Henrietta Dehmlow

Insight Into Steroid Scaffold Formation from the Structure of Human Oxidosqualene Cyclase

In higher organisms the formation of the steroid scaffold is catalysed exclusively by the membrane-bound oxidosqualene cyclase (OSC; lanosterol synthase). In a highly selective cyclization reaction OSC forms lanosterol with seven chiral centres starting from the linear substrate 2,3-oxidosqualene. Valuable data on the mechanism of the complex cyclization cascade have been collected during the past 50 years using suicide inhibitors, mutagenesis studies and homology modelling. Nevertheless it is still not fully understood how the enzyme catalyses the reaction. Because of the decisive role of OSC in cholesterol biosynthesis it represents a target for the discovery of novel anticholesteraemic drugs that could complement the widely used statins. Here we present two crystal structures of the human membrane protein OSC: the target protein with an inhibitor that showed cholesterol lowering in vivo opens the way for the structure-based design of new OSC inhibitors. The complex with the reaction product lanosterol gives a clear picture of the way in which the enzyme achieves product specificity in this highly exothermic cyclization reaction.

Endothelin-Converting Enzyme Inhibition Ameliorates Angiotensin II–Induced Cardiac Damage

Abstract—We tested the hypothesis that endothelin-converting enzyme (ECE) inhibition ameliorates end-organ damage in rats harboring both human renin and human angiotensinogen genes (dTGR). Hypertension develops in the animals, and they die by age 7 weeks of heart and kidney failure. Three groups were studied: dTGR (n=12) receiving vehicle, dTGR receiving ECE inhibitor (RO0687629; 30 mg/kg by gavage; n=10), and Sprague-Dawley control rats (SD; n=10) receiving vehicle, all after week 4, with euthanasia at week 7. Systolic blood pressure was not reduced by ECE inhibitor compared with dTGR (205±6 versus 206±6 mm Hg at week 7, respectively). In contrast, ECE inhibitor treatment significantly reduced mortality rate to 20% (2 of 10), whereas untreated dTGR had a 52% mortality rate (7 of 12). ECE inhibitor treatment ameliorated cardiac damage and reduced left ventricular ECE activity below SD levels. Echocardiography at week 7 showed reduced cardiac hypertrophy (4.8±0.2 versus 5.7±0.2 mg/g, P <0.01) and increased left ventricular cavity diameter (5.5±0.3 versus 3.1±0.1 mm, P <0.001) and filling volume (0.42±0.04 versus 0.16±0.06 mL, P <0.05) after ECE inhibitor compared with untreated dTGR. ECE inhibitor treatment also reduced cardiac fibrosis, tissue factor expression, left ventricular basic fibroblast growth factor mRNA levels, and immunostaining in the vessel wall, independent of high blood pressure. In contrast, the ECE inhibitor treatment showed no renoprotective effect. These data are the first to show that ECE inhibition reduces angiotensin II–induced cardiac damage.

Systemic bile acid sensing by G protein‐coupled bile acid receptor 1 (GPBAR1) promotes PYY and GLP‐1 release

Nutrient sensing in the gut is believed to be accomplished through activation of GPCRs expressed on enteroendocrine cells. In particular, L‐cells located predominantly in distal regions of the gut secrete glucagon‐like peptide 1 (GLP‐1) and peptide tyrosine‐tyrosine (PYY) upon stimulation by nutrients and bile acids (BA). The study was designed to address the mechanism of hormone secretion in L‐cells stimulated by the BA receptor G protein‐coupled bile acid receptor 1 (GPBAR1).

Discovery of tetrahydro-cyclopenta[b]indole as selective LXRs modulator.

A series of tetrahydro-cyclopenta[b]indoles modulating the activity of the liver-X-receptor (LXR) were derived from a high throughput screening hit. The potency and selectivity for LXRbeta versus LXRalpha was improved. One compound, administered to wild-type mice modestly increased plasma HDL-cholesterol with no change in plasma triglycerides (TG) and reduced effects on liver TG content compared to T0901317. This novel series of LXR agonists shows promise to improve therapeutic efficacy with reduced potential to increase TG.

A Novel Inhibitor of Oxidosqualene:Lanosterol Cyclase Inhibits Very Low–Density Lipoprotein Apolipoprotein B100 (ApoB100) Production and Enhances Low-Density Lipoprotein ApoB100 Catabolism Through Marked Reduction in Hepatic Cholesterol Content

Objective—Inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), an enzyme in the cholesterol synthesis pathway, has the unique ability to inhibit cholesterol synthesis while simultaneously enhancing oxysterol synthesis. Our objectives were to determine, in vivo, if a novel OSC inhibitor reduced low-density lipoprotein (LDL) cholesterol and to define the mechanism(s) involved. Methods and Results—Miniature pigs received the OSC inhibitor RO0717625 or placebo and a diet containing fat (34% of energy) and 400 mg per day of cholesterol. Treatment decreased plasma total cholesterol (−20%) and LDL cholesterol (−29%). Apolipoprotein B (apoB) kinetic parameters were determined. Very low–density lipoprotein (VLDL) apoB pool size decreased 22% because of inhibition of VLDL production (−43%). LDL apoB pool size decreased 22% because of a 1.5-fold increase in fractional catabolic rate (FCR). The increased FCR was associated with a 2-fold increase in hepatic LDL receptor mRNA. Hepatic total and microsomal cholesterol were reduced by 16% and 27%, respectively. Plasma lathosterol concentrations decreased 57%, reflecting inhibition of hepatic cholesterol synthesis. Treatment reduced plasma plant sterols and decreased postprandial cholesterol transport in chylomicrons. Conclusions—A novel OSC inhibitor, RO0717625, decreased VLDL and LDL apoB100 through decreased VLDL production and enhanced LDL clearance. Thus, OSC represents a potential therapeutic target for dyslipidemia.

2‐Phenoxy‐nicotinamides are Potent Agonists at the Bile Acid Receptor GPBAR1 (TGR5)

Potency with potential: 2-Phenoxy-nicotinamides were identified as potent agonists at the GPBAR1 receptor, a target in the treatment of obesity, type 2 diabetes and metabolic syndrome. Extensive structure-activity relationship studies supported by homology modeling and docking resulted in the identification of optimized GPBAR1 agonists, potent against both human and mouse receptors, endowed with favorable physicochemical properties and good metabolic stability.

Cytotoxic effects of combination of oxidosqualene cyclase inhibitors with atorvastatin in human cancer cells.

Ten oxidosqualene cyclase inhibitors with high efficacy as cholesterol-lowering agents and of different chemical structure classes were evaluated as potential anticancer agents against human cancer cells from various tissue origins and nontumoral human-brain-derived endothelial cells. Inhibition of cancer cell growth was demonstrated at micromolar concentrations, comparable to the concentrations of statins necessary for antitumor effect. Human glioblastoma cells were among the most sensitive cells. These compounds were also able to decrease the proliferation of angiogenic brain-derived endothelial cells, as a model of tumor-induced neovasculation. Additive effects in human glioblastoma cells were also demonstrated for oxidosqualene cyclase inhibitors in combination with atorvastatin while maintaining selectivity against endothelial cells. Thus, not only statins targeting the 3-hydroxy-3-methylglutaryl coenzyme A reductase but also inhibitors of oxidosqualene cyclase decrease tumor growth, suggesting new therapeutic opportunities of combined anti-cholesterol agents for dual treatment of glioblastoma.

Pyrido pyrimidinones as selective agonists of the high affinity niacin receptor GPR109A: optimization of in vitro activity.

Pyrido pyrimidinones are selective agonists of the human high affinity niacin receptor GPR109A (HM74A). They show no activity on the highly homologous low affinity receptor GPR109B (HM74). Starting from a high throughput screening hit the in vitro activity of the pyrido pyrimidinones was significantly improved providing lead compounds suitable for further optimization.

Oxidosqualene cyclase from Saccharomyces cerevisiae, Trypanosoma cruzi, Pneumocystis carinii and Arabidopsis thaliana expressed in yeast: a model for the development of novel antiparasitic agents.

A series of 25 compounds, some of which previously were described as inhibitors of human liver microsomal oxidosqualene cyclase (OSC), were tested as inhibitors of Saccharomyces cerevisiae, Trypanosoma cruzi, Pneumocystis carinii and Arabidopsis thaliana OSCs expressed in an OSC-defective strain of S. cerevisiae. The screening identified three derivatives particularly promising for the development of novel anti-Trypanosoma agents and eight derivatives for the development of novel anti-Pneumocystis agents.

G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey.

G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor.