Identification of glucose-dependant insulin secretion targets in pancreatic beta cells by combining defined-mechanism compound library screening and siRNA gene silencing.

Identification and validation of novel drug targets continues to be a major bottleneck in drug development, particularly for polygenic complex diseases such as type 2 diabetes. Here, the authors describe an approach that allows researchers to rapidly identify and validate potential drug targets by combining chemical tools and RNA interference technology. As a proof-of-concept study, the known mechanism Sigma LOPAC library was used to screen for glucose-dependent insulin secretion (GDIS) in INS-1 832/13 cells. In addition to several mechanisms that are known to regulate GDIS (such as cyclic adenosine monophosphate-specific phosphodiesterases, adrenoceptors, and Ca(2+) channels), the authors find that several of the dopamine receptor (DRD) antagonists significantly enhance GDIS, whereas DRD agonists profoundly inhibit GDIS. Subsequent siRNA studies in the same cell line indicate that knockdown of DRD2 enhanced GDIS. Furthermore, selective DRD2 antagonists and agonists also enhance or suppress, respectively, GDIS in isolated rat islets. The data support that the approach described here offers a rapid and effective way for target identification and validation.

Synthesis and SAR of potent and orally bioavailable tert-butylpyrrolidine archetype derived melanocortin subtype-4 receptor modulators.

Discovery of a series of tert-butyl pyrrolidine derived, potent and orally bioavailable melanocortin receptor subtype-4 (MC4R) selective modulators is disclosed.

The role of melanocortins in body weight regulation: opportunities for the treatment of obesity.

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

The role of melanocortins in body weight regulation: opportunities for the treatment of obesity.

Five G-protein-coupled melanocortin receptors (MC(1)-MC(5)) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-MSH and gamma-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC(1), MC(3) and MC(4) receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC(3) and MC(4) receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.

Pharmacokinetics and pharmacodynamics of AR9281, an inhibitor of soluble epoxide hydrolase, in single- and multiple-dose studies in healthy human subjects.

AR9281, a potent and selective inhibitor of soluble epoxide hydrolase (s-EH), is in clinical development targeting hypertension and type 2 diabetes. The safety, pharmacokinetics, and pharmacodynamics of AR9281 were evaluated in double-blind, randomized, placebo-controlled, ascending, single oral dose (10-1000 mg) and multiple dose (100-400 mg every 8 hours for 7 days) studies in healthy subjects. AR9281 was well tolerated, and no dose-related adverse events were observed during either study. The drug was rapidly absorbed with a mean terminal half-life ranging from 3 to 5 hours. The area under the plasma concentration-time curve increased in an approximately dose-proportional manner up to the 500-mg dose and exhibited a greater than dose linearity at higher doses. AR9281 directly and dose-dependently inhibited blood s-EH activity with 90% inhibition or greater over an 8-hour period at the 250-mg dose and over a 12-hour period at the 500-mg dose. Multiple doses of AR9281 ranging from 100 to 400 mg every 8 hours resulted in a sustained inhibition of s-EH activity at 90% or greater during the trough. The current studies provide proof of safety and target inhibition of AR9281 in healthy subjects. AR9281 pharmacokinetic and pharmacodynamic characteristics support a twice-daily or thrice-daily dosing regimen in patients.

A novel 3-dimensional micro-ultrasound approach to automated measurement of carotid arterial plaque volume as a biomarker for experimental atherosclerosis.

Improved methods for non-invasive in vivo assessment are needed to guide development of animal models of atherosclerosis and to evaluate target engagement and in vivo efficacy of new drugs. Using novel 3D-micro-ultrasound technology, we developed and validated a novel protocol for 3D acquisition and analysis of imaging to follow lesion progression in atherosclerotic mice. The carotid arteries of ApoE receptor knockout mice and normal control mice were imaged within the proximal 2mm from the aortic branch point. Plaque volume along that length was quantified using a semi-automated 3D segmentation algorithm. Volumes derived by this method were compared to those calculated using 3-D histology post-mortem. Bland-Altman comparison revealed close correlation between these two measures of plaque volume. Furthermore, using a segmentation technique that captures early positive and 33 week negative remodeling, we found evidence that plaque volume increases linearly over time. Each animal and each plaque served as its own control, allowing accurate comparison. The high fidelity anatomical registration of this protocol provides increased spatial resolution and therefore greater sensitivity for measurement of plaque wall size, an advance over 2-dimensional measures of intimal-medial-thickening. Further, 3-dimensional analysis ensures a point of registration that captures functional markers in addition to the standard structural markers that characterize experimental atherosclerosis. In conclusion, this novel imaging protocol provides a non-invasive, accurate surrogate marker for experimental atherosclerosis over the life of the entire lesion.

Benzyl vinylogous amide substituted aryldihydropyridazinones and aryldimethylpyrazolones as potent and selective PDE3B inhibitors.

Aryldihydropyridazinones and aryldimethylpyrazolones with 2-benzyl vinylogous amide substituents have been identified as potent PDE3B subtype selective inhibitors. Dihydropyridazinone 8a (PDE3B IC(50)=0.19 nM, 3A IC(50)=1.3 nM) was selected for in vivo evaluation of lipolysis induction, metabolic rate increase, and cardiovascular effects.