Compounds—The compounds BVT.13, BVT.762, and BVT.763 (see Fig. 1a) were synthesized as described earlier (25). GW2331, rosiglitazone, and SR12813 were synthesized in our laboratory. 22(R)-hydroxycholesterol was purchased from Sigma, and WY14.643 was purchased from Alexis.
Expression and Isolation of hPPARγ -LBD—A construct described previously and used for the crystallization of the apoPPAR-LBD (26) was redesigned by site-directed mutagenesis to harbor a factor Xa cleavage site, resulting in the deduced amino acid sequence MGHHHHHHSGSGTIEGR(Leu204-Tyr477). The expression and isolation protocol was performed as described previously (26), except for the addition of an ion exchange (Resource Q, pH 8.0) and a molecular sieving step (Superdex 75) after the first crude immobilized metal ion affinity chromatography step. Isolated PPARγ -LBD was dialyzed against 20 mM Tris/HCl, pH 8.0, and concentrated to 9 mg/ml. The engineered protease cleavage site was not used but may have facilitated crystal packing.
Crystallography—Crystals of the PPARγ -LBD in complex were grown by the hanging drop diffusion method at 18 °C and appeared in 3-5 days. The well solution contained 0.1 M Tris/HCl buffer, pH 7.5, 22% polyethylene glycol 3000, and 0.2 M calcium acetate. Typically, 3 µl of the precipitant was mixed with 3 µl of a solution containing 9 mg/ml PPARγ -LBD, 1 mM glucocorticoid receptor-interacting protein-1 (GRIP-1) coactivator peptide (KEKHKILHRLLQDS), and 1 mM ligand in the drop. Crystals were mounted in glass capillaries and diffracted at most to 2.8 Å. All data were collected at room temperature using a Rigaku RU300 rotating anode with Molecular Structure Corp. mirrors and a Raxis4 image plate detector. The data were processed with DENZO and Scalepack (27). The crystals belonged to the orthorhombic space group P212121, having the approximate unit cell lengths of 49, 67, and 123 Å, containing 1 monomer/asymmetric unit. The structures were solved by molecular replacement with coordinates from the PDB entry 3PRG [PDB] , using the AMoRe program package (28). Model building was performed using the program O (29), and the models were refined using simulated annealing and restrained B factor refinement included in CNS software (30). For all structures, both the ligand and the coactivator peptide could easily be modeled according to the difference electron density maps. The final models contain ligand, the amino acids HKILHRLLQ of the coactivator peptide, the introduced protease cleavage site, and the complete PPARγ-LBD except for 11 residues (amino acids 263-273) in the BVT.762 and BVT.763 complexes and 6 residues (amino acids 268-273) in the BVT.13 complex. Data collection and refinement statistics are given in Table I.
DNA Constructs for GAL4-LBD Fusion Analysis—The LBDs of hPPARα (amino acids 166-468), hPPAR δ (amino acids 138-441), hPPARγ (amino acids 204-477), mPPARα (amino acids 166-468), mPPAR δ (amino acids 137-440), mPPARγ (amino acids 203-505), hPXR (amino acids 107-434), hLXRα (amino acids 163-447), hLXRβ (amino acids 154-461), and hFXR (amino acids 189-469) were generated by PCR amplification using Pfu polymerase (Stratagene) and gene specific primers flanked with restriction enzymes KpnI and BamHI, respectively. The LBDs were subcloned in-frame into the pCMXGal4 vector, containing the GAL4-DBD (31). The 4xGAL4-RE luciferase reporter plasmid has been described previously (31).
Cell-based Reporter Gene Assays—Transient transfection experiments for the analysis of PPAR activation were performed in CaCo-2 subclone TC7 cells (CaCo-2/TC7, a colon adenocarcinoma cell line) in 96-well plates. For batch transfections, cells were seeded at a concentration of 4.0 x 106 cells/225 cm2 and incubated for 24 h at 37 °C in medium containing Dulbecco's modified Eagle's medium (SVA, Sweden), 10% fetal bovine serum (FBS, Hyclone), nonessential amino acids (Invitrogen) (10 ml/liter), and L-glutamine (Invitrogen) (20 ml/liter). After 24 h the medium was replaced with transfection medium containing Dulbecco's modified Eagle's medium, 10% charcoal/dextran-treated FBS, nonessential amino acids (10 ml/liter) and L-glutamine (20 ml/liter). The cells were cotransfected with 5 µg of receptor plasmid and 50 µg of reporter plasmid using Dospher (Roche Diagnostics) according to the manufacturer's instructions. After 5-6 h, medium was replaced. Following 20-24 h, cells were seeded at a concentration of 0.25 x 105 cells/well in induction medium containing Dulbecco's modified Eagle's medium, 5% charcoal/dextran FBS, nonessential amino acids (10 ml/L), and L-glutamine (20 ml/L), incubated for 5 h, and subsequently treated with the compounds in optimized serial dilutions as indicated in the legend to Fig. 2a. Following a 24-h incubation, cells were harvested in lysis buffer (0.1 M Tris/HCl, 2 mM EDTA, 0.25% Triton X-100), and the cell lysates were analyzed for luciferase activity using a Luciferase assay kit (BioThema AB, Sweden). All experiments were performed at least three times in triplicate. For curve fitting, Xlfit version 3.0.2 was used. An analysis of the effects of the 2-BABA compounds in the presence of rosiglitazone was essentially performed as described above with the following exceptions; the transfection and induction media used were Optimem (Invitrogen) with 10 or 5% charcoal/dextran-treated FBS, respectively. The transfection agent used was FuGENE 6 (Roche Diagnostics).
Transient transfection experiments for the analysis of BVT.13 selectivity were performed in CaCo-2/TC7 cells in 6-well plates. Cells were seeded at a concentration of 2 x 105 cells in each well and incubated for 24 h at 37 °C in 2 ml of growth medium containing Dulbecco's modified Eagle's medium, 10% FBS, nonessential amino acids (10 ml/liter), and L-glutamine (20 ml/liter). The medium was replaced with 2 ml of transfection medium (Optimem, Invitrogen) with 10% charcoal/dextran-treated FBS), and the cells were cotransfected with 2 µg of Gal4RE-luciferase reporter and 0.2 µg of pCMXGal4-receptor plasmid (where the LBD of the respective receptor is fused to the Gal4 DBD) using FuGENE 6 according to the manufacturer's instructions. After 20-24 h, the medium was replaced (Optimem with 2% charcoal/dextran-treated FBS), and the cells were treated with BVT.13 (10 or 1 µM) or positive controls as indicated in the legend to Fig. 3. Me2SO was used as a control. Following a 24-h incubation, cells were harvested, and the cell lysates were analyzed for luciferase activity. All experiments were performed twice in duplicate.
In Vivo Studies—Male ob/ob mice (age 10-12 weeks, C57BL/6Jbom-ob/ob (Lepob), Taconic, Denmark) were housed in individual cages and allowed free access to normal rodent (mouse) chow (R34; Lactamin, Vadstena, Sweden) and tap water. The mice were maintained at a temperature of 22 ± 3 °C and a humidity of 50 ± 20% on a fixed 12-h light/dark cycle. Before the start of treatment the animals were grouped based on nonfasting blood glucose measured from tail vein samples. Each treatment group consisted of 10 animals. The animals were orally dosed once a day for 7 days (vehicle 10% w/v polyethylene glycol 400 and 0.1% w/v Tween 80). The dose volume of BVT.13 was 10 ml/kg of body weight, yielding doses of 30, 100, and 300 mg/kg. Three animals were added to the 100-mg/kg dose group for a kinetic study. Rosiglitazone was used as a positive control at a dose of 5 mg/kg of body weight. Food and body weights were registered at the start of dosing. On day eight, orbital blood samples were taken in EDTA vials on wet ice and centrifuged at 4 °C at 400 rpm for 10 min to obtain EDTA plasma. Plasma samples were kept at -20 °C until analyzed. Plasma glucose was determined using glucose GHD Unimate 7 kit (Roche Applied Science). Plasma triglycerides and serum-free fatty acids were determined using triglycerides/GB (Roche Applied Science) and NEFAC (WAKO) assay kits. An enzyme-linked immunosorbent assay method (Mercodia) was used to measure plasma insulin. Concentrations of BVT.13, in dosing solutions and mouse plasma, were determined by on-line solid phase extraction coupled to liquid chromatography-tandem mass spectrometry detection. The procedures involving animals were in conformity with national and international laws for the care and use of laboratory animals and were approved by the local animal ethical committee.