GW9662

GW9662 binds to Cys(285) on PPARgamma which is conserved among all three PPARs. GW9662 acts as an antagonist of PPARgamma which is confirmed in an assay of adipocyte differentiation inhibition. ^[1] GW9662 prevents activation of PPARγ and inhibits growth of human mammary tumour cell lines (MCF7, MDA-MB-468, MDA-MB-231) with IC50 of 20 μM-30 μM, suggesting either the existence of PPARγ agonistic properties of GW9662 or growth-inhibitory mechanisms independent of PPARγ. Co-treatment with GW9662 (10 μM) results in statistically lower viable cell numbers after 7 days in MDA-MB-231 cells. ^[2] PPARγ1 ligands could suppress RANKL-induced osteoclast formation in primary murine myeloid (BMs) and RAW264.7 cells. Importantly, suppression by these ligands is reversed in a concentration-dependent fashion with GW 9662 (2 μM). GW 9662 (2 μM) blocks IL-4 suppression of osteoclast formation in BMs. GW 9662 (1 μM) blocks RANKL activation of NF-κB in RAW264.7 cells. ^[3] GW9662 (10 μM) inhibits hormone- and agonist-induced adipogenesis of primary preadipocytes from patients with thyroid eye disease. ^[4]

Pretreatment with LPS (1 mg/kg i.p.) significantly attenuates all markers of renal injury and dysfunction caused by ischemia/reperfusion (I/R) injury in rats. Most notably, GW9662 (1 mg/kg i.p.) abolishes the protective effects of LPS. ^[5]

• Binding assay

  The human PPARα, PPARγ, and PPARδ ligand binding domains (LBDs) are expressed in E. coli as polyhistidine-tagged fusion proteins. Receptors are immobilized on SPA beads 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 is allowed to equilibrate for at least 1 hour at room temperature, and the beads are pelleted by centrifugation at 1×10³ g. The supernate is discarded, and the beads are resuspended in the original volume of fresh assay buffer with gentle mixing. The centrifugation/resuspension procedure is repeated, and the resulting slurry of receptor-coated beads is used immediately or stored at 4 ℃ for up to 1 week before use. [3H]GW2443 are used as radioligands for determination of competition binding to PPARα, PPARγ, and PPARδ, respectively. Unless otherwise indicated, the buffer used for all assays is 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) is replaced with 50 mM Tris (pH 8).

• Cell lines

  MDA-MB-231 cells

• Concentrations

  10 μM

• Incubation Time

  10 days

• Method

  MDA-MB-231 cells are seeded at a density of 1 × 10⁵ cells per 25 cm³ tissue culture flask. After 24 h (day 0), the growth medium is replaced with fresh medium containing GW9662 (10 μM) or both together. Control flasks receives 0.1% DMSO. Cells are harvested on days 0, 3, 5, 7, 10 for each treatment condition by trypsinisation, stained using trypan blue, and the total and viable number of cells per flask calculates using a haemocytometer.

• Animal Models

  male Wistar rats

• Dosages

  1 mg/kg

• Administration

  intraperitoneal injection