Name: GW4869
Brand: Selleck Chemicals
SKU: S7609
Rating: 5 (62 reviews)

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.

GW4869 Chemical Structure

CAS: 6823-69-4

Selleck's GW4869 has been cited by 62 publications

Purity & Quality Control

Batch: Purity: >97%

97

Products often used together with GW4869

Bortezomib

GW4869 increases cortical bone volume and sensitizes the myeloma cells to bortezomib, leading to a stronger anti-tumor response when used together.

Faict S, et al. Blood cancer journal 8.11 (2018): 105.

Doxorubicin

GW4869 and PEGylated liposomal doxorubicin (PLD) cotreatment significantly increases cytotoxic cell death in U937 cells.

Hekmatirad S, et al. Front Immunol. 2021 Jun 4;12:692654.

Bisindolylmaleimide I (GF109203X)

GW4869 and Bisindolylmaleimide I can significantly restore the expression of miR-16-5p in vitro and lead to malignant pleural mesothelioma (MPM) cell death.

Desage AL, et al. Cancers (Basel). 2021 Jun 26;13(13):3205.

Bevacizumab (anti-VEGF)

GW4869 inhibits production of extracellular vesicles (EVs) and increases the effects of bevacizumab on U87 glioblastoma (GBM) cells viability.

Simon T, et al. Mol Cancer. 2018 Aug 31;17(1):132.

LY294002

GW4869 and LY294002 combination use decreases HIV-1 sequestration in polarized tonsil epithelial cells.

Yasen A, et al. Virology. 2018 Feb;515:92-107.

GW4869 Related Products

Related Targets	PLA2 PLC PLD LYPLA1 Smase	Click to Expand
Related Compound Libraries	Autophagy Compound Library Apoptosis Compound Library Ferroptosis Compound Library Pyroptosis Compound Library Mitochondria-Targeted Compound Library	Click to Expand

Signaling Pathway

Phospholipase (e.g. PLA) Signaling Pathway

Choose Selective Phospholipase (e.g. PLA) Inhibitors

Cell Data

Cell Lines	Assay Type	Concentration	Incubation Time	Activity Description	PMID
MCF7	Function assay	10-20 μM		the addition of 10 μM GW4869 significantly inhibited TNF-induced SM hydrolysis, whereas 20 μM of the compound protected completely from the loss of sphingomyelin	12154098
OPM2	Proliferation assay	0, 1.25, 2.5, 5, 10, 20, 40 μmol/L	24 h	the cell proliferation rate was increased after GW4869 treatment. The inhibition of cell proliferation was only significant when the concentration of GW4869 was 40 μmol/L or higher.	28858294
RPMI-8226	Cell viability assay		72 h	cytotoxic to a panel of MM cell lines	28211573
NCI-H929	Cell viability assay		72 h	cytotoxic to a panel of MM cell lines	28211573
JJN3	Cell viability assay		72 h	cytotoxic to a panel of MM cell lines	28211573
U266	Cell viability assay		72 h	cytotoxic to a panel of MM cell lines	28211573
Daudi	Cell viability assay		72 h	not cytotoxic to non-MM cell lines or PBMCs	28211573
Jurkat	Cell viability assay		72 h	not cytotoxic to non-MM cell lines or PBMCs	28211573
K562	Cell viability assay		72 h	not cytotoxic to non-MM cell lines or PBMCs	28211573
PBMCs	Cell viability assay		72 h	not cytotoxic to non-MM cell lines or PBMCs	28211573
Click to View More Cell Line Experimental Data

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
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].
Cell Research	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.
Experimental Result Images	Methods	Biomarkers	Images	PMID
Experimental Result Images	Immunofluorescence	Nrf2		29794115

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

References

Chemical Information & Solubility

Molecular Weight	577.5	Formula	C₃₀H₂₈N₆O₂.₂HCl.XH₂O
CAS No.	6823-69-4	SDF	Download GW4869 SDF
Smiles	C1CN=C(N1)C2=CC=C(C=C2)NC(=O)C=CC3=CC=C(C=C3)C=CC(=O)NC4=CC=C(C=C4)C5=NCCN5.Cl.Cl
Storage (From the date of receipt)	3 years-20°Cpowder	Storage of Stock Solutions Aliquot the stock solutions to avoid repeated freeze-thaw cycles	1 year-80°Cin solvent
Storage (From the date of receipt)	3 years-20°Cpowder		1 month-20°Cin solvent

In vitro
Batch:

DMSO : 1 mg/mL ( (1.73 mM)； Moisture-absorbing DMSO reduces solubility. Please use fresh DMSO.)

Water : Insoluble

Ethanol : Insoluble

Molecular Weight Calculator

In vivo Batch: Add solvents to the product individually and in order.					In vivo Formulation Calculator
	Clear solution	5% DMSO 1 95% corn oil	0.05mg/ml (0.09mM)	Taking the 1 mL working solution as an example, add 50 μL of 1 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.	In vivo Formulation Calculator
Clear solution	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O	0.05mg/ml (0.09mM)	Taking the 1 mL working solution as an example, add 50 μL of 1 mg/ml clarified DMSO stock solution to 400 μL PEG300, mix evenly to clarify it; add 50 μL Tween80 to the above system, mix evenly to clarify it; then continue to add 500 μL ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.
Clear solution	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O	0.25mg/ml (0.43mM)	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.

Preparing Stock Solutions

Molarity Calculator

Dilution Calculator Molecular Weight Calculator

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

Dosage: mg/kg Average weight of animals: g Dosing volume per animal:μL Number of animals:

Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)

% DMSO + % + % Tween 80 + % ddH₂O

%DMSO+ %

Calculation results:

Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
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