GW9662

Kinase experiment ^[1]:

Preparation method

Human PPARα, PPARγ, and PPARδ ligand binding domains (LBDs) are expressed in E. coli as polyhistidine labeled fusion proteins.

In the experimental buffer, the desired receptor (15 nM) was added to the streptavitin modified SPA bead (0.5 mg/mL) pulp to fix the receptor to the SPA bead.

The mixture was equilibrated at room temperature for at least 1 hour, then the beads were precipitated at 1x10³g rotation speed, the supernatant was removed, the beads were gently mixed, the beads were suspended in fresh experimental buffer, the centrifugation/re-suspension procedure was repeated, the resulting acceptor coated bead slurry was used immediately, or stored at 4℃ for up to 1 week before use.

Competitive binding to PPARα,PPARγ, and PPARδ was determined using [³H]GW9662 as a radioligand.

Unless otherwise indicated, all experimental buffers were 50 mM HEPES (pH 7), 50 mM NaCl, 5 mM CHAPS, 0.1 mg/mL BSA, and 10 mM DTT.In some experiments, 50 mM Tris (pH 8) was used instead of HEPES(pH 7).

Applications

GW9662 is a potent and selective PPARγ antagonist with an IC₅₀ of 3.3 nM.

Cell experiment ^[2]:

Cell lines

J774A.1 cells

Preparation method

Cells were challenged with 50 ng/ml lipopolysaccharide with(or no) 10 μM GW9662 or vehicle for 18 h.Cell culture supernatant was collected, cells were lysed for RNA separation, or cells were lysed with nitric oxide synthase (NOS) buffer added with protease inhibitors, and NOS activity was measured.

Reaction Conditions

10 μM GW9662;18 h

Applications

Treatment with GW9662 significantly attenuated LPS-induced expression of Il1b, interleukin 6 (Il6) and inducible nitric oxide synthase (iNos) as well as NO₂- formation.

Animal experiment ^[3]:

Animal models

Male Wistar rats

Preparation method

Rats were pretreated with LPS (1 mg/kg, IP, 24 hours prior to ischemia) in the absence (control) or presence of the selective PPARγ antagonist GW9662(1 mg/kg, IP, 24 and 12 hours prior to ischemia). Twenty-four hours after injection of LPS, rats were subjected to 60 minutes of bilateral renal ischemia, followed by 6 hours of reperfusion.

Dosage form

1 mg/kg GW9662; IP;24 and 12 hours prior to ischemia

Applications

Administration of the selective PPARγ antagonist GW9662 24 and 12 hours before ischemia/reperfusion (I/R) eliminated the renal protection observed in LPS preconditioning.

References:

[1]. Leesnitzer LM, Parks DJ,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. doi: 10.1021/bi0159581. PMID: 12022867.

[2].Baumann A, Burger K, et,al. GW9662, a peroxisome proliferator-activated receptor gamma antagonist, attenuates the development of non-alcoholic fatty liver disease. Metabolism. 2022 Aug;133:155233. doi: 10.1016/j.metabol.2022.155233. Epub 2022 May 30. PMID: 35654114.

[3]. Collino M, Patel NS, 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. doi: 10.1111/j.1523-1755.2005.00430.x. PMID: 16014029.