The Differential Interactions of Peroxisome Proliferator-Activated Receptor γ Ligands with Tyr473 Is a Physical Basis for Their Unique Biological Activities

Abstract

Despite their proven antidiabetic efficacy, widespread use of peroxisome proliferator-activated receptor (PPAR)␥ agonists has been limited by adverse cardiovascular effects. To overcome this shortcoming, selective PPAR␥ modulators (SPPAR␥Ms) have been identified that have antidiabetic efficacy comparable with full agonists with improved tolerability in preclinical species. The results of structural studies support the proposition that SPPAR␥Ms interact with PPAR␥ differently from full agonists, thereby providing a physical basis for their novel activities. Herein, we describe a novel PPAR␥ ligand, SPPAR␥M2. This compound was a partial agonist in a cell-based transcriptional activity assay, with diminished adipogenic activity and an attenuated gene signature in cultured human adipocytes. X-ray cocrystallography studies demonstrated that, unlike rosiglitazone, SPPAR␥M2 did not interact with the Tyr473 residue located within helix 12 of the ligand binding domain (LBD). Instead, SPPAR␥M2 was found to bind to and activate human PPAR␥ in which the Tyr473 residue had been mutated to alanine (hPPAR␥Y473A), with potencies similar to those observed with the wild-type receptor (hPPAR␥WT). In additional studies, we found that the intrinsic binding and functional potencies of structurally distinct SPPAR␥Ms were not diminished by the Y473A mutation, whereas those of various thiazolidinedione (TZD) and non-TZD PPAR␥ full agonists were reduced in a correlative manner. These results directly demonstrate the important role of Tyr473 in mediating the interaction of full agonists but not SPPAR␥Ms with the PPAR␥ LBD, thereby providing a precise molecular determinant for their differing pharmacologies.The peroxisome proliferator-activated receptors (PPARs) ␥, ␦, and ␣ compose a nuclear receptor subfamily that modulates the transcription of a large compendium of genes encoding proteins that regulate lipid metabolism, cell differentiation, and signal transduction in a ligand-dependent manner (Berger and Moller, 2002). PPAR␥ has been shown to be a master regulator of adipogenesis and nutrient metabolism in adipocytes where it is highly expressed. Thiazolidinedione (TZD) PPAR␥ full agonists have demonstrated clinical efficacy for the treatment of type 2 diabetes mellitus (T2DM) patients . However, the use of these insulin-sensitizing agents has been restricted because of their association with several adverse effects, including

INT131: A Selective Modulator of PPARγ

“…The importance of the Y473 residue in INT131 activity was further explored in a PPARγ mutant where the Y473 was mutated to alanine. Similar to a previous report, 24 the Y473A mutation caused a dramatic reduction in the ability of rosiglitazone to induce receptor activation in a cell-based reporter assay (Fig. 3d).…”

“…These observations are consistent with previous studies demonstrating the importance of helix 12 and Y473 for PPARγ agonism. 24 In contrast, Y473A mutant has no effect on INT131-induced receptor activation in the cell-based reporter assay (Fig. 3d).…”

“…This observation is similar to a previous report, which also suggested that the affinity of rosiglitazone for the mutant receptor has been greatly reduced. 24 In contrast, Y473A mutation had no significant effect on neither the efficacy nor the potency of INT131-induced receptor activation (Fig. 3d).…”

Structural design and synthesis of arylalkynyl amide-type peroxisome proliferator-activated receptor γ (PPARγ)-selective antagonists based on the helix12-folding inhibition hypothesis

“…2D). Among them, the hydrogen-bonding interaction with Tyr473 on H12 is considered to be critically important for the proper folding of H12 of the PPARg LBD, apparently serving to stabilize the closed conformation (active conformation) of H12 and thus enabling transcription of target genes [33,34]. Therefore, an effective approach for antagonist design might be to block this key interaction.…”

“…(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) [33,34]. Second, the phenylpropyl moiety of 6 (depicted in green in Fig.…”