GQ-16, a novel peroxisome proliferator-activated receptor γ (PPARγ) ligand, promotes insulin sensitization without weight gain

Abstract

The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.

FIGURE 1.

Chemical structure of GQ-16 compared with TZDs rosiglitazone and pioglitazone.

FIGURE 2.

GQ-16 is a partial agonist of PPARγ with modest adipogenic activity. A, in vitro transactivation of a PPARγ-based reporter with either rosiglitazone (squares) or GQ-16 (triangles). B and C, GQ-16 (10 μm) induces adipogenesis to a lesser extent than rosiglitazone (Rosi; 10 μm) in both C3H10T1/2 (B) and NIH-3T3-L1 cells (C), as indicated by Western blot of the adipogenic marker protein aP2 (B) and Oil Red staining (C). Error bars, S.E.

FIGURE 3.

GQ-16 improves insulin-signaling components in liver, muscle, and adipose tissue of obese Swiss mice. A–C, insulin-induced tyrosine phosphorylation of the insulin receptor (IR); D–F, insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1); G–I, insulin-induced serine phosphorylation of protein kinase B (Akt); J–L, IκBα protein levels; M–O, phosphorylation of JNK. The results of densitometry were expressed as arbitrary units. IB, immunoblot; CTL, control. Bars, mean ± S.E. (error bars).

FIGURE 4.

GQ-16 improves insulin sensitivity without evoking weight gain and inhibits Cdk5 phosphorylation of PPARγ in vitro. Shown are the short insulin tolerance test (A), intraperitoneal glucose tolerance test (B), and change in body weight (C) of mice treated with either DMSO, rosiglitazone (Rosi) (4 mg/kg/day), or GQ-16 (20 mg/kg/day) for 10 (A) or 12 days (B and C) (n = 6). The effect of rosiglitazone and GQ-16 on food intake (D) and hematocrit (E) is shown. F and G, indirect calorimetry of mice treated with either DMSO, rosiglitazone, or GQ-16 for 7 days. G, average heat over a 4-day period (n = 4). H, GQ-16 completely blocks phosphorylation of PPARγ by Cdk5 in vitro. PCV, packed cell volume. Shown is mean ± S.E. (error bars). Analysis was by one-way analysis of variance. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

GQ-16 interacts with PPARγ via a distinctive binding mode. A, although GQ-16 (purple) induces an overall structure similar to that of rosiglitazone (blue, GQ-16·PPARγ; white, rosiglitazone·PPARγ) (Protein Data Bank entry 2PRG), GQ-16 binds to PPARγ in a different orientation than traditional TZDs, such as rosiglitazone. GQ-16 makes no direct contacts with residues of helix 12 (shown in red), a hallmark of traditional TZDs. The Cdk5 recognition site, which includes Ser-273, is shown in magenta. B, electron density of GQ-16 (SA-Composite Omit map; F_o − F_c) contoured at 2 s. C, superposition of GQ-16 and other reported PPARγ·ligand complex structures showing that GQ-16 uses a unique binding mode relative to other ligands. D and E, comparison of ligand-protein contacts between PPARγ and GQ-16 (D) or rosiglitazone (E) (polar contacts are shown as dotted lines).

FIGURE 6.

GQ-16 strongly protects the helix 3 and β-sheet regions from hydrogen/deuterium exchange. A, comparison of hydrogen/deuterium exchange levels after 30 min. In the absence of ligand, high levels of exchange are observed throughout the LBD. B, both rosiglitazone and GQ-16 strongly protect the overall LBD from exchange, although the pattern produced by each is distinct (A). C–E, although both ligands protect helix 12 similarly (C), GQ-16 more strongly protects the β-sheet (D) and helix 3 (E) regions from hydrogen/deuterium exchange.

FIGURE 7.

Molecular dynamics simulations predict that GQ-16 stabilizes the active conformation of helix 12 less than rosiglitazone. A and B, unlike rosiglitazone (A), GQ-16 (B) does not directly contact helix 12; instead a water molecule mediates an interaction between Tyr-501 of helix 12 and a carbonyl of GQ-16. C, this water-mediated interaction with helix 12 is weaker than the direct rosiglitazone-Tyr-501 interaction and exists less than 60% of the time during simulations. Rosiglitazone makes a direct contact with helix 12 via a hydrogen bond with the hydroxyl group of Tyr-501 (B) that is quite stable as the bond is maintained >90% of the time during simulations. D, helix 12 samples a broader ensemble of conformations when bound to GQ-16 relative to rosiglitazone (RSG).