Discovery by Virtual Screening of an Inhibitor of CDK5-Mediated PPARγ Phosphorylation

Abstract

Thiazolidinedione PPARγ agonists such as rosiglitazone and pioglitazone are effective antidiabetic drugs, but side effects have limited their use. It has been posited that their positive antidiabetic effects are mainly mediated by the inhibition of the CDK5-mediated Ser273 phosphorylation of PPARγ, whereas the side effects are linked to classical PPARγ agonism. Thus compounds that inhibit PPARγ Ser273 phosphorylation but lack classical PPARγ agonism have been sought as safer antidiabetic therapies. Herein we report the discovery by virtual screening of 10, which is a potent PPARγ binder and in vitro inhibitor of the CDK5-mediated phosphorylation of PPARγ Ser273 and displays negligible PPARγ agonism in a reporter gene assay. The pharmacokinetic properties of 10 are compatible with oral dosing, enabling preclinical in vivo testing, and a 7 day treatment demonstrated an improvement in insulin sensitivity in the ob/ob diabetic mouse model.

Figure 1

Representative PPARγ ligands: full agonists (1 and 2) and partial agonists (3–6) and inhibitors of PPARγ phosphorylation (7–9).

Figure 2

Space-filling models of full agonist 1 (red, panel A) and partial agonist 6 (green, panel B) in complex with the PPARγ LBD, exemplifying the lack of H12 interaction that is characteristic of PPARγ partial agonists (generated from PDB 2PRGand2Q6S).

Scheme 1. Synthesis of 10

Reagents and conditions: (a) (i) KMnO₄, K₂CO₃, H₂O, reflux, overnight, (ii) hydrazine sulfate, H₂O, reflux, 2 h, 32% over two steps; (b) (i) conc. H₂SO₄, EtOH, reflux, overnight, (ii) 4-(bromomethyl)-1,2-dichlorobenzene, Cs₂CO₃, MeCN, 2 h, (iii) 2 M aq NaOH, THF rt, overnight, 68% over three steps.

Figure 3

Concentration–response data for 10 in the human PPARγ reporter gene agonist (green circles, n = 4, no curve fitted to data) and binding (blue squares, n = 15) assays. Error bars indicate the SD from the mean of individual assay occasions for the reporter gene assay and the SD from the mean of aggregated data from all screening occasions for the binding assay.

Figure 4

(A) X-ray crystal structure of the PPARγ LBD in complex with 10 (orange, PDB7QB1), showing a lack of interaction with PPARγ H12 and proximity to the H2–H2′ loop containing Ser273 (highlighted in red). (B) Close-up of the binding mode of 10 showing the H-bond interaction (dotted purple line) between the carboxylate of 10 and the Ser370 backbone NH.

Figure 5

Differential HDX-MS data of the 10-induced stabilization of the PPARγ LBD (mapped onto PDB 2PRG). Deeper shades of blue indicate greater ligand-induced stabilization, and regions of the protein for which no peptides were detected in MS are colored in tan. Compound 10 is displayed as a green space-filling model.

Figure 6

In vivo pharmacodynamic (PD) effects of 10 in ob/ob mouse model of diabetes. Compounds 1 and 10 were dosed by oral gavage at 10 and 100 μmol/kg/day for 7 days. Effects on (A) terminal fasting plasma glucose, (B) insulin, and (C) body weight. 100 μmol/kg/day of 10 resulted in reduced plasma insulin, corresponding to an increase in insulin sensitivity. Data are presented as the mean ± SEM. Data normality was assessed using the D’Agostino and Pearson test. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison’s test between the ob/ob vehicle group and the substance-treated groups. For data sets that did not meet equality of variances, statistical analysis was performed using the Brown–Forsythe test and Welch’s one-way ANOVA followed by Dunnett’s T3 multiple comparison test. A value of P < 0.05 was considered statistically significant.