c-Jun N-terminal kinase pathways in diabetes.

Type 2 diabetes develops from insulin resistance and has become a worldwide epidemic. The c-Jun N-terminal kinases have been considered as signaling molecules linking inflammation and insulin resistance. Genetic disruption of c-Jun N-terminal kinase-1 gene prevents the development of insulin resistance in obese and diabetic mice. Inhibition of c-Jun N-terminal kinases by a small cell-permeable peptide improves insulin sensitivity in mice. Hepatic inhibition of c-Jun N-terminal kinases using a dominant-negative protein or knockdown of c-Jun N-terminal kinase-1 gene by RNA interference reduces blood glucose and insulin levels and enhances hepatic insulin signaling in mice. Recent evidence demonstrates that the hepatic c-Jun N-terminal kinase pathway plays an important role in lipid and lipoprotein homeostasis in mice. This review discusses recent advances in our understanding of the role of c-Jun N-terminal kinase pathway in metabolic control and its potential as a target for the treatment of type 2 diabetes.

Liver-specific knockdown of JNK1 up-regulates proliferator-activated receptor gamma coactivator 1 beta and increases plasma triglyceride despite reduced glucose and insulin levels in diet-induced obese mice.

The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor gamma coactivator 1 beta, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.

Zotarolimus, a novel sirolimus analogue with potent anti-proliferative activity on coronary smooth muscle cells and reduced potential for systemic immunosuppression.

Sirolimus (rapamycin) is an immunosuppressant used in preventing allograft rejection and in drug-eluting stents to prevent restenosis after angioplasty. Zotarolimus, an analogue of sirolimus, was designed to have a shorter in vivo half-life. Zotarolimus was found to be mechanistically similar to sirolimus in having high-affinity binding to the immunophilin FKBP12 and comparable potency for inhibiting in vitro proliferation of both human and rat T cells. Rat pharmacokinetic studies with intravenous dosing demonstrated terminal elimination half-lives of 9.4 hours and 14.0 hours for zotarolimus and sirolimus, respectively. Given orally, T1/2 values were 7.9 hours and 33.4 hours, respectively. Consistent with its shorter duration, zotarolimus showed a corresponding and statistically significant 4-fold reduction in potency for systemic immunosuppression in 3 rat disease models. Pharmacokinetic studies in cynomolgus monkey underpredicted the half-life difference between zotarolimus and sirolimus apparent from recent clinical data. In vitro inhibition of human coronary artery smooth muscle cell proliferation by zotarolimus was comparable to sirolimus. Drug-eluting stents for local delivery of zotarolimus to the vessel wall of coronary arteries are in clinical development. The pharmacological profile of zotarolimus suggests it may be advantageous for preventing restenosis with a reduced potential for causing systemic immunosuppression or other side effects.

Discovery and structure-activity relationships of piperidinone- and piperidine-constrained phenethylamines as novel, potent, and selective dipeptidyl peptidase IV inhibitors.

Dipeptidyl peptidase IV (DPP4) inhibitors are emerging as a new class of therapeutic agents for the treatment of type 2 diabetes. They exert their beneficial effects by increasing the levels of active glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are two important incretins for glucose homeostasis. Starting from a high-throughput screening hit, we were able to identify a series of piperidinone- and piperidine-constrained phenethylamines as novel DPP4 inhibitors. Optimized compounds are potent, selective, and have good pharmacokinetic profiles.

Pyrrolidine-constrained phenethylamines: The design of potent, selective, and pharmacologically efficacious dipeptidyl peptidase IV (DPP4) inhibitors from a lead-like screening hit.

A novel series of pyrrolidine-constrained phenethylamines were developed as dipeptidyl peptidase IV (DPP4) inhibitors for the treatment of type 2 diabetes. The cyclohexene ring of lead-like screening hit 5 was replaced with a pyrrolidine to enable parallel chemistry, and protein co-crystal structural data guided the optimization of N-substituents. Employing this strategy, a >400x improvement in potency over the initial hit was realized in rapid fashion. Optimized compounds are potent and selective inhibitors with excellent pharmacokinetic profiles. Compound 30 was efficacious in vivo, lowering blood glucose in ZDF rats that were allowed to feed freely on a mixed meal.

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.

Hemodynamic effects of potent and selective JNK inhibitors in anesthetized rats: implication for targeting protein kinases in metabolic diseases.

The hemodynamic effects of a series of potent and selective 4-aminopyridine carboxamide-based pan-JNK inhibitors were assessed in an anesthetized rat model. The effects of these agents on mean arterial pressure, heart rate, cardiac contractility, and peripheral vascular resistance are described, and the implication for targeting protein kinases in metabolic diseases is discussed.

Discovery of ((4R,5S)-5-amino-4-(2,4,5- trifluorophenyl)cyclohex-1-enyl)-(3- (trifluoromethyl)-5,6-dihydro- [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)methanone (ABT-341), a highly potent, selective, orally efficacious, and safe dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes.

Dipeptidyl peptidase IV (DPP4) deactivates glucose-regulating hormones such as GLP-1 and GIP, thus, DPP4 inhibition has become a useful therapy for type 2 diabetes. Optimization of the high-throughput screening lead 6 led to the discovery of 25 (ABT-341), a highly potent, selective, and orally bioavailable DPP4 inhibitor. When dosed orally, 25 dose-dependently reduced glucose excursion in ZDF rats. Amide 25 is safe in a battery of in vitro and in vivo tests and may represent a new therapeutic agent for the treatment of type 2 diabetes.

Discovery of 2-[4-{{2-(2S,5R)-2-cyano-5-ethynyl-1-pyrrolidinyl]-2-oxoethyl]amino]- 4-methyl-1-piperidinyl]-4-pyridinecarboxylic acid (ABT-279): a very potent, selective, effective, and well-tolerated inhibitor of dipeptidyl peptidase-IV, useful for the treatment of diabetes.

Dipeptidyl peptidase-IV (DPP-IV) inhibitors are poised to be the next major drug class for the treatment of type 2 diabetes. Structure-activity studies of substitutions at the C5 position of the 2-cyanopyrrolidide warhead led to the discovery of potent inhibitors of DPP-IV that lack activity against DPP8 and DPP9. Further modification led to an extremely potent (Ki(DPP)(-)(IV) = 1.0 nM) and selective (Ki(DPP8) > 30 microM; Ki(DPP9) > 30 microM) clinical candidate, ABT-279, that is orally available, efficacious, and remarkably safe in preclinical safety studies.

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.

Discovery, structure-activity relationship, and pharmacological evaluation of (5-substituted-pyrrolidinyl-2-carbonyl)-2-cyanopyrrolidines as potent dipeptidyl peptidase IV inhibitors.

A series of (5-substituted pyrrolidinyl-2-carbonyl)-2-cyanopyrrolidine (C5-Pro-Pro) analogues was discovered as dipeptidyl peptidase IV (DPPIV) inhibitors as a potential treatment of diabetes and obesity. X-ray crystallography data show that these inhibitors bind to the catalytic site of DPPIV with the cyano group forming a covalent bond with the serine residue of DPPIV. The C5-substituents make various interactions with the enzyme and affect potency, chemical stability, selectivity, and PK properties of the inhibitors. Optimized analogues are extremely potent with subnanomolar K(i)'s, are chemically stable, show very little potency decrease in the presence of plasma, and exhibit more than 1,000-fold selectivity against related peptidases. The best compounds also possess good PK and are efficacious in lowering blood glucose in an oral glucose tolerance test in ZDF rats.

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.

Crystal structures of DPP-IV (CD26) from rat kidney exhibit flexible accommodation of peptidase-selective inhibitors.

Dipeptidyl peptidase IV (DPP-IV) belongs to a family of serine peptidases, and due to its indirect regulatory role in plasma glucose modulation, DPP-IV has become an attractive pharmaceutical target for diabetes therapy. DPP-IV inactivates the glucagon-like peptide (GLP-1) and several other naturally produced bioactive peptides that contain preferentially a proline or alanine residue in the second amino acid sequence position by cleaving the N-terminal dipeptide. To elucidate the details of the active site for structure-based drug design, we crystallized a natural source preparation of DPP-IV isolated from rat kidney and determined its three-dimensional structure using X-ray diffraction techniques. With a high degree of similarity to structures of human DPP-IV, the active site architecture provides important details for the design of inhibitory compounds, and structures of inhibitor-protein complexes offer detailed insight into three-dimensional structure-activity relationships that include a conformational change of Tyr548. Such accommodation is exemplified by the response to chemical substitution on 2-cyanopyrrolidine inhibitors at the 5 position, which conveys inhibitory selectivity for DPP-IV over closely related homologues. A similar conformational change is also observed in the complex with an unrelated synthetic inhibitor containing a xanthine core that is also selective for DPP-IV. These results suggest the conformational flexibility of Tyr548 is unique among protein family members and may be utilized in drug design to achieve peptidase selectivity.

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.

Expression, purification, and enzymatic characterization of the dual specificity mitogen-activated protein kinase phosphatase, MKP-4.

Mitogen-activated protein kinase phosphatase-4 (MKP-4) is a dual specificity phosphatase, which acts as a negative regulator of insulin-stimulated pathways. Here, we describe expression, purification, and biochemical characterization of MKP-4. We used the Baculovirus expression system and purification with a combination of affinity and gel filtration chromatography to generate pure MKP-4 and MKP-4/p38 complex. Both MKP-4 and the MKP-4/p38 complex exhibited moderate activity toward the surrogate substrates p-nitrophenyl phosphate, 6, 8-difluoro-4-methylumbelliferyl phosphate, and 3-O-methylfluorescein phosphate. The phosphatase activity could be inhibited by peroxovanate, a potent inhibitor of protein tyrosine phosphatases. We further determined kinetic parameters for the MKP-4 and the MKP-4/p38 by using spectrophotometric and fluorescence intensity methods. The MKP-4/p38 complex was found to provide substantially higher phosphatase activity than MKP-4 alone, similar to what has been shown for MKP-3. Our data allow the configuration of screens for modulators of MKP-4 activity.

Isoxazole carboxylic acids as protein tyrosine phosphatase 1B (PTP1B) inhibitors.

Guided by X-ray crystallography, we have extended the structure-activity relationship (SAR) study on an isoxazole carboxylic acid-based PTP1B inhibitor (1) and more potent and equally selective (>20-fold selectivity over the highly homologous T-cell PTPase, TCPTP) PTP1B inhibitors were identified. Inhibitor 7 demonstrated good cellular activity against PTP1B in COS 7 cells.

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.

Identification of a monoacid-based, cell permeable, selective inhibitor of protein tyrosine phosphatase 1B.

Monoacid-based PTP1B inhibitors with improved physiochemical properties have been investigated. A (2-hydroxy-phenoxy) acetic acid-based phosphotyrosyl mimetic has been linked with an optimized second arylphosphate binding site ligand to produce compound 20 with low micromolar potency against PTP1B, good selectivity over TCPTP (20-fold) and high cell permeability in the Caco-2 system.