A peroxisome proliferator-activated receptor gamma ligand inhibits adipocyte differentiation

Abstract

The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate glucose and lipid homeostasis. The PPARgamma subtype plays a central role in the regulation of adipogenesis and is the molecular target for the 2, 4-thiazolidinedione class of antidiabetic drugs. Structural studies have revealed that agonist ligands activate the PPARs through direct interactions with the C-terminal region of the ligand-binding domain, which includes the activation function 2 helix. GW0072 was identified as a high-affinity PPARgamma ligand that was a weak partial agonist of PPARgamma transactivation. X-ray crystallography revealed that GW0072 occupied the ligand-binding pocket by using different epitopes than the known PPAR agonists and did not interact with the activation function 2 helix. In cell culture, GW0072 was a potent antagonist of adipocyte differentiation. These results establish an approach to the design of PPAR ligands with modified biological activities.

Figure 1

GW0072 is a PPARγ ligand with a unique binding mode. (A) Chemical structures of the TZD rosiglitazone and the thiazolidine acetamide GW0072. (B) Cocrystal structure of the PPARγ ligand-binding domain with GW0072. The polypeptide backbone is illustrated as a yellow worm schematic, and GW0072 is shown as a Van der Waals space-filling representation with each atom type colored: carbon, green; oxygen, red; nitrogen, blue; and sulfur, yellow. (C) The PPARγ polypeptide backbone atoms from the cocrystal structure with GW0072 superimposed on the cocrystal structure with rosiglitazone (6). The polypeptide backbone of the GW0072 cocrystal structure is shown as a thin yellow ribbon; GW0072 as well as H323, H449, and Y473 of the protein complex are shown as carbon, green; oxygen, red; nitrogen, blue; and sulfur, yellow. Rosiglitazone and the same three amino acids for its respective protein complex are colored red. Differences between the rosiglitazone and GW0072 cocrystal structures exist in the side-chain amino acid positions and not in the overall polypeptide conformation except for the loop between helix 2′ and helix 3, which is the result of crystal packing. GW0072 occupies an epitope of the ligand-binding pocket of PPARγ that precludes it from interactions with Y473, H323, and H449.

Figure 2

GW0072 is a PPARγ ligand with a unique functional profile. (A) Dose response on the PPARγ-GAL4 chimera for GW0072 (○) and GW0072 plus 100 nM rosiglitazone (●). Reporter activity was expressed as the % of the maximal activation by 1 μM rosiglitazone. GW0072 demonstrates competitive antagonism of rosiglitazone but retains weak agonist activity at μM concentrations. (B) Activity on full-length PPARγ2 for 100 nM rosiglitazone (TZD), 10 μM GW0072 (GW), and 100 nM rosiglitazone plus 10 μM GW0072 (TZD + GW). Vehicle was 0.1% DMSO. Reporter activity was expressed as the % of the maximal activation by 1 μM rosiglitazone. (C–F) The functional activity of GW0072 is paralleled by its effects on coactivator recruitment to PPARγ2 in a mammalian two-hybrid assay. GW0072 (GW) (10 μM) antagonizes recruitment of the coactivators CBP and SRC1 promoted by 1 μM rosiglitazone (TZD). GW0072 (GW) (10 μM) does not recruit the corepressors NCoR or SMRT.

Figure 3

GW0072 is a potent antagonist of adipocyte differentiation. (A–D) Oil-red O staining of 10T1/2 cells incubated for 6 days with 0.1% DMSO (vehicle), 1 μM rosiglitazone (TZD), 10 μM GW0072 (GW), or 1 μM rosiglitazone plus 10 μM GW0072 (TZD + GW). (E) Northern blot analysis of 10T1/2 cells for adipocyte-specific genes after treatment of cells for 3 and 6 days. aP2, adipocyte fatty acid binding protein.

Figure 4

GW0072 defines an additional class of nuclear receptor ligands. Agonist ligands (gray) shift the AF-2 helix (yellow) into a position that stabilizes recruitment of coactivator (green) to the receptor ligand-binding domain (white). Antagonist ligands (gray) bind to the receptor by using the same epitopes, but their larger size shifts the AF-2 helix into a position that displaces the coactivator. Antagonist ligands also recruit corepressor (red) to the receptor ligand-binding domain. The partial agonist GW0072 (gray) binds to its receptor by using different epitopes, such that it does not directly interact with the AF-2 helix.