RESUMO
Obesity is one of the main risk factors for type 2 diabetes, and peroxisome proliferator-activated receptor γ (PPARγ) is considered a promising pathway on insulin sensitivity and adipose tissue metabolism. The search for molecules acting as insulin sensitizers have increased, especially for molecules that block PPARγ-Ser273 phosphorylation, without reaching full agonism. We evaluated the in vivo effects of AM-879, a PPARγ non-agonist, and found that AM-879 exerts different effects in mice depending on the dose. At lower doses, this ligand decreased BAT, increased leptin and Crh expression. However, at a higher dose, it promoted improvement on insulin sensitivity, ameliorates expression of metabolism-related genes, decreased the expression of genes related to liver toxicity, maintaining body weight and adipocyte size. These results present a new lead molecule to ameliorates insulin resistance and confirm AM-879 as a PPARγ non-agonist which blocks Ser273 phosphorylation as a good strategy to modulate insulin sensitivity without developing the adverse effects promoted by PPARγ full agonists.
RESUMO
Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor with a key role in metabolic processes and is target of CDK5 kinase phosphorylation at S245 (S273 in PPARγ isoform 2), thereby inducing insulin resistance. A remarkable effort has been addressed to find PPARγ ligands that inhibit S245 phosphorylation, but the poor understanding in this field challenges the design of such ligands. Here, through computational and biophysical methods, we explored an experimentally validated model of PPARγ-CDK5 complex, and we presented K261, K263 or K265, which are conserved in mammals, as important anchor residues for this interaction. In addition, we observed, from structural data analysis, that PPARγ ligands that inhibit S245 phosphorylation are not in direct contact with these residues; but induce structural modifications in PPARγ:CDK5/p25 interface. In summary, our PPARγ and CDK5/p25 interaction analyses open new possibilities for the rational design of novel inhibitors that impair S245 phosphorylation.
Assuntos
Quinase 5 Dependente de Ciclina/química , Complexos Multiproteicos/química , PPAR gama/química , Conformação Proteica , Animais , Sítios de Ligação/genética , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Humanos , Ligantes , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação , Ligação ProteicaRESUMO
Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH3). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH3, increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH3. We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes.