Discovery and characterization of non-ATP site inhibitors of the mitogen activated protein (MAP) kinases.

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38α (involved in the formation of TNFα and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38α both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.

Discovery of a new class of 4-anilinopyrimidines as potent c-Jun N-terminal kinase inhibitors: Synthesis and SAR studies.

A new series of 4-anilinopyrimidines has been synthesized and evaluated as JNK1 inhibitors. SAR studies led to the discovery of potent JNK1 inhibitors with good enzymatic activity as well as cellular potency represented by compound 2b. Kinase selectivity profile and the crystal structure of 2b are also described.

Developmental switch from prolonged insulin action to increased insulin sensitivity in protein tyrosine phosphatase 1B-deficient hepatocytes.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. The purpose of this study was to evaluate the differences in insulin sensitivity between neonate and adult hepatocytes lacking PTP1B. Immortalized neonatal hepatocytes and primary neonatal and adult hepatocytes have been generated from PTP1B(-/-) and wild-type mice. PTP1B deficiency in immortalized neonatal hepatocytes prolonged insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and IR substrates (IRS) -1, -2 compared with wild-type control cells. Endogenous IR and IRS-2 were down-regulated, whereas IRS-1 was up-regulated in PTP1B(-/-) neonatal hepatocytes and livers of PTP1B(-/-) neonates. Insulin-induced activation of phosphatidylinositol 3-kinase/Akt pathway was prolonged in PTP1B(-/-) immortalized neonatal hepatocytes. However, insulin sensitivity was comparable to wild-type hepatocytes. Rescue of PTP1B in deficient cells suppressed the prolonged insulin signaling, whereas RNA interference in wild-type cells promoted prolonged signaling. In primary neonatal PTP1B(-/-) hepatocytes, insulin prolonged the inhibition of gluconeogenic mRNAs, but the sensitivity to this inhibition was similar to wild-type cells. By contrast, in adult PTP1B-deficient livers, p85alpha was down-regulated compared with the wild type. Moreover, primary hepatocytes from adult PTP1B(-/-) mice displayed enhanced Akt phosphorylation and a more pronounced inhibition of gluconeogenic mRNAs than wild-type cells. Hepatic insulin sensitivity due to PTP1B deficiency is acquired through postnatal development. Thus, changes in IR and IRS-2 expression and in the balance between regulatory and catalytic subunits of phosphatidylinositol 3-kinase are necessary to achieve insulin sensitization in adult PTP1B(-/-) hepatocytes.

Aminopyridine carboxamides as c-Jun N-terminal kinase inhibitors: targeting the gatekeeper residue and beyond.

The structure-activity relationships of 5,6-positions of aminopyridine carboxamide-based c-Jun N-terminal Kinase (JNK) inhibitors were explored to expand interaction with the kinase specificity and ribose-binding pockets. The syntheses of analogues and the impact of structural modification on in vitro potency and cellular activity are described.

Discovery of potent, highly selective, and orally bioavailable pyridine carboxamide c-Jun NH2-terminal kinase inhibitors.

C-Jun NH2 terminal kinases (JNKs) are important cell signaling enzymes. JNK1 plays a central role in linking obesity and insulin resistance. JNK2 and JNK3 may be involved in inflammatory and neurological disorders, respectively. Small-molecule JNK inhibitors could be valuable tools to study the therapeutic benefits of inhibiting these enzymes and as leads for potential drugs targeting JNKs. In this report, we disclose a series of potent and highly selective JNK inhibitors with good pharmacokinetic profiles.

Aminopyridine-based c-Jun N-terminal kinase inhibitors with cellular activity and minimal cross-kinase activity.

The c-Jun N-terminal kinases (JNK-1, -2, and -3) are members of the mitogen activated protein (MAP) kinase family of enzymes. They are activated in response to certain cytokines, as well as by cellular stresses including chemotoxins, peroxides, and irradiation. They have been implicated in the pathology of a variety of different diseases with an inflammatory component including asthma, stroke, Alzheimer's disease, and type 2 diabetes mellitus. In this work, high-throughput screening identified a JNK inhibitor with an excellent kinase selectivity profile. Using X-ray crystallography and biochemical screening to guide our lead optimization, we prepared compounds with inhibitory potencies in the low-double-digit nanomolar range, activity in whole cells, and pharmacokinetics suitable for in vivo use. The new compounds were over 1,000-fold selective for JNK-1 and -2 over other MAP kinases including ERK2, p38alpha, and p38delta and showed little inhibitory activity against a panel of 74 kinases.

PKCtheta is a key player in the development of insulin resistance.

Activation of PKCtheta is associated with lipid-induced insulin resistance and PKCtheta knockout mice are protected from the lipid-induced defects. However, the exact mechanism by which PKCtheta contributes to insulin resistance is not known. To investigate whether an increase in PKCtheta expression leads to insulin resistance, C2C12 skeletal muscle cells were transfected with PKCtheta DNA and treated with different concentrations of insulin for 10 min. PKCtheta overexpression induced reduction of IRS-1 protein levels with a decrease in insulin-induced p85 binding to IRS-1, phosphorylation of PKB and its substrates, p70 and GSK3. Pretreatment of these cells with GF-109203X (a non-specific PKC inhibitor, IC50 for PKCtheta = 10 nM) recovered insulin signaling. PKCtheta was found to be expressed in liver and treatment of human hepatoma cells (HepG2) with high insulin and glucose resulted in an increase in PKCtheta expression that correlated with a decrease in IRS-1 protein levels and the development of insulin resistance. Reduction of PKCtheta expression using RNAi technology significantly inhibited the degradation of IRS-1 and enhanced insulin-induced IRS-1 tyrosine phosphorylation, p85 association to IRS-1 and PKB phosphorylation. In conclusion, by overexpressing PKCtheta or using RNAi technology to downregulate PKCtheta, we have demonstrated that PKCtheta has a key role in the development of insulin resistance. These findings suggest that PKCtheta mediates not only insulin resistance in muscle but also in liver, which may contribute to the development of whole body insulin resistance and diabetes.

Synthesis and SAR of 1,9-dihydro-9-hydroxypyrazolo[3,4-b]quinolin-4-ones as novel, selective c-Jun N-terminal kinase inhibitors.

A novel class of 1,9-dihydro-9-hydroxypyrazolo[3,4-b]quinolin-4-ones as c-Jun-N-terminal kinase (JNK) inhibitors is described. These compounds were synthesized via the condensation of 2-nitrobenzaldehydes and hydroxypyrazoles. The structure-activity relationships (SAR) and kinase selectivity profile of the inhibitors are also discussed. Compound 16 was identified as a potent JNK inhibitor with good cellular potency.

Reduction of PTP1B induces differential expression of PI3-kinase (p85alpha) isoforms.

Protein tyrosine phosphatase 1B (PTP1B) inhibition increases insulin sensitivity and normalizes blood glucose levels in animals. The molecular events associated with PTP1B inhibition that increase insulin sensitivity remain controversial. Insulin resistant, diabetic ob/ob mice, dosed with PTP1B antisense for 3 weeks exhibited a decrease in PTP1B protein levels and a change in the expression level of p85alpha isoforms in liver, characterized by a reduction in p85alpha and an upregulation of the p50alpha and p55alpha isoforms. Transfection of mouse hepatocytes with PTP1B antisense caused a downregulation PTP1B and p85alpha protein levels. Furthermore, transfection of mouse hepatocytes with PTP1B siRNA downregulated p85alpha protein expression and enhanced insulin-induced PKB phosphorylation. Treatment of mouse hepatocytes with p85alpha antisense oligonucleotide caused a reduction of p85alpha and an increase in p50alpha and p55alpha isoforms and enhanced insulin-stimulated PKB activation. These results demonstrate that PTP1B inhibition causes a direct differential regulation of p85alpha isoforms of PI3-kinase in liver and that reduction of p85alpha may be one mechanism by which PTP1B inhibition improves insulin sensitivity and glucose metabolism in insulin-resistant states.

PTP1B antisense-treated mice show regulation of genes involved in lipogenesis in liver and fat.

Protein tyrosine phosphatases are important regulators of insulin signal transduction. Our studies have shown that in insulin resistant and diabetic ob/ob and db/db mice, reducing the levels of protein tyrosine phosphatase 1B (PTP1B) protein by treatment with a PTP1B antisense oligonucleotide resulted in improved insulin sensitivity and normalized plasma glucose levels. The mechanism by which PTP1B inhibition improves insulin sensitivity is not fully understood. We have used microarray analysis to compare gene expression changes in adipose tissue, liver and muscle of PTP1B antisense-treated ob/ob mice. Our results show that treatment with PTP1B antisense resulted in the downregulation of genes involved in lipogenesis in both fat and liver, and a downregulation of genes involved in adipocyte differentiation in fat, suggesting that PTP1B antisense acts through a different mechanism than thiazolidinedione (TZD) treatment. In summary, microarray results suggest that reduction of PTP1B may alleviate hyperglycemia and enhance insulin sensitivity by a different mechanism than TZD treatment.

Reduction of protein tyrosine phosphatase 1B increases insulin-dependent signaling in ob/ob mice.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin receptor (IR) signal transduction and a drug target for treatment of type 2 diabetes. Using PTP1B antisense oligonucleotides (ASOs), effects of decreased PTP1B levels on insulin signaling in diabetic ob/ob mice were examined. Insulin stimulation, prior to sacrifice, resulted in no significant activation of insulin signaling pathways in livers from ob/ob mice. However, in PTP1B ASO-treated mice, in which PTP1B protein was decreased by 60% in liver, similar stimulation with insulin resulted in increased tyrosine phosphorylation of the IR and IR substrate (IRS)-1 and -2 by threefold, fourfold, and threefold, respectively. IRS-2-associated phosphatidylinositol 3-kinase activity was also increased threefold. Protein kinase B (PKB) serine phosphorylation was increased sevenfold in liver of PTP1B ASO-treated mice upon insulin stimulation, while phosphorylation of PKB substrates, glycogen synthase kinase (GSK)-3alpha and -3beta, was increased more than twofold. Peripheral insulin signaling was increased by PTP1B ASO, as evidenced by increased phosphorylation of PKB in muscle of insulin-stimulated PTP1B ASO-treated animals despite the lack of measurable effects on muscle PTP1B protein. These results indicate that reduction of PTP1B is sufficient to increase insulin-dependent metabolic signaling and improve insulin sensitivity in a diabetic animal model.

Reduction of protein-tyrosine phosphatase-1B increases insulin signaling in FAO hepatoma cells.

Protein-tyrosine phosphatase-1B (PTP1B) has been implicated as a negative regulator of insulin signaling. PTP1B dephosphorylates the insulin receptor and insulin receptor substrates (IRS-1/2), inhibiting the insulin-signaling pathway. PTP1B has been reported to be elevated in diabetes and insulin-resistant states. Conversely, PTP1B null mice have increased insulin sensitivity. To further investigate the effect of PTP1B reduction on insulin signaling, FAO rat hepatoma cells were transfected, by electroporation, with a specific PTP1B antisense oligonucleotide (ASO), or a control oligonucleotide. The PTP1B ASO caused a 50-70% reduction in PTP1B protein expression as measured by Western blot analysis. Upon insulin stimulation, an increase in the phosphorylation of the insulin receptor and insulin receptor substrates was observed, without any change in protein expression levels. Reduction of PTP1B expression in FAO cells also caused an increase in insulin-stimulated phosphorylation of PKB and GSK3, without any change in protein expression. These results demonstrate that reduction of PTP1B can modulate key insulin signaling events downstream of the insulin receptor.

Putting thought to paper: a microARCS protease screen.

In micro-arrayed compound screening (microARCS), an agarose gel is used as a reaction vessel that maintains humidity and compound location as well as being a handling system for reagent addition. Two or more agarose gels may be used to bring test compounds, targets, and reagents together, relying on the pore size of the gel matrix to regulate diffusion of reactants. It is in the microenvironment of the agarose matrix that all the components of an enzymatic reaction interact and result in inhibitable catalytic activity. In an effort to increase the throughput of microARCS-based screens, reduce the effort involved in manipulating agarose gels, and reduce costs, blotter paper was used rather than a second agarose gel to introduce a substrate to a gel containing a target enzyme. In this assay, the matrix of the blotter paper did not prevent the substrate from diffusing into the enzyme gel. The compound density of the microARCS format, the ease of manipulating sheets of paper for reagent addition, and a scheduled protocol for running multiple gels allowed for a throughput capacity of more than 200,000 tests per hour. A protease assay was developed and run in the microARCS format at a rate of 200,000 tests per hour using blotter paper to introduce the substrate. Picks in the primary screen were retested in the microARCS format at a density of 384 compounds per sheet. IC(50) values were confirmed in a 96-well plate format. The screen identified several small molecule inhibitors of the enzyme. The details of the screening format and the analysis of the hits from the screen are presented.

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice.

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA(1C). Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50alpha, were increased and PI3-kinase p85alpha expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes.

Protein tyrosine phosphatase 1B reduction regulates adiposity and expression of genes involved in lipogenesis.

Protein tyrosine phosphatase 1B (PTP1B) has been implicated as a negative regulator of insulin action. Overexpression of PTP1B protein has been observed in insulin-resistant states associated with obesity. Mice lacking a functional PTP1B gene exhibit increased insulin sensitivity and are resistant to weight gain. To investigate the role of PTP1B in adipose tissue from obese animals, hyperglycemic obese (ob/ob) mice were treated with PTP1B antisense oligonucleotide (ISIS-113715). A significant reduction in adiposity correlated with a decrease of PTP1B protein levels in fat. Antisense treatment also influenced the triglyceride content in adipocytes, correlating with a downregulation of genes encoding proteins involved in lipogenesis, such as sterol regulatory element-binding protein 1 and their downstream targets spot14 and fatty acid synthase, as well as other adipogenic genes, lipoprotein lipase, and peroxisome proliferator-activated receptor gamma. In addition, an increase in insulin receptor substrate-2 protein and a differential regulation of the phosphatidylinositol 3-kinase regulatory subunit (p85alpha) isoforms expression were found in fat from antisense-treated animals, although increased insulin sensitivity measured by protein kinase B phosphorylation was not observed. These results demonstrate that PTP1B antisense treatment can modulate fat storage and lipogenesis in adipose tissue and might implicate PTP1B in the enlargement of adipocyte energy stores and development of obesity.