SE/squalene epoxidase
NB-598 Maleate (10 μM) decreases total cholesterol levels in MIN6 cells by 36±7%. NB-598 Maleate significantly lowered cholesterol in the PM, ER, and SG by 49±2%, 46±7%, and 48±2%, respectively. NB598 at concentrations up to 10 μM did not influence the acute outward KV of the current or the voltage dependency of the enhanced activation. , but it will be removed by current [1]. NB-598 maleic acid (10 μM) inhibits the production of sterols and sterol esters from [14C]acetic acid without influencing the synthesis of other electrolytes such as phospholipids (PL), free ions (FFA), and triglycerides (TG). In the absence of exogenous electrolyte phospholipids, NB-598 maleic acid can lower ACAT activity by 31% in the presence of 600 PM sleep in vivo, and NB-598 maleic acid staining can reduce ACAT activity by 22% [2]. NB-598 maleic acid colors HepG evenly 2 cells into sleep and sleep body triglycerides [3].

NB-598 Maleate (10 μM) decreases total cholesterol levels in MIN6 cells by 36±7%. NB-598 Maleate significantly lowered cholesterol in the PM, ER, and SG by 49±2%, 46±7%, and 48±2%, respectively. NB598 at concentrations up to 10 μM did not influence the acute outward KV of the current or the voltage dependency of the enhanced activation. , but it will be removed by current [1]. NB-598 maleic acid (10 μM) inhibits the production of sterols and sterol esters from [14C]acetic acid without influencing the synthesis of other electrolytes such as phospholipids (PL), free ions (FFA), and triglycerides (TG). In the absence of exogenous electrolyte phospholipids, NB-598 maleic acid can lower ACAT activity by 31% in the presence of 600 PM sleep in vivo, and NB-598 maleic acid staining can reduce ACAT activity by 22% [2]. NB-598 maleic acid colors HepG evenly 2 cells into sleep and sleep body triglycerides [3].

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NB-598 (10 μM, 48 h incubation) significantly decreased total cholesterol levels by 36% in MIN6 β-cells, 40% in mouse pancreatic islets, and 52% in human pancreatic islets compared to controls.
In MIN6 cells, cholesterol reduction was also observed in subcellular compartments: plasma membrane (49%), endoplasmic reticulum (46%), and insulin secretory granules (48%).
NB-598 dose-dependently inhibited both basal (1 mM glucose) and glucose-stimulated (16.7 mM glucose) insulin secretion from mouse pancreatic islets, with 10 μM reducing glucose-stimulated secretion by 75%.
Total insulin content of islets was not affected by NB-598 treatment.
Electron microscopy showed no change in insulin granule morphology after NB-598 treatment.
NB-598 (10 μM, 48 h) markedly inhibited voltage-gated Ca²⁺ (Caᵥ) channel currents in mouse β-cells, reducing peak current amplitude at +10 mV from −13.3 pA/pF (control) to −2.1 pA/pF.
NB-598 increased the inactivation of voltage-gated K⁺ (Kᵥ) channels without affecting peak current density or activation voltage dependence.
NB-598 also decreased Kₐₜₚ current density by 57% at −140 mV.
In single β-cells, NB-598 impaired exocytosis independently of Caᵥ channels, as shown by reduced slow burst and sustained components of membrane capacitance increases following photolytic Ca²⁺ uncaging.
Protein expression of Caᵥ1.2, Kᵥ2.1, and SNARE proteins (syntaxin 1A, SNAP-25, VAMP-2) was not altered by NB-598, but these proteins were redistributed out of cholesterol-rich membrane raft fractions.
The effects of NB-598 on insulin secretion, Caᵥ currents, Kᵥ inactivation, and exocytosis were partially or fully reversed by cholesterol repletion with soluble cholesterol. [1]

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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MIN6 cells were cultured in DMEM with 25 mM glucose and 10% fetal bovine serum, passaged every 4–5 days. For cholesterol inhibition, cells were incubated with 10 μM NB-598 in medium supplemented with 10% delipidated FBS for 48 hours.
Mouse and human pancreatic islets were isolated by collagenase digestion and cultured in RPMI 1640 medium with 11 mM glucose and 10% FBS. Islets were treated with NB-598 in delipidated FBS medium for 48 hours.
Total cholesterol was extracted from cells or islets using a chloroform-methanol mixture, dried, and dissolved in immunoprecipitation buffer. Cholesterol content was measured using a fluorescence assay kit.
For insulin secretion, mouse islets were pre-incubated in low glucose (1 mM) Krebs-Ringer bicarbonate buffer, then stimulated with high glucose (16.7 mM) for 1 hour. Secreted insulin in supernatant was measured by radioimmunoassay and normalized to total islet insulin content.
For electrophysiology, dispersed islet cells from transgenic mice expressing GFP under the mouse insulin promoter were used. Whole-cell patch clamp was performed to record Caᵥ, Kᵥ, and Kₐₜₚ currents.
For exocytosis measurement independent of Caᵥ channels, β-cells were loaded with caged Ca²⁺ (NP-EGTA) via patch pipette. Exocytosis was triggered by UV flash photolysis, and membrane capacitance changes were recorded.
For membrane raft isolation, MIN6 cells were lysed in cold Triton X-100-containing buffer, and lysates were subjected to sucrose gradient ultracentrifugation. Fractions were collected and analyzed by Western blot for ion channels and SNARE proteins. [1]

[1]. Xia F, et al. Inhibition of cholesterol biosynthesis impairs 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.

NB-598 is a squalene cyclooxygenase inhibitor used for long-term inhibition of endogenous cholesterol biosynthesis in pancreatic β-cells. Chronic cholesterol depletion inhibits Ca²⁺ channel function and disrupts exocytosis mechanisms, thereby impairing insulin secretion, possibly due to the redistribution of ion channels and SNARE proteins in cholesterol-rich membrane raft microdomains. Cellular cholesterol dysregulation may contribute to β-cell dysfunction and the pathogenesis of type 2 diabetes. [1]

C₃₁H₃₅NO₅S₂

565.74

565.196

155294-62-5

NB-598;131060-14-5;NB-598 hydrochloride;136719-25-0

71576679

Typically exists as solid at room temperature

7.195

2

8

12

39

747

0

O=C(O)/C=C\C(O)=O.CC(C)(C)C#C/C=C/CN(CC)CC1=CC(OCC2=CC(C3=CSC=C3)=CS2)=CC=C1

QGUWPAYJKVJWEK-JVTXGDFZSA-N

InChI=1S/C27H31NOS2.C4H4O4/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;5-3(6)1-2-4(7)8/h6,8-12,15-17,20-21H,5,14,18-19H2,1-4H3;1-2H,(H,5,6)(H,7,8)/b8-6+;2-1-

(Z)-but-2-enedioic acid;(E)-N-ethyl-6,6-dimethyl-N-[[3-[(4-thiophen-3-ylthiophen-2-yl)methoxy]phenyl]methyl]hept-2-en-4-yn-1-amine

NB598; NB 598; NB-598; NB-598 (Maleate); NB-598 Maleate; 155294-62-5; 136719-26-1; (Z)-but-2-enedioic acid;(E)-N-ethyl-6,6-dimethyl-N-[[3-[(4-thiophen-3-ylthiophen-2-yl)methoxy]phenyl]methyl]hept-2-en-4-yn-1-amine; NB598 Maleate salt; NB-598xMaleicacidsalt; NB-598 xMaleic acid salt;

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 : ~100 mg/mL (~176.76 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (4.42 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 ultrasonication.
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 3: ≥ 2.5 mg/mL (4.42 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.)