SE/squalene epoxidase

In MIN6 cells, NB598 (10 μM) decreased total cholesterol levels by 36±7%. In PM, ER, and SG, NB598 dramatically decreased cholesterol by 49±2%, 46±7%, and 48±2%, respectively. Under both basal (1 mM glucose) and glucose-stimulated (16.7 mM glucose) conditions, NB598 dose-dependently decreases insulin secretion. At doses up to 10 μM, NB598 enhances current deactivation but has no effect on peak outward KV current or the voltage dependency of activation [1]. The synthesis of sterols and sterol esters from [14C]acetate is inhibited by NB-598 (10 μM), but other lipids including phospholipids (PL), free fatty acids (FFA), and triacylglycerols (TG) are not affected. NB-598 decreased ACAT activity by 31% when exogenous liposomal cholesterol was not present. Even at 600 PM liposomal cholesterol concentration, NB-598 caused a 22% reduction in ACAT activity[2]. HepG2 cells' release of triacylglycerol and cholesterol into the culture medium is inhibited by NB-598 [3].

NB-598, (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3'-bith iophen-5-yl)methoxy]benzene-methanamine, was found to inhibit human microsomal squalene epoxidase (from Hep G2 cells) in a competitive manner. NB-598 inhibited cholesterol synthesis from [14C]acetate dose dependently in Hep G2 cells and increased the intracellular radioactivity of squalene. A single oral administration of NB-598 inhibited cholesterol synthesis from [14C]acetate in rats. Moreover, multiple oral administration of NB-598 to dogs decreased serum total and low density lipoprotein cholesterol levels and increased serum squalene levels. After termination of treatment, the reduced serum cholesterol and increased squalene levels returned to their control values[4].

NB-598, a specific inhibitor of squalene epoxidase, suppressed the secretion of cholesterol and triacylglycerol from HepG2 cells into the medium. L-654,969, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibited the secretion of cholesterol as potently as NB-598, but did not suppress the secretion of triacylglycerol. Both compounds decreased the intracellular cholesterol content almost equally, and neither of the compounds reduced the intracellular triacylglycerol content. The suppression of lipid secretion by NB-598 was associated with a significant reduction in apolipoprotein (apo) B secretion into the medium. Therefore, the suppression of lipid secretion by NB-598 may be caused by a reduction in the number of triacylglycerol-rich lipoprotein particles. In contrast, the suppression of cholesterol secretion by L-654,969 may be due to a modulation of lipoprotein lipid composition, since this agent did not reduce the secretion of apo B or triacylglycerol. The secretion of apo A-I was unaffected by either NB-598 or L-654,969. Pulse chase studies using [35S]methionine showed that the suppression of apo B secretion by NB-598 depended on an enhancement of intracellular degradation of apo B. These results indicate that the secretion of apo B from HepG2 cells is not regulated by the lipid synthesis alone, and suggest that the mechanism of the hypolipidemic effect of NB-598 involves the suppression of triacylglycerol-rich lipoprotein secretion from the liver as well as an inhibition of cholesterol synthesis in the liver[3].

Subcellular fractionation of plasma membranes, endoplasmic reticulum, and insulin secretory granules[1]
MIN6 cells (4 × 108) were cultured for 48 h at 37 C in the culture medium supplemented with 10% delipidated FBS, in the absence or presence of 10 μM NB598. The cells were harvested and homogenized in fractionation buffers: 50 mM 2-(N-morpholino) ethane sulfonic acid (MES), 250 mM sucrose (pH 7.2) for plasma membrane (PM) and endoplasmic reticulum (ER); 10 mM 3[N-morholino]propanesulfonic acid-Tris, 270 mM sucrose (pH 6.8) for insulin secretory granules (SG). Fractionations for PM and ER were performed by sucrose density gradient ultracentrifugation established by Ramanadham et al. Insulin secretory granules were fractionated with Histodenz gradient ultracentrifugation followed by Percoll purification, as established by Brunner et al. The isolated subcellular fractions were stored at −20 C for protein concentration determination and cholesterol extraction.

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Cholesterol content assay[1]
MIN6 cells (5 × 105) or 20 pancreatic islets from mouse or human were cultured for 48 h at 37 C in the relative culture media supplemented with 10% delipidated FBS, in the absence or presence of 10 μM cholesterol biosynthesis inhibitor NB598. Cells and islets were collected and washed with PBS. Cholesterol was extracted by adding 50 μl of 2:1 chloroform-methanol mixture, followed by 100 μl of PBS. To extract cholesterol from subcellular fractions, 50 μl of 2:1 chloroform-methanol mixture was added to different compartments. The top water phase was removed after centrifugation for 3 min at 10,000 rpm. Cholesterol sample was dried and dissolved in 10–40 μl of immunoprecipitation buffer containing (in millimoles) 150 NaCl, 20 Tris-HCl, 5 MgSO4, 1 EDTA, 1 EGTA, and 1% Triton X-100. Cholesterol content was measured using a fluorescence assay kit, following the manufacturer’s instructions.

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Caco-2 cells grown on membrane filters were used as a model to study the effects of NB-598, an inhibitor of squalene epoxidase, on cholesterol absorption from the intestinal epithelia. NB-598 (10 microM) inhibited the synthesis of sterol and sterol ester from [14C]acetate without affecting the synthesis of other lipids such as phospholipids (PL), free fatty acids (FFA) and triacylglycerol (TG). When labeled lipid was apically loaded as a micellar lipid solution into Caco-2 cell cultures, NB-598 reduced basolaterally secreted radioactivity in cholesterol, cholesterol ester, PL and TG. Furthermore, NB-598 suppressed the basolateral secretion of apolipoprotein (apo) B. When microsomes prepared from control Caco-2 cells were incubated with 10 microM NB-598, acyl CoA:cholesterol acyltransferase (ACAT) activity was inhibited slightly. After incubating Caco-2 cells with 10 microM NB-598, a slight reduction in cellular ACAT activity was also observed. These results suggest that suppression of the secretion of particles containing apo B and reduction of cellular ACAT activity in the intestinal epithelia are part of the mechanism of the cholesterol-lowering effect of NB-598[2].

[1]. Xia F, et al. Inhibition of cholesterol biosynthesis impairs insulin secretion and voltage-gated calcium channel function in pancreatic beta-cells. Endocrinology. 2008 Oct;149(10):5136-45.
[2]. Horie M, et al. Effects of NB-598, a potent squalene epoxidase inhibitor, on the apical membrane uptake of cholesterol and basolateral membrane secretion of lipids in Caco-2 cells. Biochem Pharmacol. 1993 Jul 20;46(2):297-305.
[3]. Horie M, et al. An inhibitor of squalene epoxidase, NB-598, suppresses the secretion of cholesterol and triacylglycerol and simultaneously reduces apolipoprotein B in HepG2 cells. Biochim Biophys Acta. 1993 May 20;1168(1):45-51.
[4]. NB-598: a potent competitive inhibitor of squalene epoxidase. J Biol Chem . 1990 Oct 25;265(30):18075-8.

Insulin secretion from pancreatic beta-cells is mediated by the opening of voltage-gated Ca2+ channels (CaV) and exocytosis of insulin dense core vesicles facilitated by the secretory soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein machinery. We previously observed that beta-cell exocytosis is sensitive to the acute removal of membrane cholesterol. However, less is known about the chronic changes in endogenous cholesterol and its biosynthesis in regulating beta-cell stimulus-secretion coupling. We examined the effects of inhibiting endogenous beta-cell cholesterol biosynthesis by using the squalene epoxidase inhibitor, NB598. The expression of squalene epoxidase in primary and clonal beta-cells was confirmed by RT-PCR. Cholesterol reduction of 36-52% was observed in MIN6 cells, mouse and human pancreatic islets after a 48-h incubation with 10 mum NB598. A similar reduction in cholesterol was observed in the subcellular compartments of MIN6 cells. We found NB598 significantly inhibited both basal and glucose-stimulated insulin secretion from mouse pancreatic islets. CaV channels were markedly inhibited by NB598. Rapid photolytic release of intracellular caged Ca2+ and simultaneous measurements of the changes in membrane capacitance revealed that NB598 also inhibited exocytosis independently from CaV channels. These effects were reversed by cholesterol repletion. Our results indicate that endogenous cholesterol in pancreatic beta-cells plays a critical role in regulating insulin secretion. Moreover, chronic inhibition of cholesterol biosynthesis regulates the functional activity of CaV channels and insulin secretory granule mobilization and membrane fusion. Dysregulation of cellular cholesterol may cause impairment of beta-cell function, a possible pathogenesis leading to the development of type 2 diabetes. Endocrinology . 2008 Oct;149(10):5136-45.

C27H32CLNOS2

486.13

449.185

C, 72.12; H, 6.95; N, 3.11; O, 3.56; S, 14.26

131060-14-5

NB-598 Maleate;155294-62-5;NB-598 hydrochloride;136719-25-0

6443223

Off-white to light yellow solid powder

1.122g/cm3

530.7ºC at 760mmHg

-60ºC

274.8ºC

2.4E-11mmHg at 25°C

1.596

7.483

0

4

10

31

628

0

CCN(C/C=C/C#CC(C)(C)C)CC1=CC=CC(OCC2=CC(C3=CSC=C3)=CS2)=C1

WDXQLZXORYGXJN-WVLIHFOGSA-N

InChI=1S/C27H31NOS2.ClH/c1-5-28(14-8-6-7-13-27(2,3)4)18-22-10-9-11-25(16-22)29-19-26-17-24(21-31-26)23-12-15-30-20-23/h6,8-12,15-17,20-21H,5,14,18-19H2,1-4H31H/b8-6+ SMILES

(E)-N-ethyl-6,6-dimethyl-N-[[3-[(4-thiophen-3-ylthiophen-2-yl)methoxy]phenyl]methyl]hept-2-en-4-yn-1-aminehydrochloride

NB-598; NB 598; CHEMBL27885; Benzenemethanamine, 3-([3,3'-bithiophen]-5-ylmethoxy)-N-[(2E)-6,6-dimethyl-2-hepten-4-yn-1-yl]-N-ethyl-; (E)-N-(3-([3,3'-Bithiophen]-5-ylmethoxy)benzyl)-N-ethyl-6,6-dimethylhept-2-en-4-yn-1-amine; (E)-N-ethyl-6,6-dimethyl-N-[[3-[(4-thiophen-3-ylthiophen-2-yl)methoxy]phenyl]methyl]hept-2-en-4-yn-1-amine; (E)-3-[(3,3'-Bithiophen)-5-ylmethoxy]-N-(6,6-dimethyl-2-hepten-4-ynyl)-N-ethylbenzylamine; NB598.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~111.19 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.56 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.56 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)