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.
Background and Purpose 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). Experimental Approach A novel, selective, orally bioavailable, and potent GPBAR1 agonist, RO5527239, was synthesized in order to investigate L‐cell secretion in vitro and in vivo in mice and monkey. In analogy to BA, RO5527239 was conjugated with taurine to reduce p.o. bioavailability yet retaining its potency. Using RO5527239 and tauro‐RO5527239, the acute secretion effects on L‐cells were addressed via different routes of administration. Key Results GPBAR1 signalling triggers the co‐secretion of PYY and GLP‐1, and leads to improved glucose tolerance. The strong correlation of plasma drug exposure and plasma PYY levels suggests activation of GPBAR1 from systemically accessible compartments. In contrast to the orally bioavailable agonist RO5527239, we show that tauro‐RO5527239 triggers PYY release only when applied intravenously. Compared to mice, a slower and more sustained PYY secretion was observed in monkeys. Conclusion and Implications Selective GPBAR1 activation elicits a strong secretagogue effect on L‐cells, which primarily requires systemic exposure. We suggest that GPBAR1 is a key player in the intestinal proximal‐distal loop that mediates the early phase of nutrient‐evoked L‐cell secretion effects.
The binding structures of 11 human oxidosqualene cyclase inhibitors designed as cholesterol-lowering agents were determined for the squalene-hopene cyclase from Alicyclobacillus acidocaldarius, which is the only structurally known homologue of the human enzyme. The complexes were produced by cocrystallization, and the structures were elucidated by X-ray diffraction analyses. All inhibitors were bound in the large active center cavity. The detailed binding structures are presented and discussed in the light of the IC50 values of these 11 as well as 17 other inhibitors. They provide a consistent picture for the inhibition of the bacterial enzyme and can be used to adjust and improve homology models of the human enzyme. The detailed active center structures of the two enzymes are too different to show an IC50 correlation.
New orally active non-terpenoic inhibitors of human 2,3-oxidosqualene cyclase (hOSC) are reported. The starting point for the optimization process was a set of compounds derived from a fungicide project, which in addition to showing high affinity for OSC from Candida albicans showed also high affinity for human OSC. Common structural elements of these inhibitors are an amine residue and an electrophilic carbonyl C atom embedded in a benzophenone system, which are at a distance of about 10.7 A. Considering that the keto moiety is in a potentially labile position, modifications of the substitution pattern at the benzophenone as well as annelated heteroaryl systems were explored. Our approach combined testing of the compounds first for increased binding affinity and for increased stability in vitro. Most promising compounds were then evaluated for their efficacy in lowering plasma total cholesterol (TC) and plasma low-density lipoprotein cholesterol (LDL-C) in hyperlipidemic hamsters. In this respect, the most promising compounds are the benzophenone derivative 1.fumarate and the benzo[d]isothiazol 24.fumarate, which lowered TC by 40% and 33%, respectively.
Endothelin-1 (ET-1) is mitogenic and/or antiapoptotic in human cancers, and antagonists to ET-1 receptors are under evaluation for cancer treatment. Inhibition of ET-1 activation by the endothelin-converting enzymes 1(a)(-)(d) (ECE-1(a)(-)(d); EC 3.4.24.71) represents another approach to block the ET-1 effect in cancer. To evaluate this potential, we synthesized and characterized a series of low nanomolar nonpeptidic thiol-containing ECE-1 inhibitors, and evaluated their effect, as well as the effect of inhibitors for the related metalloproteases neprilysin (NEP; EC 3.4.24.11) and angiotensin-converting enzyme (ACE; EC 3.4.15.1), on human glioblastoma cell growth. Only ECE-1 inhibitors inhibited DNA synthesis by human glioblastoma cells. Exogenous addition of ET-1 or bigET-1 to glioblastoma cells did not counterbalance the growth inhibition elicited by ECE-1 inhibitors, suggesting that ECE-1 inhibitors block the proliferation of human glioblastoma cells most likely via a mechanism not involving extracellular production of ET-1. This class of molecules may thus represent novel therapeutic agents for the potential treatment of human cancer.
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 (nϭ12) receiving vehicle, dTGR receiving ECE inhibitor (RO0687629; 30 mg/kg by gavage; nϭ10), and Sprague-Dawley control rats (SD; nϭ10) receiving vehicle, all after week 4, with euthanasia at week 7. Systolic blood pressure was not reduced by ECE inhibitor compared with dTGR (205Ϯ6 versus 206Ϯ6 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 (4.8Ϯ0.2 versus 5.7Ϯ0.2 mg/g, PϽ0.01) and increased left ventricular cavity diameter (5.5Ϯ0.3 versus 3.1Ϯ0.1 mm, PϽ0.001) and filling volume (0.42Ϯ0.04 versus 0.16Ϯ0.06 mL, PϽ0.05) 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. Key Words: angiotensin II Ⅲ enzymes Ⅲ fibrosis Ⅲ hypertrophy A ngiotensin (Ang) II-related vascular effects are partially mediated by endothelin-1 (ET-1). Long-term Ang II infusion induces preproendothelin mRNA expression. 1 In rats transgenic for both the human renin and human angiotensinogen genes (dTGR), hypertension as well as severe heart and kidney damage develop, largely independent of blood pressure elevation. The rats die by age 7 weeks. 2 The ET-1 A and B (ETA/B) receptor blocker bosentan inhibits the activation of both nuclear factor-kappa B (NF-B) and transcription factor activator protein (AP)-1 in the kidney and the heart, independent of blood pressure reduction in these rats. 3 Bohlender et al 4 studied the same rat strain and showed that a specific ETA receptor blocker is effective, particularly when combined with an Ang II receptor blocker. ET-1 is a 21-amino acid peptide that was first isolated from porcine endothelial cells. 5 Two structurally related peptides differing by 2 (ET-2) and 6 (ET-3) amino acids were subsequently identified. The endothelin precursors are processed by 2 proteases that create mature active forms, termed preproendothelins. The preproendothelins are cleaved at dibasic sites by furin-like endopeptidases to produce inactive intermediates termed big endothelins. Big endothelins are cleaved to form the final products. A family of membrane-bound zinc metalloproteases from the neprilysin superfamily conducts the last...
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 (Ϫ20%) and LDL cholesterol (Ϫ29%). Apolipoprotein B (apoB) kinetic parameters were determined. Very low-density lipoprotein (VLDL) apoB pool size decreased 22% because of inhibition of VLDL production (Ϫ43%). 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. Key Words: oxidosqualene:lanosterol cyclase Ⅲ apoB kinetics Ⅲ LDL receptor Ⅲ ABCG5 Ⅲ ABCG8 L andmark trials using 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) revealed reductions in cardiovascular mortality and morbidity associated with low-density lipoprotein (LDL) cholesterol lowering. 1 Statins are well tolerated, although reductions in nonsterol intermediates in the cholesterol-synthesis pathway, such as isoprenoids and coenzyme Q, may theoretically be associated with adverse clinical events. 2,3 This has stimulated the development of compounds that inhibit cholesterol biosynthesis yet act distal to the synthesis of these nonsterol intermediates. 2,3-oxidosqualene:lanosterol cyclase (OSC; enzyme collection 5.4.99.7, also known as lanosterol synthase), a microsomal enzyme, represents a unique target for cholesterol-lowering drugs. 4,5 OSC is downstream of isoprenoid synthesis. Furthermore, compounds that decrease plasma concentrations of atherogenic lipoproteins by more than 1 mechanism are likely to be more efficacious.OSC catalyzes the highly selective cyclization of 2,3-monoepoxysqualene (MOS) to lanosterol, the first sterol to be formed. 4 OSC also catalyzes cyclization of 2,3;22,23-diepoxysqualene (DOS), which itself is derived from MOS, to 24(S),25-epoxylanosterol, the immediate precursor of 24(S),25-epoxycholesterol. Synthesis of 24(S),25-epoxycholesterol is favored over cholesterol synthesis under conditions of partial OSC inhibition, whereas complete OSC inhibition results in decreased synthesis of choleste...
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