GW4869

Catalog No.S7609 Batch:S760903

Technical Data

Formula

C₃₀H₂₈N₆O₂.₂HCl.XH₂O

Molecular Weight

577.5

CAS No.

6823-69-4

Solubility (25°C)*

In vitro

DMSO (warmed with 50ºC water bath)

3.25 mg/mL (5.62 mM)

Water

Insoluble

Ethanol

Insoluble

In vivo (Add solvents to the product individually and in order)

Clear solution	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O	0.25mg/ml	Taking the 1 mL working solution as an example, add 50 μL of clarified DMSO stock solution of 5 mg/ml to 400 μL of PEG300, mix evenly to clarify it; add 50 μL of Tween80 to the above system, mix evenly to clarify it; then continue to add 500 μL of ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.
Clear solution	5% DMSO 1 95% Corn oil	0.162mg/ml	Taking the 1 mL working solution as an example, add 50 μL of 3.25 mg/ml clear DMSO stock solution to 950 μL of corn oil and mix evenly. The mixed solution should be used immediately for optimal results.

* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
* Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description

GW4869 (GW69A, GW554869A) is a neutral, noncompetitive inhibitor of sphingomyelinase (SMase) with an IC50 of 1 μM. It is selective for N-SMase, and does not inhibit acid SMase at up to at least 150 μM, also is a commonly used exosome inhibitor.

Targets

SMase ^[1]
(Cell-free assay)

1 μM

In vitro

GW4869 inhibits N-SMase not only in vitro but also in a cellular model. GW4869 does not significantly impair TNF-induced NF-κB translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 is able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects are accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. At up to 150 μM, GW4869 does not inhibit the cloned human A-SMase. GW4869 shows no or minor inhibitory activity versus other hydrolytic enzymes, such as bacterial phosphatidylcholine-PLC and bovine protein phosphatase 2A, and it shows significantly higher activity versus the rat brain enzyme compared with the human lyso-PAF PLC^[1].

In vivo

Systemic administration of GW4869 does not alter the ceramide or sphingomylein content of liver, heart or skeletal muscle but does decrease the ceramide content and increase the sphingomyelin content in brain. Inhibition of nSMase2 with GW4869 slowed learning. Mice administered GW4869 do not progressively decrease latency to locate the hidden platform with repeated training trials, suggesting that they has difficulty learning to use spatial cues to navigate the pool^[2]. Intraperitoneal injection of GW4869 reduces the levels of brain and serum exosomes, brain ceramide, and Aβ1-42 plaque load. GW4869 reduces amyloid plaque formation in vivo by preventing exosome secretion. GW4869 may have a low toxicity at levels needed to achieve a biological effect from nSMase2 inhibition^[3].

Protocol (from reference)

Cell Assay:^{^[1]}	Cell lines MCF7 cells Concentrations 10-20 μM Incubation Time 30 min Method MCF7 human breast cancer cells are routinely cultured in RPMI 1640 containing 10% FBS at 37 °C in 5% CO2. Unless otherwise indicated, for treatment, cells are seeded at 1.7 × 10⁶ cells/10-cm culture dish in 10 ml of complete medium; after 24 h, the medium is replaced with 7 ml of RPMI 1640 containing 2% FBS and 25 mM Hepes, pH 7.5, and the cells are rested for 2 h prior to treatment. GW4869 is routinely stored at −80 °C as a 1.5 mM stock suspension in Me2SO. Right before use, the suspension is solubilized by the addition of 5% methane sulfonic acid (MSA) (2.5 μl of 5% MSA in sterile double-distilled H2O are added to 50 μl of GW4869 stock suspension; therefore, the concentration of the GW4869 stock solution at the time of the experiments is 1.43 mM). The suspension is mixed and warmed up at 37 °C until clear. Cells are preincubated with the inhibitor for 30 min prior to treatment with TNF. Control cells are treated with Me2SO containing 5% MSA, similarly to the samples receiving the GW4869 solution. When different doses of GW4869 are tested, amounts of vehicle solution are added in order to equal the volume of GW4869 used for the highest dose.
Animal Study:^{^[2]}	Animal Models Mice with a complete loss of nSMase2 activity (background: C57BL/6J mice) Dosages 1.25 mg/kg Administration i.p.

Cell Assay:^{^[1]}

Cell lines
MCF7 cells
Concentrations
10-20 μM
Incubation Time
30 min
Method
MCF7 human breast cancer cells are routinely cultured in RPMI 1640 containing 10% FBS at 37 °C in 5% CO2. Unless otherwise indicated, for treatment, cells are seeded at 1.7 × 10⁶ cells/10-cm culture dish in 10 ml of complete medium; after 24 h, the medium is replaced with 7 ml of RPMI 1640 containing 2% FBS and 25 mM Hepes, pH 7.5, and the cells are rested for 2 h prior to treatment. GW4869 is routinely stored at −80 °C as a 1.5 mM stock suspension in Me2SO. Right before use, the suspension is solubilized by the addition of 5% methane sulfonic acid (MSA) (2.5 μl of 5% MSA in sterile double-distilled H2O are added to 50 μl of GW4869 stock suspension; therefore, the concentration of the GW4869 stock solution at the time of the experiments is 1.43 mM). The suspension is mixed and warmed up at 37 °C until clear. Cells are preincubated with the inhibitor for 30 min prior to treatment with TNF. Control cells are treated with Me2SO containing 5% MSA, similarly to the samples receiving the GW4869 solution. When different doses of GW4869 are tested, amounts of vehicle solution are added in order to equal the volume of GW4869 used for the highest dose.

Animal Study:^{^[2]}

Animal Models
Mice with a complete loss of nSMase2 activity (background: C57BL/6J mice)
Dosages
1.25 mg/kg
Administration
i.p.

References

Customer Product Validation

: Data from [Data independently produced by , , Cancer Lett, 2018, 433:210-220]

Selleck's GW4869 has been cited by 80 publications

GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia [ Signal Transduct Target Ther, 2024, 9(1):253]	PubMed: 39327432
Synergistic Viro-chemoimmunotherapy in Breast Cancer Enabled by Bioengineered Immunostimulatory Exosomes and Dual-Targeted Coxsackievirus B3 [ ACS Nano, 2024, 18(5):4241-4255]	PubMed: 38278522
Targeting exosomal double-stranded RNA-TLR3 signaling pathway attenuates morphine tolerance and hyperalgesia [ Cell Rep Med, 2024, 5(10):101782]	PubMed: 39413734
Cold exposure-induced plasma exosomes impair bone mass by inhibiting autophagy [ J Nanobiotechnology, 2024, 22(1):361]	PubMed: 38910236
Epithelium-derived exosomes promote silica nanoparticles-induced pulmonary fibroblast activation and collagen deposition via modulating fibrotic signaling pathways and their epigenetic regulations [ J Nanobiotechnology, 2024, 22(1):331]	PubMed: 38867284
Aberrant regulation of serine metabolism drives extracellular vesicle release and cancer progression [ Cell Rep, 2024, 43(8):114517]	PubMed: 39024098
Exosomal circRHCG promotes breast cancer metastasis via facilitating M2 polarization through TFEB ubiquitination and degradation [ NPJ Precis Oncol, 2024, 8(1):22]	PubMed: 38287113
Prophages divert Staphylococcus aureus defenses against host lipids [ J Lipid Res, 2024, S0022-2275(24)00198-6]	PubMed: 39505263
Aberrant NSUN2-mediated m5C modification of exosomal LncRNA MALAT1 induced RANKL-mediated bone destruction in multiple myeloma [ Commun Biol, 2024, 7(1):1249]	PubMed: 39358426
Alpha-hederin reprograms multi-miRNAs activity and overcome small extracellular vesicles-mediated paclitaxel resistance in NSCLC [ Front Pharmacol, 2024, 15:1257941]	PubMed: 38362150

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SHIPPING AND STORAGE
Selleck products are transported at room temperature. If you receive the product at room temperature, please rest assured, the Selleck Quality Inspection Department has conducted experiments to verify that the normal temperature placement of one month will not affect the biological activity of powder products. After collecting, please store the product according to the requirements described in the datasheet. Most Selleck products are stable under the recommended conditions.

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