PPARγ: (IC50 = 3.3 nM)
PPARα: (IC50 = 32 nM)
PPARδ: (IC50 = 2000 nM)

GW9662 has the ability to suppress radioligand binding to PPARγ, PPARα, and PPARδ, with corresponding pIC50 values of 8.48±0.27 (IC50=3.3 nM; n = 10), 7.49±0.17 (IC50=32 nM; n = 9), and 5.69±0.17 (IC50=2000 nM; n = 3). The binding studies with PPARα and PPARδ show that GW9662 is 10- and 600-fold less effective, respectively, with a nanomolar IC50 against PPARγ. GW9662 effectively and selectively blocks full-length PPARγ in cell-based reporter assays[1]. When paired with either 50 μM BRL 49653 (P=0.001) or 10 μM GW9662 (P=0.01) alone, co-treatment with both 50 μM BRL 49653 and 10 μM GW9662 leaves a significantly less number of viable cells after 7 days[2].

Both BADGE- and GW9662(1 mg/kg, ip)-treated mice had significantly greater bone marrow (BM) nucleated cell counts than the aplastic anemia (AA) group[3]. In rats, GW9662 (1 mg/kg, ip) significantly reduces the renoprotective benefits of lipopolysaccharide (LPS)[4].

Binding Assays. [1]
SPAs for all three PPARs were performed as previously described for PPARγ. In brief, the human PPARα, PPARγ, and PPARδ ligand binding domains (LBDs) were expressed in E. coli as polyhistidine-tagged fusion proteins. Receptors were immobilized on SPA beads (Amersham Pharmacia) by addition of the desired receptor (15 nM) to a slurry of streptavidin-modifed SPA beads (0.5 mg/mL) in assay buffer. The mixture was allowed to equilibrate for at least 1 h at room temperature, and the beads were pelleted by centrifugation at 1000g. The supernate was discarded, and the beads were resuspended in the original volume of fresh assay buffer with gentle mixing. The centrifugation/resuspension procedure was repeated, and the resulting slurry of receptor-coated beads was used immediately or stored at 4 °C for up to 1 week before use. [3H]GW2331, [3H]rosiglitazone, and [3H]GW2443 were used as radioligands for determination of competition binding to PPARα, PPARγ, and PPARδ, respectively. Unless otherwise indicated, the buffer used for all assays was 50 mM HEPES (pH 7), 50 mM NaCl, 5 mM CHAPS, 0.1 mg/mL BSA, and 10 mM DTT. For some experiments, the HEPES (pH 7) was replaced with 50 mM Tris (pH 8).

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Preparation of GW9662-Modified PPARγ for Mass Spectral Analysis. [1]
A stock solution of GW9662 in dimethyl sulfoxide was added to a 20 μM solution of PPARγ in 50 mM Tris (pH 8), 50 mM NaCl, 5 mM CHAPS, 0.1 mg/mL BSA, and 10 mM DTT. The final concentration of GW9662 was 40 μM. The solution was incubated at 4 °C followed by mass spectral analysis as described below.

Cell-Based Reporter Assays.[1]
The ability of GW9662 to activate PPAR-mediated reporter gene transcription was assessed using GAL4 chimeras of the human receptors and a (UAS)5-tk-SPAP reporter plasmid as previously described for PPARγ, PPARα, and PPARδ. GW9662 antagonism of ligand-induced gene transcription was measured as previously described. Antagonism of agonist-induced reporter gene transcription was done by titrating varying concentrations of GW9662 in the presence of a constant concentration of activating ligand. The activating ligands used were 100 nM rosiglitazone for PPARγ, 8 nM GW7647 for PPARα, and 0.55 μM GW2433 for PPARδ, respectively.
The effects of GW9662 on activation of PPARγ, PPARα, and PPARδ were also assessed using full-length human receptors and a reporter construct, (L-FABP)4-tk-Dual-LUC, containing four copies of the L-FABP PPRE upstream of the minimal herpesvirus thymidine kinase promoter and a luciferase reporter gene. The receptor plasmids contained the appropriate full-length PPAR cDNA plus 9 base pairs of Kozak consensus sequence cloned into the TOPO site of pcDNA3.1-TOPO. Briefly, HEK293 cells were cultured in Minimum Essential Media containing 10% fetal calf serum, 1% penicillin/streptomycin, and 1% fungizone in a humidified incubator (5% CO2 in air) at 37 °C. The cells were seeded at 2 × 104 cells per well in 96 well culture plates the day prior to assay execution. Transfection was accomplished using PolyFect according to the manufacturers' instructions. Transfection mixtures for each well contained 0.167 μg of PPAR plasmid, 0.167 μg of LFABP reporter, and 0.167 μg of a renilla luciferase plasmid as transfection control. Cells were incubated with the transfection mixture for 5 h before treatment with either compound or vehicle for 48 h. Culture plates were assayed using the Dual Luciferase assay system according to the manufacturers' instructions.

Treatment of mice[3]
PPARγ antagonists, BADGE or GW9662, were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. The aliquots were diluted with PBS to a final concentration of 10% DMSO and administrated by daily intraperitoneal injection at 30 mg/kg for BADGE, or at 1 mg/kg for GW9662, from one day prior to the experiment and continued for up to 2 weeks. In the FVB AA model, some mice were injected with cyclosporine A (CsA, 50 mg/kg/day) starting 1 hour after the LN injection, and continued for 5 days as immunosuppression. At the end of the experiments, the mice were euthanized by CO2 inhalation.
Methods, including peripheral blood (PB) and BM cell counting, flow cytometry, RNA isolation and gene expression analysis by PCR array, protein extraction and immunoblotting, cytokine measurement, histology using confocal microscopy, calcium flux assay and cell culture are detailed in the Online Supplementary Methods.

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Dissolved in 10% (v/v) DMSO; 1 mg/kg; i.p. injection

Male Wistar rats

[1]. Leesnitzer LM, et al. Functional consequences of cysteine modification in the ligand binding sites of peroxisome proliferator activated receptors by GW9662. Biochemistry. 2002 May 28;41(21):6640-50.
[2]. Seargent JM, et al. GW9662, a potent antagonist of PPARgamma, inhibits growth of breast tumor cells and promotes the anticancer effects of the PPARgamma agonist BRL 49653, independently of PPARgamma activation. Br J Pharmacol. 2004 Dec;143(8):933-7.
[3]. Sato K, et al. PPARγ antagonist attenuates mouse immune-mediated bone marrow failure by inhibition of T cell function.Haematologica. 2016 Jan;101(1):57-67.
[4]. Collino M, et al. The selective PPARgamma antagonist GW9662 reverses the protection of LPS in a model of renal ischemia-reperfusion. Kidney Int. 2005 Aug;68(2):529-36

In the course of a high throughput screen to search for ligands of peroxisome proliferator activated receptor-gamma (PPARgamma), we identified GW9662 using a competition binding assay against the human ligand binding domain. GW9662 had nanomolar IC(50) versus PPARgamma and was 10- and 600-fold less potent in binding experiments using PPARalpha and PPARdelta, respectively. Pretreatment of all three PPARs with GW9662 resulted in the irreversible loss of ligand binding as assessed by scintillation proximity assay. Incubation of PPAR with GW9662 resulted in a change in the absorbance spectra of the receptors consistent with covalent modification. Mass spectrometric analysis of the PPARgamma ligand binding domain treated with GW9662 established Cys(285) as the site of covalent modification. This cysteine is conserved among all three PPARs. In cell-based reporter assays, GW9662 was a potent and selective antagonist of full-length PPARgamma. The functional activity of GW9662 as an antagonist of PPARgamma was confirmed in an assay of adipocyte differentiation. GW9662 showed essentially no effect on transcription when tested using both full-length PPARdelta and PPARalpha. Time-resolved fluorescence assays of ligand-modulated receptor heterodimerization, coactivator binding, and corepressor binding were consistent with the effects observed in the reporter gene assays. Control activators increased PPAR:RXR heterodimer formation and coactivator binding to both PPARgamma and PPARdelta. Corepressor binding was decreased. In the case of PPARalpha, GW9662 treatment did not significantly increase heterodimerization and coactivator binding or decrease corepressor binding. The experimental data indicate that GW9662 modification of each of the three PPARs results in different functional consequences. The selective and irreversible nature of GW9662 treatment, and the observation that activity is maintained in cell culture experiments, suggests that this compound may be a useful tool for elucidation of the role of PPARgamma in biological processes.[1]

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Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, is activated by several compounds, including the thiazolidinediones. In addition to being a therapeutic target for obesity, hypolipidaemia and diabetes, perturbation of PPARgamma signalling is now believed to be a strategy for treatment of several cancers, including breast. Although differential expression of PPARgamma is observed in tumours compared to normal tissues and PPARgamma agonists have been shown to inhibit tumour cell growth and survival, the interdependence of these observations is unclear. This study demonstrated that the potent, irreversible and selective PPARgamma antagonist GW9662 prevented activation of PPARgamma and inhibited growth of human mammary tumour cell lines. Controversially, GW9662 prevented rosiglitazone-mediated PPARgamma activation, but enhanced rather than reversed rosiglitazone-induced growth inhibition. As such, these data support the existence of PPARgamma-independent pathways and question the central belief that PPARgamma ligands mediate their anticancer effects via activation of PPARgamma.[2]

C13H9CLN2O3

276.68

276.0301

C, 56.43; H, 3.28; Cl, 12.81; N, 10.13; O, 17.35

22978-25-2

GW9662-d5;2117730-84-2

644213

Typically exists as solids (or liquids in special cases) at room temperature

1.4±0.1 g/cm3

360.9±32.0 °C at 760 mmHg

171-175 °C

172.0±25.1 °C

0.0±0.8 mmHg at 25°C

1.676

2.76

74.92

O=C(NC1=CC=CC=C1)C2=CC([N+]([O-])=O)=CC=C2Cl

DNTSIBUQMRRYIU-UHFFFAOYSA-N

InChI=1S/C13H9ClN2O3/c14-12-7-6-10(16(18)19)8-11(12)13(17)15-9-4-2-1-3-5-9/h1-8H,(H,15,17)

2-Chloro-5-nitro- N -phenylbenzamide

GW-9662; GW 9662; GW9662;

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.04 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 (9.04 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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Solubility in Formulation 3: ≥ 0.5 mg/mL (1.81 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 4: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL

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