Background
GW9662 is a specific inhibitor of peroxisome proliferator activated receptor-gamma (PPAR-gamma) with an IC50 of 3.3 nM, GW9662 acts 10 to 600 times more selectively on PPARγ in cells than on PPARα and PPARδ[1-3].
Treatment with GW9662 significantly attenuated LPS-induced expression of Il1b, interleukin 6 (Il6) and inducible nitric oxide synthase (iNos) as well as NO2-formation in J774A.1 cells [4]. DMBA-induced mammary alveolar lesions (MAL) were significantly inhibited by PPARγ antagonist GW9662[5]. GW9662(2.5/5 μM) prevented disruption of the Colon carcinoma cells cycle induced by resveratrol and consequently abrogated resveratrol-induced apoptosis[6].
GW9662(p.o;3 mg/kg/day ;2 weeks) worsened the brain injury in alcohol-fed reperfusion mice[7]. Pretreatment with LPS significantly attenuated all markers of renal injury and dysfunction caused by I/R. Most notably, GW9662(1 mg/kg GW9662; IP;24 and 12 hours prior to ischemia) abolished the protective effects of LPS[8].
GW9662是一种特异性的过氧化物酶体增殖物激活受体-γ(PPAR-γ)抑制剂,其IC50为3.3 nM。在细胞中,GW9662相较于PPARα和PPARδ选择性地作用于PPARγ,选择性作用范围为10至600倍[1-3]。
使用GW9662可显著减轻LPS诱导的J774A.1细胞中Il1b、白细胞介素6(Il6)和诱导型一氧化氮合酶(iNos)的表达以及NO2-生成[4]。使用GW9662可显著抑制DMBA诱导的乳腺腺泡病变(MAL)[5]。GW9662(2.5/5μM)防止了白藜芦醇引起的结肠癌细胞周期紊乱,抵消引起的细胞凋亡[6]。
GW9662(p.o;3 mg/kg/day ;2 weeks)加剧了饮酒再灌注小鼠的脑损伤[7]。LPS预处理可显著减弱I/R引起的肾损伤和功能障碍的所有标志物。GW9662(1 mg/kg GW9662; IP;24 and 12 hours prior to ischemia)使LPS的保护效果消失[8]。
参考文献:
[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]. Lea MA, Sura M, et,al. Inhibition of cell proliferation by potential peroxisome proliferator-activated receptor (PPAR) gamma agonists and antagonists. Anticancer Res. 2004 Sep-Oct;24(5A):2765-71. PMID: 15517883.
[3]. Nielsen R, Gr?ntved L, et,al. Peroxisome proliferator-activated receptor subtype- and cell-type-specific activation of genomic target genes upon adenoviral transgene delivery. Mol Cell Biol. 2006 Aug;26(15):5698-714. doi: 10.1128/MCB.02266-05. PMID: 16847324; PMCID: PMC1592764.
[4]. 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.
[5]. Mehta RG, Peng X, et,al. PPARγ antagonist GW9662 induces functional estrogen receptor in mouse mammary organ culture: potential translational significance. Mol Cell Biochem. 2013 Jan;372(1-2):249-56. doi: 10.1007/s11010-012-1466-9. Epub 2012 Sep 24. PMID: 23001870.
[6]. Aires V, Brassart B, et,al. A role for peroxisome proliferator-activated receptor gamma in resveratrol-induced colon cancer cell apoptosis. Mol Nutr Food Res. 2014 Sep;58(9):1785-94. doi: 10.1002/mnfr.201300962. Epub 2014 Jun 30. PMID: 24975132.
[7]. Sun H, Xiong W, et,al. Low-dose alcohol consumption protects against transient focal cerebral ischemia in mice: possible role of PPARγ. PLoS One. 2012;7(7):e41716. doi: 10.1371/journal.pone.0041716. Epub 2012 Jul 27. PMID: 22848576; PMCID: PMC3407212.
[8]. 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.
Protocol
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Kinase experiment [1]:
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Preparation method
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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).
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Applications
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GW9662 is a potent and selective PPARγ antagonist with an IC50 of 3.3 nM.
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Cell experiment [2]:
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Cell lines
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J774A.1 cells
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Preparation method
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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.
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Reaction Conditions
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10 μM GW9662;18 h
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Applications
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Treatment with GW9662 significantly attenuated LPS-induced expression of Il1b, interleukin 6 (Il6) and inducible nitric oxide synthase (iNos) as well as NO2- formation.
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Animal experiment [3]:
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Animal models
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Male Wistar rats
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Preparation method
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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.
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Dosage form
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1 mg/kg GW9662; IP;24 and 12 hours prior to ischemia
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Applications
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Administration of the selective PPARγ antagonist GW9662 24 and 12 hours before ischemia/reperfusion (I/R) eliminated the renal protection observed in LPS preconditioning.
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参考文献:
[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.
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