A New Class of Peroxisome Proliferator-activated Receptor Agonists with a Novel Binding Epitope Shows Antidiabetic Effects*


A New Class of Peroxisome Proliferator-activated Receptor Agonists with a Novel Binding Epitope Shows Antidiabetic Effects*

Tove Östberg{ddagger}§¶, Stefan Svensson¶||, Göran Selén**, Jonas Uppenberg||, Markus Thor{ddagger}{ddagger}, Maj Sundbom**, Mona Sydow-Bäckman§§, Anna-Lena Gustavsson||, and Lena Jendeberg**¶¶

From the {ddagger}Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, SE-171 77 Stockholm, Sweden, the ||Department of Structural Chemistry, **Department of Biology, {ddagger}{ddagger}Department of Medicinal Chemistry, §§Department of Assay Development and Screening, Biovitrum, SE-112 76 Stockholm, Sweden


The peroxisome proliferator-activated receptors (PPARs) areligand-activated transcription factors belonging to the NR1subfamily of nuclear receptors. The PPARs play key roles inthe control of glucose and lipid homeostasis, and the syntheticisoform-specific PPAR agonists are used clinically to improveinsulin sensitivity and to lower serum triglyceride levels.All of the previously reported PPAR agonists form the same characteristicinteractions with the receptor, which have been postulated tobe important for the induction of agonistic activity. Here wedescribe a new class of PPAR{alpha}/{gamma} modulators, the 5-substituted2-benzoylaminobenzoic acids (2-BABAs). As shown by x-ray crystallography,the representative compounds BVT.13, BVT.762, and BVT.763, utilizea novel binding epitope and lack the agonist-characteristicinteractions. Despite this, some compounds within the 2-BABAfamily are potent agonists in a cell-based reporter gene assay.Furthermore, BVT.13 displays antidiabetic effects in ob/ob mice.We concluded that the 2-BABA binding mode can be used to designisoform-specific PPAR modulators with biological activity invivo.

Source: J. Biol. Chem., Vol. 279, Issue 39, 41124-41130, September 24, 2004


The peroxisome proliferator-activated receptors (PPARs)1 are ligand-activated transcription factors that can be activated by a range of fatty acids and their eicosanoid derivatives, and they function as regulators in the biosynthesis, metabolism, and storage of these compounds (1-8). The PPARs bind DNA as heterodimers with the retinoid X receptors to the peroxisome proliferator response elements. Peroxisome proliferator response elements have been identified in the promoter region of a number of genes involved in lipid and carbohydrate metabolism, and the binding site preferences for each PPAR subtype have been shown to differ slightly (9). The three human isoforms of PPARα, -δ, and -γ (NR1C1, NR1C2, and NR1C3, respectively) show distinct patterns of tissue distribution and ligand preference and control different biological activities. PPARα is a regulator of fatty acid catabolism in the liver (10, 11), whereas PPARγ plays a key role in adipogenesis (12). All three isoforms are expressed in macrophages where they are implicated in the control of cholesterol efflux (13, 14). The use of synthetic PPAR ligands has demonstrated the importance of these receptors in the regulation of lipid and glucose homeostasis and today PPARs are established molecular targets for the treatment of type 2 diabetes and cardiovascular disease. The thiazolidinediones (TZDs), used pharmaceutically as insulin sensitizers, are known activators of PPARγ (15, 16), whereas hypolipidemic fibrate drugs to some extent exert their effect via PPARα (1, 17, 18). With the growing understanding of PPAR biology, it has become evident that novel drugs modulating PPAR activity could improve present diabetes treatment and have implications in the treatment of other diseases (19, 20).

Crystal structures of PPARα, -δ, and -γ  ligand-binding domains (LBDs), in complex with various agonists (5, 21-23), reveal a common binding mode where the ligands form specific hydrogen bonds with residues in, and in the vicinity of, helix 12, also known as activation function 2 (AF2). It is generally believed that agonist binding to NRs induces changes in the dynamics and position of helix 12, which in turn facilitates the recruitment of coactivator proteins resulting in an activation of the transcriptional machinery. The ligand-binding pockets of PPAR LBDs are larger than for other NRs and, as shown for the PPARγ-GW0072 complex (24), allow ligand binding at epitopes distal to helix 12. The GW0072 compound is a poor transactivator and can antagonize TZD-driven adipocyte differentiation (24). Taken together, these observations suggest that the specific hydrogen bonding interactions seen between the agonists and AF2 could act as a necessary molecular switch for transactivation to occur (22, 24).

In opposition to this hypothesis we here describe a new class of PPAR agonists, the 5-substituted 2-benzoylaminobenzoic acids (2-BABAs), which act by binding at the entrance of the ligand pocket and activate the receptor without a direct interaction with helix 12. The 2-BABA compound BVT.13, which selectively activates PPARγ with a similar maximal efficacy as rosiglitazone, was shown to have antidiabetic effects in ob/ob mice.

Experimental Procedures


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{gamma}-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.



Identification of a Novel Class of PPAR Ligands—The parent 2-BABA scaffold (BVT.142) was identified as a PPARγ ligand and activator as described previously (25). An initial expansion around this scaffold yielded 250 compounds, where the agonists BVT.13, BVT.762, and BVT.763 were substituted in the 5-position with 2-pyrimidinyloxy, 2-thienylmethoxy, and 2-(3-thienyl)ethoxy, respectively (Fig. 1a). These compounds were chosen as good representatives based on both chemical and preclinical properties.

Structural Analysis of Cocrystal Complexes—Initial docking studies of the 2-BABA scaffold to the apoPPARγ-LBD structure (26) in combination with the bioisosteric properties of the TZD headgroup and carboxylate group of the 2-BABA compounds suggested an anchoring mode similar to that of eicosapentaenoic acid to PPARδ (5), with the carboxylic headgroup interacting with Tyr-473 in helix 12. To further elucidate this hypothesis, the crystal structures of BVT.13, BVT.762, and BVT.763 in complex with PPARγ-LBD and a GRIP-1-derived coactivator peptide were solved by molecular replacement. Resolutions of the complexes were 2.8, 3.15, and 2.9 Å, respectively (Table I). The overall fold of the PPARγ-LBD shows only minor differences as compared with the previously determined apoPPARγ structure (26). The major differences are in the region connecting helix 2' and helix 3, a region with high mobility. The 2-BABA complexes superimpose well with the apoPPARγ-LBD structure, resulting in a root mean square deviation of 0.8 Å over 260 C-α atoms (Protein Data Bank accession code 3PRG [PDB] ). Surprisingly, the crystal structures reveal that the three agonists lack a direct interaction with helix 12. The 2-BABA ligands are located in an elongated conformation of the ligand-binding pocket (Fig. 1, b and c) and they occupy a region in proximity with and approximately parallel to helix 3 and in proximity to β-strand 3 and helices 5 and 2'.

The interactions of the molecule with the protein can be divided into four groups. The first group is formed by the interactions to the dichloro-phenyl moiety. This moiety is located in a predominantly hydrophobic pocket of the receptor. Principal interactions are hydrophobic in nature and formed by the side chains of Arg-288, Ala-292, Ile-326, Met-329, Leu-330, and Leu-333 (Fig. 1d). The second group of interactions is formed between the carbonyl oxygen on the peptide linker of the ligand and the side chain sulfur and the backbone carbonyl oxygen of Cys-285. The third group is made up of interactions between the central benzoic acid moiety and the protein. This moiety is located in a narrow groove of the protein formed by the side chains of Cys-285, Arg-288, Ile-341, and Met-364, and the backbone atoms of Gly-284, Cys-285, and Ser-342. The interactions are hydrophobic in nature with the exception of a distinct hydrogen bond formed between the backbone nitrogen of Ser-342 and one of the carboxylate oxygens of the ligand (Fig. 1d). The fourth group of interactions is formed between the heterocyclic ring and the protein. These are also predominantly hydrophobic in nature, where the protein atoms involved belong to the side chains and backbone of Leu-255, Arg-280, and Ile-281.

The pyrimidine ring of BVT.13 has additional interactions with Phe-264 and His-266, residues that are situated in the part of the loop connecting helix 2' and helix 3, which is structured in the BVT.13 complex but not in the other two complexes. The GRIP-1 coactivator peptide is bound in a helical conformation in the predominantly hydrophobic groove formed by helices 3-5 and 12 (AF2), a binding mode identical to that seen for the steroid receptor coactivator 1 helix in the PPARγ-rosiglitazone complex (21). As described previously, Lys-301 and Glu-471 of the LBD stabilize the dipole moment of the coactivator helix by forming a charge clamp (21).

Ligand-induced Transcriptional Activation—To evaluate the ability of BVT.13, BVT.762, and BVT.763 to transcriptionally activate PPARγ and PPAR{alpha}, a cell-based reporter gene assay was employed. CaCo-2/TC7 cells were transiently transfected with chimeric Gal4-DBD/PPAR-LBD receptors and a 4xGAL4-RE reporter plasmid and subsequently treated with BVT.13, BVT.762, BVT.763 or rosiglitazone in optimized serial dilutions. A strong ligand-dependent activation of PPARγ, with a relative efficacy of ~80% compared with rosiglitazone, was seen for all three compounds (Fig. 2a). The calculated EC50 values were 1.3, 0.3, and 0.8 µM, respectively (25). BVT.762 and BVT.763 induced the transcriptional activity of PPARα to a similar efficacy as seen for PPARγ but displayed EC50 values of 5 and 3.8 µM, respectively (25). Interestingly, BVT.13 failed to bind and activate PPARα (25). Furthermore, the 2-BABA compounds were not able to antagonize the activity of 30 nM rosiglitazone in the cell-based reporter gene assay (Fig. 2b). To further determine the specificity of BVT.13, a set of NR LBDs were analyzed in cell-based reporter gene assays. Transcriptional activation by BVT.13 was shown to be selective for the mouse and human PPARγ isoforms (Fig. 3).

Pharmacological Activity of BVT.13—Next, the antidiabetic and antilipidemic effects of BVT.13 in mice were examined. Male ob/ob mice were treated once daily with BVT.13 doses of 30, 100, or 300 mg/kg for 7 days. Plasma concentrations of BVT.13 at the dose of 100 mg/kg were above the lowest limit of quantification for the analytical method at all time points, implicating that the mice were exposed to BVT.13 during the whole 24-h dosing interval. The average steady-state concentration at 100 mg/kg/day was determined in a kinetic study. Using an unbound fraction of 0.6%, as determined for BVT.13 in vitro, the calculations yielded an approximate steady-state unbound concentration of 1 µM. The body weight of both the BVT.13- and rosiglitazone-treated animals increased significantly compared with the vehicle-treated animals, although no difference in food intake was observed (data not shown). After 7 days, levels of plasma glucose, triglycerides, insulin, and free fatty acids were measured. Significant reductions of fasting plasma glucose and triglycerides were seen after treatment with BVT.13 at the dose of 300 mg/kg compared with vehicle treatment. The reductions were comparable with those seen after treatment with rosiglitazone at 5 mg/kg (Fig. 4, a and b). The lower doses of BVT.13 did not significantly reduce the plasma glucose and triglyceride levels (Fig. 4, a and b). The fasting plasma insulin and free fatty acids were significantly reduced after treatment with BVT.13 at the two higher doses (100 and 300 mg/kg) compared with vehicle treatment. The two higher doses of BVT.13 resulted in plasma insulin and free fatty acid levels comparable with those seen after treatment with rosiglitazone (Fig. 4, c and d).



In the early 1980s a new class of thiazolidinedione-based insulin sensitizers (TZDs) was reported. These compounds, termed glitazones, were later shown to be potent and selective PPARγ agonists and are now widely used for the treatment of type 2 diabetes. PPARs modulate multiple aspects of lipid and carbohydrate metabolism and are therefore relevant targets for treating several important aspects of type 2 diabetes and the metabolic syndrome. However, some TZDs were associated with cases of liver toxicity, which resulted in the withdrawal of troglitazone from the market. This and other side effects such as fluid retention and weight gain, associated with the commercially available rosiglitazone and pioglitazone, demonstrate the need to develop new compounds with improved efficacy and reduced side-effect profiles.

In this report we presented a new class of PPARα/γ modulators, the 2-BABAs. A set of representative compounds, termed BVT.13, BVT.762, and BVT.763, were shown by x-ray crystallography to utilize a novel binding epitope not involving the classical agonist-characteristic interactions. Exemplified compounds within the 2-BABA family displayed agonistic activity in a PPARγ cell-based reporter gene assay and failed to antagonize the rosiglitazone-induced activity at an approximate EC50 concentration of 30 nM. In vivo, the specific PPARγ agonist BVT.13, displayed antidiabetic effects in ob/ob mice by significantly reducing the plasma levels of glucose, insulin, triglycerides, and free fatty acids.

Our current understanding of the ligand-induced transcription of nuclear receptors originates from mutational studies, highlighting the importance of the C-terminal part of the ligand binding domain, which has been denoted AF2 (32). The increasing number of LBD structures has more recently contributed to the understanding of this mechanism on a molecular level. Taken together, these observations resulted in the conclusion that NR agonists stabilize the active conformation of the receptor primarily by locking helix 12 (AF2) in a conformation that allows coactivators to bind to a specific recognition site, mainly formed by helices 3, 4, and 12. Direct interactions between the agonist and amino acid residues of helix 12 have been suggested as the structural basis for transcriptional activation of many receptors (22, 24). One exception is the retinoid X receptor where the natural ligand 9-cis-retinoic acid only interacts indirectly with helix 12 via residues Cys-269 and Ala-272 of helix 3 and Trp-305 of helix 5 (33), localized in the vicinity of helix 12. The previously reported holoPPAR complexes share a conserved network of hydrogen bonds between the ligand and the side chains of residues His-323, His-449, and Tyr-473 (PPARγ nomenclature), which therefore has been suggested as crucial for the ligand-dependent activation of the PPARs (5, 21, 23, 34). Surprisingly, hydrogen bonding to this triad is not conserved for the 2-BABA compounds, which suggests that these interactions are not necessary for the induction of transcriptional activation by PPARα and -γ. Furthermore, the 2-BABAs are situated at the entrance of the active site and do not interact directly with helix 12. There are three unrelated crystallographic apo structures of PPARγ (21, 26), of which two closely resemble the agonistic structures in terms of helix 12 position and interactions, thus giving no hints of conformational changes related to ligand activation. In the third structure helix 12 adopts a different conformation, slightly protruding away from the LBD, and could therefore more easily be interpreted as an inactive state. Furthermore, NMR studies of the PPAR{alpha} and the PPARγ LBDs have shown that the apo forms of the receptors are in an equilibrium of conformations rather than adopting one single stable conformation (23, 35, 36). Altogether, this suggests that the unliganded LBD of PPARs can adopt the active conformation as well as a number of inactive conformations. Despite the lack of direct interactions with AF2, the binding of the 2-BABA compounds seem to push the equilibrium of conformational states toward an active state, which suggests an alternative activation mechanism for PPARγ by an overall stabilization of the LBD. This activation mechanism is further supported by the observation that a partial PPARγ agonist, nTZDpa, only partially stabilizes the receptor (35). As judged by superpositioning of the holoPPAR complexes, all agonists induce highly similar conformations of the LBD despite their different interactions with the receptor (Fig. 5). Large variations are shown in three clustered regions, H2 to the N-terminal part of H3, the N-terminal part of H7, and the C-terminal part of H11 to the N-terminal part of H12. These regions show higher B factors than the LBD in general, and the differences seen could be because of a higher degree of mobility in these regions. It has been reported that PPAR agonists differ in their ability to induce recruitment of specific coactivators (37). Again the PPARγ complexes do not give a straight answer to this selectivity, and it is possible that selectivity is generated by subtle differences in the dynamics of the LBD not detectable in these crystal structures.

The cell-based reporter gene assay revealed that all three substances, BVT.13, BVT.762, and BVT.763, are in fact potent activators of PPARγ. BVT.762 and BVT.763 were also shown to activate PPARα, whereas BVT.13 failed to bind and transcriptionally activate PPARα (25). Structural alignments of the PPAR subtypes revealed one noteworthy amino acid difference in the vicinity of the heterocyclic substituent where residue Gly-284 in PPARγ has been substituted by a cysteine in PPARα and an arginine in PPARδ. The cysteine and the arginine side chains may restrict the space available for short and bulky substituents, providing an explanation for the selectivity of BVT.13.

In accordance with the in vitro data of PPARγ selective agonist activity, the administration of BVT.13 led to a significant reduction in plasma levels of glucose, insulin, triglycerides, free fatty acids, and cholesterol (Fig. 4 and data not shown). Furthermore, a significant increase in body weight was observed for both the BVT.13- and the rosiglitazone-treated animals compared with the vehicle-treated, although no difference in food intake was observed (data not shown). Weight increase is commonly associated with PPARγ agonist treatment and generally associated with the pharmacological activity mediated through PPARγ agonism (38). The ob/ob mouse, as a model for type 2 diabetes, is known to respond well to the PPARγ agonistic TZDs, which was also confirmed here by the rosiglitazone-treated group. The kinetic study using 100 mg/kg of BVT.13, yielded an approximate average steady-state unbound concentration of 1 µM. This dose corresponds to an approximate EC50 concentration, as determined for BVT.13 in the cell-based reporter gene assay. This suggests that at least the two higher dose groups were sufficiently exposed to BVT.13 to respond through PPARγ activation. We believe that the antidiabetic effects observed are likely because of PPARγ activation by BVT.13.

We concluded that the 2-BABA binding mode can be used to design isoform-specific PPAR modulators with biological activity in vivo and that direct interaction with helix 12 is not a necessity for transcriptional activation. With respect to the large ligand binding pockets of the PPARs (1250-1500 Å3), our observations present new possibilities for the design of PPAR modulators interacting with the receptor through binding sites distal to the classical activation site. This might also have implications for the transcriptional activation of other orphan NRs.


We thank Katarina Krook, Malin Warolén, Göran Bertilsson, and Jessica Heidrich for technical assistance with cloning, Sven-Åke Franzén, Andrea Varadi, and Marianne Israelsson for DNA sequence analysis, Bengt Lindqvist and Graeme Dykes for chemical synthesis, Björn Norrlind for formulations, Yuko Rönquist-Nii for bioanalysis, and Peter Gozzi for pharmacokinetic analysis, Gunnel Klingström, Catarina Larsson, and Ursula Multan for clinical chemistry. The TC7 subclone of CaCo-2 cells was kindly provided by Dr. Monique Rousset, Institut National de la Santé et de la Recherche Médicale U178, Villejuif, France.


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mcith_BC-peroxisomeF01.jpg Figure 1 Molecular basis for 2-BABA interaction with PPAR{gamma} a, chemical structures of BVT.13, BVT.762, and BVT.763. b, ribbons drawing of PPAR-gamma LBD in complex with BVT.13 (light blue), BVT.762 (magenta), and BVT.763 (yellow). The GRIP-1 coactivator peptide is depicted in orange. c, close-up of the PPAR-gamma ligand binding pocket with BVT.13 (light blue), BVT.762 (magenta), BVT.763 (yellow), and rosiglitazone (red). d, amino acid residues involved in BVT.13 binding. A Fo - Fc omit map is shown contoured at 2.5 sigma.

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mcith_BC-peroxisomeF02.gif Figure 2 Transcriptional activation of PPAR{gamma} by BVT.13, BVT.762, BVT.763, and rosiglitazone. CaCo-2/TC7 cells were transiently transfected with a 4xGAL4-RE luciferase reporter and a GAL4-PPAR{gamma} LBD fusion construct and subsequently treated with BVT.13, BVT.762, BVT.763, and rosiglitazone in optimized serial dilutions (a) or BVT.13, BVT.762, and BVT.763 in optimized serial dilutions in the presence of 30 nM rosiglitazone (b), as indicated in the figures. Data are shown as the -fold inductions of agonist-induced luciferase activity divided by the luciferase activity of the vehicle.

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mcith_BC-peroxisomeF03.jpg Figure 3 Selectivity of BVT.13 determined for a set of NR LBDs. Data are shown as the -fold inductions of agonist-induced luciferase activity divided by the luciferase activity of the vehicle. The following reference compounds were used as positive controls at the indicated concentrations, for hPPAR-alpha and mPPAR-alpha, 10 µM WY14643; for hPPAR-delta and mPPAR-delta, 10 µM GW2331; for hPPAR{gamma} and mPPAR-gamma, 1 µM rosiglitazone; for PXR and FXR 1 µM SR12813; for LXR{alpha} and LXR-beta, 10 µM 22(R)-hydroxycholesterol.

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mcith_BC-peroxisomeF04.jpg Figure 4 Effects of BVT.13 administration to ob/ob mice after 7 days of treatment. Fasting plasma levels of glucose (a), insulin (b), trigycerides (c), and free fatty acids (d). Ten mice/dose group were used. A statistical analysis of the plasma glucose levels was performed by one-way analysis of variance followed by Bonferroni's multiple comparison test. A statistical analysis of plasma insulin, triglyceride, and free fatty acid levels was performed by the Kruskal-Wallis test followed by Dunn's multiple comparison test. The corresponding p values for comparison with the vehicle group are indicated in the figures. {alpha}, analytes were below the detection limit of the standard protocol in four samples and therefore were not included in the statistical analysis.

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mcith_BC-peroxisomeF05.gif Figure 5 Ribbons representation of superpositioned PPAR{gamma} structures. Coordinates are from 1WM0 (light blue), 1PRG (dark blue), 2PR

G (green), 4PRG (a subunit, yellow), 1FM6 (red), 1fm9 (pink), 1K74 (light gray), 1KNU (black), and 1NYX (gold). Selected secondary elements are annotated with numbers positioned at their N-terminal ends.


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  TABLE I. Crystallographic data

PPARγ -LBD-BVT.13 complex

PPARγ -LBD-BVT.762 complex

PPARγ -LBD-BVT.763 complex

Data collection      
Resolution (Å) 2.9 3.15 2.90
I/-sigma (in highest resolution shell) 13.4 (3.2) 20.3 (3.2) 33.3 (2.8)
Completeness (in highest shell) 86.9 (91.1) 89.0 (93.6) 94.4 (96.5)
Rsym (in highest shell)a 0.095 (0.400) 0.089 (0.314) 0.068 (0.333)
Rfactor 0.191 0.222 0.224
Rfree 0.295 0.308 0.292
Ramachandran angle distribution: core, allowed, generously allowed, disallowed (%)b

87.0, 12.2, 0.8, 0

83.9, 15.3, 0.8, 0

87.1, 12.5, 0.4, 0

a, where (I(h)i) is the average intensity of reflection h, Σh is the sum over all reflections, and Σi is the sum of all measurements of reflection h.

b Values and definitions are from Procheck (39).

Source: J. Biol. Chem., Vol. 279, Issue 39, 41124-41130, September 24, 2004