Marco Giudici

Synthesis, Characterization and Biological Evaluation of Ureidofibrate-Like Derivatives Endowed with Peroxisome Proliferator-Activated Receptor Activity

A series of ureidofibrate-like derivatives was prepared and assayed for their PPAR functional activity. A calorimetric approach was used to characterize PPARγ-ligand interactions, and docking experiments and X-ray studies were performed to explain the observed potency and efficacy. R-1 and S-1 were selected to evaluate several aspects of their biological activity. In an adipogenic assay, both enantiomers increased the expression of PPARγ target genes and promoted the differentiation of 3T3-L1 fibroblasts to adipocytes. In vivo administration of these compounds to insulin resistant C57Bl/6J mice fed a high fat diet reduced visceral fat content and body weight. Examination of different metabolic parameters showed that R-1 and S-1 are insulin sensitizers. Notably, they also enhanced the expression of hepatic PPARα target genes indicating that their in vivo effects stemmed from an activation of both PPARα and γ. Finally, the capability of R-1 and S-1 to inhibit cellular proliferation in colon cancer cell lines was also evaluated.

Linking epigenetics to lipid metabolism: Focus on histone deacetylases

Abstract A number of recent studies revealed that epigenetic modifications play a central role in the regulation of lipid and of other metabolic pathways such as cholesterol homeostasis, bile acid synthesis, glucose and energy metabolism. Epigenetics refers to aspects of genome functions regulated in a DNA sequence-independent fashion. Chromatin structure is controlled by epigenetic mechanisms through DNA methylation and histone modifications. The main modifications are histone acetylation and deacetylation on specific lysine residues operated by two different classes of enzymes: Histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. The interaction between these enzymes and histones can activate or repress gene transcription: Histone acetylation opens and activates chromatin, while deacetylation of histones and DNA methylation compact chromatin making it transcriptionally silent. The new evidences on the importance of HDACs in the regulation of lipid and other metabolic pathways will open new perspectives in the comprehension of the pathophysiology of metabolic disorders.

The sirtuin class of histone deacetylases: Regulation and roles in lipid metabolism

After the completion of the human genome sequence and that from many other organisms, last decade has witnessed a spectacular gain of knowledge on gene functions. These studies provided new insights on the roles of genes in physiology and disease. Nonetheless, the availability of genetically modified models and of “omics” technologies such as next generation sequencing unveiled clear evidences on epigenetic regulation of many cellular functions. At this regard, sirtuins, belonging to class III histone deacetylase family, have emerged as regulators of metabolism as well as other cellular processes and seem ideally suited as targets of future therapeutical interventions. This review deals on general aspects of the biology of sirtuins and focuses on their relevance in lipid metabolism in different tissues, pointing to their exploitation as potential pharmacological targets of compounds that could be used as new therapeutic alternatives in several disorders ranging from type 2 diabetes and obesity to age‐related cardiovascular and neurodegenerative diseases.

Molecular determinants for nuclear receptors selectivity: chemometric analysis, dockings and site-directed mutagenesis of dual peroxisome proliferator-activated receptors α/γ agonists.

A series of previously synthesized chiral derivatives of clofibric and phenylacetic acids, acting as dual agonists towards the peroxisome proliferator-activated receptors (PPARs) α and γ, was taken into account, and the efficacy of these compounds was analyzed by means of 2D-, 3D-QSAR and docking studies with the goal to gain deeper insights into the three-dimensional determinants governing ligands selectivity for PPARs. By multiregressional analysis a correlation between the lipophilicity and PPARα activity was found, whereas for PPARγ the correlation was achieved once efficacy was related to the presence of polar groups on agonists scaffold. Docking of these compounds further corroborated this hypothesis, and then provided a valid support for subsequent chemometric analysis and pharmacophore models development for both receptors subtypes. Computational results suggested site directed mutagenesis experiments which confirmed the importance of amino acid residues in PPAR activity, allowing the identification of critical hotspots most likely taking over PPARs selectivity.

Attenuation of diet-induced obesity and induction of white fat browning with a chemical inhibitor of histone deacetylases

Background/objectives:In the last decade, a strict link between epigenetics and metabolism has been demonstrated. Histone deacetylases (HDACs) have emerged as key epigenetic regulators involved in metabolic homeostasis in normal and pathologic conditions. Here we investigated the effect of the class I HDAC inhibitor MS-275 in a model of obesity induced by a high-fat diet (HFD).Methods:C57BL6/J male mice were fed HFD for 17 weeks and then randomized in two groups, treated intraperitoneally with vehicle dimethylsulfoxide (DMSO) or with the class I selective HDAC inhibitor MS-275 every other day for 22 days. Glucose tolerance test and measurement of body temperature during cold exposure were performed. Adipose tissues and liver were phenotypically characterized through histological analysis. Gene and protein expression analysis of brown and white adipose tissues (WATs) were performed.Results:MS-275 treated mice showed 10% reduction of body weight, lower adipocyte size and improved glucose tolerance. Inhibition of class I HDAC determined reduction of adipocyte size and of fat mass, paralleled by higher expression of adipose functionality markers and by increased rate of lipolysis and fatty acid β-oxidation. MS-275 also promoted thermogenic capacity, related to ‘browning’ of visceral and subcutaneous WAT, showing increased expression of uncoupling protein 1. In brown adipose tissue, we observed limited effects on gene expression and only reduction of brown adipocyte size.Conclusions:This study provides evidence that class I HDAC inhibition stimulated functionality and oxidative potential of adipose tissue, improving glucose tolerance and ameliorating the metabolic profile in diet-induced obese mice.

Fenofibrate attenuates cardiac and renal alterations in young salt-loaded spontaneously hypertensive stroke-prone rats through mitochondrial protection

Objectives: The simultaneous presence of cardiac and renal diseases is a pathological condition that leads to increased morbidity and mortality. Several lines of evidence have suggested that lipid dysmetabolism and mitochondrial dysfunction are pathways involved in the pathological processes affecting the heart and kidney. In the salt-loaded spontaneously hypertensive stroke-prone rat (SHRSP), a model of cardiac hypertrophy and nephropathy that shows mitochondrial alterations in the myocardium, we evaluated the cardiorenal effects of fenofibrate, a peroxisome proliferator-activated receptor alpha (PPAR&agr;) agonist that acts by modulating mitochondrial and peroxisomal fatty acid oxidation. Methods: Male SHRSPs aged 6–7 weeks were divided in three groups: standard diet (n = 6), Japanese diet with vehicle (n = 6), and Japanese diet with fenofibrate 150 mg/kg/day (n = 6) for 5 weeks. Cardiac and renal functions were assessed in vivo by MRI, ultrasonography, and biochemical assays. Mitochondria were investigated by transmission electron microscopy, succinate dehydrogenase (SDH) activity, and gene expression analysis. Results: Fenofibrate attenuated cardiac hypertrophy, as evidenced by histological and MRI analyses, and protected the kidneys, preventing morphological alterations, changes in arterial blood flow velocity, and increases in 24-h proteinuria. Cardiorenal inflammation, oxidative stress, and cellular senescence were also inhibited by fenofibrate. In salt-loaded SHRSPs, we observed severe morphological mitochondrial alterations, reduced SDH activity, and down-regulation of genes regulating mitochondrial fatty-acid oxidation (i.e. PPAR&agr;, SIRT3, and Acadm). These changes were counteracted by fenofibrate. In vitro, a direct protective effect of fenofibrate on mitochondrial membrane potential was observed in albumin-stimulated NRK-52E renal tubular epithelial cells. Conclusion: The results suggest that the cardiorenal protective effects of fenofibrate in young male salt-loaded SHRSPs are explained by its capacity to preserve mitochondrial function.

Identification of the First PPARα/γ Dual Agonist Able To Bind to Canonical and Alternative Sites of PPARγ and To Inhibit Its Cdk5-Mediated Phosphorylation

A new series of derivatives of the PPARα/γ dual agonist 1 allowed us to identify the ligand ( S)-6 as a potent partial agonist of both PPARα and γ subtypes. X-ray studies in PPARγ revealed two different binding modes of ( S)-6 to the canonical site. However, ( S)-6 was also able to bind an alternative site as demonstrated by transactivation assay in the presence of a canonical PPARγ antagonist and supported from docking experiments. This compound did not activate the PPARγ-dependent program of adipocyte differentiation inducing a very less severe lipid accumulation compared to rosiglitazone but increased the insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Finally, ( S)-6 inhibited the Cdk5-mediated phosphorylation of PPARγ at serine 273 that is currently considered the mechanism by which some PPARγ partial agonists exert antidiabetic effects similar to thiazolidinediones, without showing their typical side effects. This is the first PPARα/γ dual agonist reported to show this inhibitory effect representing the potential lead of a new class of drugs for treatment of dyslipidemic type 2 diabetes.

Site-Directed Mutagenesis to Study the Role of Specific Amino Acids in the Ligand Binding Domain of PPARs

The role of certain amino acids in the interactions of ligands with their cognate nuclear receptors is usually achieved by the resolution of the crystal structure of the receptor complexed with the ligand. As a complementary functional approach, site-directed mutagenesis, a technique broadly used in molecular biology, allows the assessment of the role of a specific amino acid in determining the interaction with a specific ligand. This method makes it possible to evaluate several mutations of a key amino acid for ligand binding and to determine the relationship between protein structure and ligand interaction. Here, we describe an application of this technique to evaluate different point mutations on the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) in the absence or presence of chemically different ligands.