T0070907

T0070907 is a potent and selective PPARγ antagonist. With an apparent binding affinity (concentration at 50% inhibition of [³H] rosiglitazone binding or IC50) of 1 nM, T0070907 covalently modifies PPARγ on cysteine 313 in helix 3 of human PPARγ2. T0070907 blocks PPARγ function in both cell-based reporter gene and adipocyte differentiation assays. Consistent with its role as an antagonist of PPARγ, T0070907 blocks agonist-induced recruitment of coactivator-derived peptides to PPARγ in a homogeneous time-resolved fluorescence-based assay and promotes recruitment of the transcriptional corepressor NCoR to PPARγ in both glutathione S-transferase pull-down assays and a PPARγ/retinoid X receptor (RXR) α-dependent gel shift assay. Studies with mutant receptors suggest that T0070907 modulates the interaction of PPARγ with these cofactor proteins by affecting the conformation of helix 12 of the PPARγ ligand-binding domain. Interestingly, whereas the T0070907-induced NCoR recruitment to PPARγ/RXRα heterodimer can be almost completely reversed by the simultaneous treatment with RXRα agonist LGD1069, T0070907 treatment has only modest effects on LGD1069-induced coactivator recruitment to the PPARγ/RXRα heterodimer. ^[1] T0070907 treatment inhibits proliferation, invasion and migration but does not significantly affect apoptosis. Molecular inhibition using a dominant negative (Δ462) receptor yields similar results. T007 also mediates a dose-dependent decrease in phosphorylation of PPARγ, and its ability to bind to DNA, and may directly affect mitogen-activated protein kinase signaling. ^[2]

Lipopolysaccharide preconditioning significantly attenuates the development of renal dysfunction, hepatocellular injury, and circulatory failure as well as the increase in the plasma levels of interleukin-1 [beta] caused by severe endotoxemia. T0070907 can attenuate all of these beneficial effects afforded by preconditioning with lipopolysaccharide ^[3]

• Ligand Binding Assay

  To determine the binding affinity of T0070907 to the PPARs, scintillation proximity assay (SPA) is performed with the following modifications. A 90-μl reaction contains SPA buffer (10 mm KH₂PO₄, 10 mm KH₂PO₄, 2 mm EDTA, 50 mm NaCl, 1 mm dithiothreitol, 2 mmCHAPS, 10% (v/v) glycerol, pH 7.1), 50 ng of GST-PPARγ (or 150 ng of GST-PPARα, GST-PPARδ), 5 nm ³H-labeled radioligands, and 5 μl of T0070907 in Me₂SO. After incubation for 1 h at room temperature, 10 μl of polylysine-coated SPA beads (at 20 mg/ml in SPA buffer) are added, and the mixtureis incubated for 1 h before reading in Packard Topcount. [³H]Rosiglitazone is used for PPARγ, and [³H]GW2433 is used for PPARα and PPARδ.

• Cell lines

  MCF-7 cells

• Concentrations

  20 μM and higher concentrations

• Incubation Time

  48 h

• Method

  MTS assay

• Animal Models

  Preconditioning is performed by administering a low dose (1 mg/kg) of Escherichia coli LPS (serotype 0.127:B8) intraperitoneally 24 hr before the induction of severe endotoxemia.

• Dosages

  1 mg/kg

• Administration

  intraperitoneally