Screening for PPAR Non-Agonist Ligands Followed by Characterization of a Hit, AM-879, with Additional No-Adipogenic and cdk5-Mediated Phosphorylation Inhibition Properties

Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor, playing key roles in maintenance of adipose tissue and in regulation of glucose and lipid homeostasis. This receptor is the target of thiazolidinediones, a class of antidiabetic drugs, which improve insulin sensitization and regulate glycemia in type 2 diabetes. Despite the beneficial effects of drugs, such as rosiglitazone and pioglitazone, their use is associated with several side effects, including weight gain, heart failure, and liver disease, since these drugs induce full activation of the receptor. By contrast, a promising activation-independent mechanism that involves the inhibition of cyclin-dependent kinase 5 (CDK5)-mediated PPARγ phosphorylation has been related to the insulin-sensitizing effects induced by these drugs. Thus, we aimed to identify novel PPARγ ligands that do not possess agonist properties by conducting a mini-trial with 80 compounds using the sequential steps of thermal shift assay, 8-anilino-1-naphthalenesulfonic acid fluorescence quenching, and a cell-based transactivation assay. We identified two non-agonist PPARγ ligands, AM-879 and P11, and one partial-agonist, R32. Using fluorescence anisotropy, we show that AM-879 does not dissociate the NCOR corepressor in vitro, and it has only a small effect on TRAP coactivator recruitment. In cells, AM-879 could not induce adipocyte differentiation or positively regulate the expression of genes associated with adipogenesis. In addition, AM-879 inhibited CDK5-mediated phosphorylation of PPARγ in vitro. Taken together, these findings supported an interaction between AM-879 and PPARγ; this interaction was identified by the analysis of the crystal structure of the PPARγ:AM-879 complex and evidenced by AM-879’s mechanism of action as a putative PPARγ non-agonist with antidiabetic properties. Moreover, we present an optimized assay pipeline capable of detecting ligands that physically bind to PPARγ but do not cause its activation as a new strategy to identify ligands for this nuclear receptor.

PPARγ S273 Phosphorylation Modifies the Dynamics of Coregulator Proteins Recruitment

“…A better understanding of this mechanism of action opens new pathways for anti-diabetic drug development. Previous studies show that there is a range of molecules that can bind to PPARg preventing Ser273 phosphorylation, without cause the high activation characteristic of strong agonists (20)(21)(22)27) and these results opened a new target possibility, the PPARg:coregulator interaction. Inhibitors of this interaction can act either by binding to the binding groove formed by the IDs or by binding to the receptor's H12 (60).…”

“…pET-28a_PPARgLBD (aa207-aa477), pET-28a_PPARgS237D, and pET-28a_PPARgS237A expression and purification was performed as previously described in (27). pET-15_NCoR (aa2059-aa2297) expression was performed in Escherichia coli BL21 (DE3) strain.…”

“…CDK5 mediated phosphorylation of PPARg and of the PPARg complexes with PGC1-a, TIF2, SMRT and NCoR from pull down assays were measured by luminescent detection of ADP produced in the in vitro phosphorylation reaction, as it was described in (27,28). We used ADP-Glo ™ kinase assay (Promega) following manufacturer's instructions, in which 15 mM of purified PPARg LBD and the pull down purified complexes PPARg + PGC1-a, PPARg + TIF2, PPARg + SMRT and PPARg + NCoR were incubated with 25 ng of purified CDK5/p35, at room temperature for 15 min, in the kinase assay reaction buffer (200mM Tris-HCl, pH 7.4, 100mM MgCl2 and 0.5 mg/ml BSA, SignalChem kinase assay buffer III) added 10 mM of ATP, in 12.5 ml of reaction volume.…”

“…Among them is the insulin-sensitizer class of drugs TZDs, which owns familiar anti-diabetic actions but presents negative side effects due to its strong agonism. On the other hand, some partial agonists, such as MRL24 (20), SR1664 (21), GQ-16 (22), UHC1 (23), F12016 (24), L312 (25), Chelerythrine (26), and AM-879 (27), have been identified to inhibit this PTM without the agonist activity. Structural data analysis showed that PPARg ligands that inhibit S273 phosphorylation do not make direct contact with this residue, but induces structural modifications in PPARg:CDK5 interaction interface.…”

GQ‐130, a novel analogue of thiazolidinedione, improves obesity‐induced metabolic alterations in rats: Evidence for the involvement of PPARβ/δ pathway

“…PPARγ LBD AAs 207‐477 or PPARβ/δ LBD AAs 207‐477 constructions inserted into pET 28a(+) vectors, were expressed in Escherichia coli strain BL21(DE3). The expression of both proteins was performed as described in Ribeiro Filho et al After expression, the proteins were purified by affinity chromatography followed by size exclusion chromatography. Affinity chromatography was performed using a Talon Superflow resin (Clontech) and protein elution was carried out with a 50 mmol/L Tris‐HCl pH 8.0, 300 mmol/L NaCl, 5% glycerol, 300 mmol/L imidazole and 2 mmol/L β‐mercaptoethanol buffer for PPARγ and a 20 mmol/L HEPES pH 7.5, 300 mmol/L NaCl, 5% glycerol, 300 mmol/L imidazole and 2 mmol/L β‐mercaptoethanol buffer for PPARβ/δ.…”