An integrative genomics approach to infer causal associations between gene expression and disease.

A key goal of biomedical research is to elucidate the complex network of gene interactions underlying complex traits such as common human diseases. Here we detail a multistep procedure for identifying potential key drivers of complex traits that integrates DNA-variation and gene-expression data with other complex trait data in segregating mouse populations. Ordering gene expression traits relative to one another and relative to other complex traits is achieved by systematically testing whether variations in DNA that lead to variations in relative transcript abundances statistically support an independent, causative or reactive function relative to the complex traits under consideration. We show that this approach can predict transcriptional responses to single gene-perturbation experiments using gene-expression data in the context of a segregating mouse population. We also demonstrate the utility of this approach by identifying and experimentally validating the involvement of three new genes in susceptibility to obesity.

11ß-HSD1 inhibition reduces atherosclerosis in mice by altering proinflammatory gene expression in the vasculature.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is implicated in the etiology of metabolic syndrome. We previously showed that pharmacological inhibition of 11ß-HSD1 ameliorated multiple facets of metabolic syndrome and attenuated atherosclerosis in ApoE-/- mice. However, the molecular mechanism underlying the atheroprotective effect was not clear. In this study, we tested whether and how 11ß-HSD1 inhibition affects vascular inflammation, a major culprit for atherosclerosis and its associated complications. ApoE-/- mice were treated with an 11ß-HSD1 inhibitor for various periods of time. Plasma lipids and aortic cholesterol accumulation were quantified. Several microarray studies were carried out to examine the effect of 11ß-HSD1 inhibition on gene expression in atherosclerotic tissues. Our data suggest 11ß-HSD1 inhibition can directly modulate atherosclerotic plaques and attenuate atherosclerosis independently of lipid lowering effects. We identified immune response genes as the category of mRNA most significantly suppressed by 11ß-HSD1 inhibition. This anti-inflammatory effect was further confirmed in plaque macrophages and smooth muscle cells procured by laser capture microdissection. These findings in the vascular wall were corroborated by reduction in circulating MCP1 levels after 11ß-HSD1 inhibition. Taken together, our data suggest 11ß-HSD1 inhibition regulates proinflammatory gene expression in atherosclerotic tissues of ApoE-/- mice, and this effect may contribute to the attenuation of atherosclerosis in these animals.

Systems analysis of eleven rodent disease models reveals an inflammatome signature and key drivers.

Common inflammatome gene signatures as well as disease-specific signatures were identified by analyzing 12 expression profiling data sets derived from 9 different tissues isolated from 11 rodent inflammatory disease models. The inflammatome signature significantly overlaps with known drug targets and co-expressed gene modules linked to metabolic disorders and cancer. A large proportion of genes in this signature are tightly connected in tissue-specific Bayesian networks (BNs) built from multiple independent mouse and human cohorts. Both the inflammatome signature and the corresponding consensus BNs are highly enriched for immune response-related genes supported as causal for adiposity, adipokine, diabetes, aortic lesion, bone, muscle, and cholesterol traits, suggesting the causal nature of the inflammatome for a variety of diseases. Integration of this inflammatome signature with the BNs uncovered 151 key drivers that appeared to be more biologically important than the non-drivers in terms of their impact on disease phenotypes. The identification of this inflammatome signature, its network architecture, and key drivers not only highlights the shared etiology but also pinpoints potential targets for intervention of various common diseases.

11beta-HSD1 inhibition ameliorates metabolic syndrome and prevents progression of atherosclerosis in mice.

The enzyme 11beta-hydroxysteroid dehydrogenase (HSD) type 1 converts inactive cortisone into active cortisol in cells, thereby raising the effective glucocorticoid (GC) tone above serum levels. We report that pharmacologic inhibition of 11beta-HSD1 has a therapeutic effect in mouse models of metabolic syndrome. Administration of a selective, potent 11beta-HSD1 inhibitor lowered body weight, insulin, fasting glucose, triglycerides, and cholesterol in diet-induced obese mice and lowered fasting glucose, insulin, glucagon, triglycerides, and free fatty acids, as well as improved glucose tolerance, in a mouse model of type 2 diabetes. Most importantly, inhibition of 11beta-HSD1 slowed plaque progression in a murine model of atherosclerosis, the key clinical sequela of metabolic syndrome. Mice with a targeted deletion of apolipoprotein E exhibited 84% less accumulation of aortic total cholesterol, as well as lower serum cholesterol and triglycerides, when treated with an 11beta-HSD1 inhibitor. These data provide the first evidence that pharmacologic inhibition of intracellular GC activation can effectively treat atherosclerosis, the key clinical consequence of metabolic syndrome, in addition to its salutary effect on multiple aspects of the metabolic syndrome itself.

Bicyclo[2.2.2]octyltriazole inhibitors of 11ß-hydoxysteroid dehydrogenase type 1. Pharmacological agents for the treatment of metabolic syndrome.

Following the discovery of a metabolic 'soft-spot' on a bicyclo[2.2.2]octyltriazole lead, an extensive effort was undertaken to block the oxidative metabolism and improve PK of this potent HSD1 lead. In this communication, SAR survey focusing on various alkyl chain replacements will be detailed. This effort culminated in the discovery of a potent ethyl sulfone inhibitor with an improved PK profile across species and improved physical properties.

4-Methyl-5-phenyl triazoles as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type I.

4-Methyl-5-phenyl-(1,2,4)-triazoles were identified as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). They were active in vitro and in an in vivo mouse pharmacodynamic (PD) model. The synthesis and structure activity relationships are presented.

Phenylcyclobutyl triazoles as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type I.

3-(Phenylcyclobutyl)-1,2,4-triazoles were identified as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). These were active both in vitro and in an in vivo mouse pharmacodynamic (PD) model. Fluorine substitution of the cyclobutane ring improved the pharmacokinetic profile significantly. The synthesis and structure-activity relationships are presented.

Bis-aryl triazoles as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1.

3-Aryl-5-phenyl-(1,2,4)-triazoles were identified as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). They are active in both in vitro and an in vivo mouse pharmacodynamic (PD) model. The synthesis and structure activity relationships are presented.

Depot-specific modulation of rat intraabdominal adipose tissue lipid metabolism by pharmacological inhibition of 11beta-hydroxysteroid dehydrogenase type 1.

The metabolic consequences of visceral obesity have been associated with amplification of glucocorticoid action by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in adipose tissue. This study aimed to assess in a rat model of diet-induced obesity the effects of pharmacological 11beta-HSD1 inhibition on the morphology and expression of key genes of lipid metabolism in intraabdominal adipose depots. Rats fed a high-sucrose, high-fat diet were treated or not with a specific 11beta-HSD1 inhibitor (compound A, 3 mg/kg.d) for 3 wk. Compound A did not alter food intake or body weight gain but specifically reduced mesenteric adipose weight (-18%) and adipocyte size, without significantly affecting those of epididymal or retroperitoneal depots. In mesenteric fat, the inhibitor decreased (to 25-50% of control) mRNA levels of genes involved in lipid synthesis (FAS, SCD1, DGAT1) and fatty acid cycling (lipolysis/reesterification, ATGL and PEPCK) and increased (30%) the activity of the fatty acid oxidation-promoting enzyme carnitine palmitoyltransferase 1. In striking contrast, in the epididymal depot, 11beta-HSD1 inhibition increased (1.5-5-fold) mRNA levels of those genes related to lipid synthesis/cycling and slightly decreased carnitine palmitoyltransferase 1 activity, whereas gene expression remained unaffected in the retroperitoneal depot. Compound A robustly reduced liver triacylglycerol content and plasma lipids. The study demonstrates that pharmacological inhibition of 11beta-HSD1, at a dose that does not alter food intake, reduces fat accretion specifically in the mesenterical adipose depot, exerts divergent intraabdominal depot-specific effects on genes of lipid metabolism, and reduces steatosis and lipemia.

Preliminary report: pharmacologic 11beta-hydroxysteroid dehydrogenase type 1 inhibition increases hepatic fat oxidation in vivo and expression of related genes in rats fed an obesogenic diet.

This study aimed to explore in a model of diet-induced steatosis the impact of pharmacologic 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) inhibition, under conditions of unchanged ingestive behavior, on liver fat oxidation. Male Sprague-Dawley rats were fed an obesogenic diet and were continuously treated or not with an 11beta-HSD1 inhibitor (Compound A, 3 mg/[kg d]; Merck Research Laboratories, Rahway, NJ), after which liver expression of oxidative genes and in vivo hepatic fat oxidation were quantified. Treatment with Compound A reduced liver triglyceride concentration (-28%), increased hepatic expression of several genes coding for enzymes of mitochondrial and peroxisomal beta-oxidation, and concomitantly enhanced in vivo liver fat oxidation (+38%). The study demonstrates, under conditions that avoided changes in food intake seen in gene knockout or higher-dose pharmacologic models, the efficacy of 11beta-HSD1 inhibition to up-regulate hepatic fat oxidation gene expression, which functionally translates into enhanced hepatic lipid oxidation in vivo.

Identification and validation of genes affecting aortic lesions in mice.

Atherosclerosis represents the most significant risk factor for coronary artery disease (CAD), the leading cause of death in developed countries. To better understand the pathogenesis of atherosclerosis, we applied a likeli-hood-based model selection method to infer gene-disease causality relationships for the aortic lesion trait in a segregating mouse population demonstrating a spectrum of susceptibility to developing atherosclerotic lesions. We identified 292 genes that tested causal for aortic lesions from liver and adipose tissues of these mice, and we experimentally validated one of these candidate causal genes, complement component 3a receptor 1 (C3ar1), using a knockout mouse model. We also found that genes identified by this method overlapped with genes progressively regulated in the aortic arches of 2 mouse models of atherosclerosis during atherosclerotic lesion development. By comparing our gene set with findings from public human genome-wide association studies (GWAS) of CAD and related traits, we found that 5 genes identified by our study overlapped with published studies in humans in which they were identified as risk factors for multiple atherosclerosis-related pathologies, including myocardial infarction, serum uric acid levels, mean platelet volume, aortic root size, and heart failure. Candidate causal genes were also found to be enriched with CAD risk polymorphisms identified by the Wellcome Trust Case Control Consortium (WTCCC). Our findings therefore validate the ability of causality testing procedures to provide insights into the mechanisms underlying atherosclerosis development.

11beta-HSD1 inhibition improves triglyceridemia through reduced liver VLDL secretion and partitions lipids toward oxidative tissues.

Tissue-specific alterations in 11beta-hydroxysteroid dehydrogenase (HSD) type 1 activity, which amplifies glucocorticoid action, are thought to contribute to some of the metabolic complications of obesity. The present study tested whether hypertriglyceridemia is one such complication by investigating the effects of an 11beta-HSD1 inhibitor (compound A, 3 mgxkg(-1)xday(-1), 21 days) on triglyceride (TG) metabolism in a rat model of diet-induced obesity. The dose of compound A used did not affect food intake or final body weight. Compound A improved fasting triglyceridemia (-42%) through a robust reduction (-41%) in hepatic TG secretion rate, without change in plasma TG clearance rate. Uptake of TG-derived fatty acids was, however, increased in oxidative tissues, including red gastrocnemius (+47%), heart (+39%), and brown adipose tissue (BAT, +46%) at the expense of the liver, with a concomitant increase in plasma membrane fatty acid-binding protein. Lipid oxidation products were increased in red gastrocnemius (+35%) and heart (+33%), as were levels of uncoupling protein 1 mRNA in BAT (+48%), and carnitine palmitoyltransferase 1 activity tended to be increased in some oxidative tissues. These findings demonstrate that pharmacological inhibition of 11beta-HSD1 at a dose that does not affect food intake improves triglyceridemia by reducing hepatic very low density lipoprotein-TG secretion, with a shift in the pattern of TG-derived fatty acid uptake toward oxidative tissues, in which lipid accumulation is prevented by increased lipid oxidation.

Inhibition of 11beta-HSD1 as a novel treatment for the metabolic syndrome: do glucocorticoids play a role?

The metabolic syndrome (syndrome X) is a cluster of risk factors and a common cause of cardiovascular disease in humans. Although the underlying mechanism for metabolic syndrome is still poorly understood, recent clinical data and studies with transgenic animals implicate elevated intracellular glucocorticoid tone in the etiology of metabolic syndrome. Development of selective inhibitors of 11beta-hydroxysteroid dehydrogenase (11beta-HSD)-1 and their use in rodent animal disease models encompassing several aspects of metabolic syndrome indicate the possibility of therapeutic intervention. This review will focus on recent advances in our understanding of the role of 11beta-HSD1 in metabolic disorders and other disease processes.

Discovery of 4-heteroarylbicyclo[2.2.2]octyltriazoles as potent and selective inhibitors of 11beta-HSD1: novel therapeutic agents for the treatment of metabolic syndrome.

Replacement of the pentyl chain on our original bicyclo[2.2.2]octyltriazole leads 1 and 2 has led to the discovery that heteroaryl substituted bicyclo[2.2.2]octyltriazoles are potent and selective 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1) inhibitors with excellent pharmacokinetic profiles.

Adamantyl triazoles as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1.

Adamantyl triazoles were identified as selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). They are active both in in vitro and in in vivo pharmacodynamic models. The synthesis and structure-activity relationships of these inhibitors are presented.