GSK3787

GSK3787 irreversibly antagonizes human and mouse PPARδ that covalently modifies Cys249 within the ligand binding pocket. GSK3787 (1 μM) effectively antagonizes the agonist GW0742 stimulated transcription of CPT1a and PDK4 in human skeletal muscle cells, and effectively antagonize the basal gene expression of CPT1a. GSK 3787 shows no effective antiproliferative activity against colorectal cancer cells. ^[1]

GSK3787 (1 μM) completely antagonizes 50 nM GW0742-induced PPARβ/δ-dependent Angptl4 gene expression in wild-type fibroblasts and in keratinocytes. GSK3787 (1 μM) largely antagonizes 50 nM GW0742-induced Angptl4 and Adrp mRNAs expression in skin cancer cell A341. GSK3787 (1 μM) largely antagonizes GW0742-induced Angptl4 mRNAs expression in cancer cell lines MCF7 (breast), Huh7 (liver), and HepG2 (liver) cells but not in H1838 or A549 cells (lung). GSK3787 (1 μM) largely antagonizes GW0742-induced increase of Adrp mRNA in Huh7 and HepG2 cells but not in H1838 cells. GSK3787 does not antagonize basal expression of either of these two PPARβ/δtarget genes in these cells. Neither GW0742 nor GSK3787 has any effect on cell proliferation of these cells at concentrations ranging from 0.1 to 10 μM. GSK3787 is able to modulate the association of both PPARβ/δ and PPARγ coregulator peptides in response to ligand activation. Efficacy of GSK3787 to modulate PPARγ activity is markedly lower than the efficacy of GSK3787 to act as a PPARβ/δ antagonist. ^[2]

GSK3787 antagonizes ligand-induced PPARβ/δ-dependent gene expression in vivo. Oral administration of GSK3787 has no effect on the expression of Angptl4 and Adrp mRNA in mouse colon epithelium. Coadministration of GSK3787 (10 mg/kg) effectively prevents the GW0742-induced expression of both Angptl4 and Adrp mRNA in wild-type mouse colon epithelium, which is correlated with reduced promoter occupancy of PPARβ/δ on the Angptl4 and Adrp genes. Administration of GSK3787 had no effect on glucose tolerance. ^[2]

• Saturation binding assay

  Using 96-well culture plates, 50 nM purified human PPARG LBD and the desired concentration of [³H]GW3738 are diluted to a total volume of 100 μL with buffer consisting of 50 mM KCl, 5 mM EDTA, 10 mM dithiothreitol (DTT), 50 mM HEPES, at pH 7. Saturation binding assays are conducted using concentrations of [³H]GW3738 ranging from 1 to 250 nM. Nonspecific binding at each concentration of [³H]GW3738 is estimated in parallel incubations containing 50 μM unlabeled GW 3738. Plates are incubated for 2 h at room temperature. Free ligand is separated from receptor-bound ligand by size exclusion chromatography using commercially available 96-well format spin columns. Samples (50 μL) from each well of a single test plate are loaded onto a buffer- and temperature-equilibrated 96-well gel filtration block. The block is placed on top of a clean microtiter plate and centrifuged at 1100 g for 4 min. Scintillation fluid (180 ul) is added to each well of the plate containing the eluent, and the plates are sealed and allowed to equilibrate for at least 4 h before counting in a counter. The amount of nonspecific binding at each concentration of [³H]GW3738 is subtracted from all wells and plots of [³H]GW3738 concentration versus counts per minute (cpm) bound are constructed. The K_d values for [³H]GW3738 are determined from a nonlinear least squares fit of the data to a simple binding model.