this system, we cloned and characterized a novel cDNA (5HT1Hc) from the parasitic nematode Haemonchus contortus that has high amino acid sequence homology with known G-protein coupled 5HT1-receptors from invertebrates and vertebrates. Transcript expression studies in four development stages (egg, L1/L2, L3, and adult) revealed the presence of the mRNA in the L1/L2, L3, and adult stages. Membranes from insect cells (Sf9) expressing the 5HT1Hc-receptor cDNA displayed nanomolar binding affinity to serotonin and a unique pharmacological profile distinct from known invertebrate and mammalian 5HT-receptors. Receptor signaling studies with mammalian AV12 cells expressing the 5HT1Hc-receptor and the promiscuous G-protein, Galpha15, demonstrated dose-dependent intracellular signals with serotonin acting as an agonist. Together, these studies describe a novel invertebrate 5HT-receptor with high affinity for the indolealkylamine, serotonin, and pharmacological properties that do not conform to any known members of this superfamily of metabotropic receptors.

To investigate the roles of insulin receptor substrate 3 (IRS-3) and IRS-4 in the insulin-like growth factor 1 (IGF-1) signaling cascade, we introduced these proteins into 3T3 embryonic fibroblast cell lines prepared from wild-type (WT) and IRS-1 knockout (KO) mice by using a retroviral system. Following transduction of IRS-3 or IRS-4, the cells showed a significant decrease in IRS-2 mRNA and protein levels without any change in the IRS-1 protein level. In these cell lines, IGF-1 caused the rapid tyrosine phosphorylation of all four IRS proteins. However, IRS-3- or IRS-4-expressing cells also showed a marked decrease in IRS-1 and IRS-2 phosphorylation compared to the host cells. This decrease was accounted for in part by a decrease in the level of IRS-2 protein but occurred with no significant change in the IRS-1 protein level. IRS-3- or IRS-4-overexpressing cells showed an increase in basal phosphatidylinositol 3-kinase activity and basal Akt phosphorylation, while the IGF-1-stimulated levels correlated well with total tyrosine phosphorylation level of all IRS proteins in each cell line. IRS-3 expression in WT cells also caused an increase in IGF-1-induced mitogen-activated protein kinase phosphorylation and egr-1 expression ( approximately 1.8- and approximately 2.4-fold with respect to WT). In the IRS-1 KO cells, the impaired mitogenic response to IGF-1 was reconstituted with IRS-1 to supranormal levels and was returned to almost normal by IRS-2 or IRS-3 but was not improved by overexpression of IRS-4. These data suggest that IRS-3 and IRS-4 may act as negative regulators of the IGF-1 signaling pathway by suppressing the function of other IRS proteins at several steps.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that has been implicated as a driving oncogene in a number of cancers, including non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma and inflammatory myofibroblastic tumors (IMT). Numerous genetic aberrations at the ALK locus are observed in cancer including point mutations, amplifications, translocations and inversions. Inversions are exemplified by inv(2)(p21;p23), which leads to the constitutively active oncogenic fusion protein EML4-ALK present in ∼5% of NSCLC. Crizotinib, a dual cMet/ALK kinase inhibitor, was recently approved by the FDA for locally advanced or metastatic NSCLC that is ALK-positive, thereby validating ALK as therapeutic target. Here we describe the pharmacological characterization of a novel series of potent, selective and orally bioavailable ALK kinase inhibitors. Members of this series inhibit wild type ALK, NPM-ALK fusion and crizotinib resistant ALK[L1196M] kinase activity at sub-nanomolar concentrations, displaying up to ∼200 fold increased inhibitory activity over crizotinib. Kinase profiling indicate that members of this series display increased selectivity scores relative crizotinib. In Karpas-299 cells, selected compounds inhibited both pY1604 ALK activation (IC50 = 2 nM) and cell proliferation (IC50 = 1 nM). Members of this series were also evaluated in the EML4-ALK expressing NSCLC cell line H3122, and displayed equipotent inhibition of pY1604 ALK activation and inhibition of cell proliferation (both IC50 = 1 nM). Members of this class did not inhibit growth of an ALK negative lymphoma cell line (HT). The in vivo activity of this series was examined in the Karpas-299 ALCL xenograft model. Compound was dosed daily (PO) at 10, 30 and 60 mg/kg. Tumor growth inhibition was observed at all dose levels, and the highest dose level resulted in significant tumor regression (96%, p80% pALK inhibition was observed at the lowest doses tested, and complete inhibition was seen at doses of 30 mg/kg and above. In a direct comparison, members of this series achieved ALK inhibition in these tumors at a ∼15 fold lower plasma concentration than crizotinib. A PK/PD time course study was performed in the Karpas-299 model. A single 60 mg/kg dose of an inhibitor was able to maintain >90% ALK inhibition in tumors up to 24 hours post-dose, indicating significant tumor penetration and sustained ALK kinase inhibition. In conclusion, the described compounds are potent and selective inhibitors of ALK kinase, possess an impressive efficacy profile and drug-like pharmacokinetic properties. These features together indicate the potential for significant advantages over crizotinib. There is a compelling case for their clinical evaluation in patients with ALK-driven cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1795. doi:1538-7445.AM2012-1795

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

Corrigendum to ‘‘Structural modifications of N-arylamide oxadiazoles: Identification of N-arylpiperidine oxadiazoles as potent and selective agonists of CB2” [Bioorg. Med. Chem. Lett. 18 (2008) 4267–4274] Erin F. DiMauro *, John L. Buchanan , Alan Chen , Renee Emkey , Stephen A. Hitchcock , Liyue Huang , Ming Y. Huang , Brett Janosky , Josie H. Lee , Xingwen Li , Matthew W. Martin , Susan A. Tomlinson , Ryan D. White , Xiao Mei Zheng , Vinod F. Patel , Robert T. Fremeau Jr. e

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.