RESUMEN

Recent investigations showed that anandamide, the main endogenous ligand of CB1 and CB2 cannabinoid receptors, possesses analgesic, antidepressant and anti-inflammatory effects. In the perspective to treat inflammatory bowel disease (IBD), our approach was to develop new selective CB2 receptor agonists without psychotropic side effects associated to CB1 receptors. In this purpose, a new series of 3-carboxamido-5-aryl-isoxazoles, never described previously as CB2 receptor agonists, was designed, synthesized and evaluated for their biological activity. The pharmacological results have identified great selective CB2 agonists with in vivo anti-inflammatory activity in a DSS-induced acute colitis mouse model.

Asunto(s)

Antiinflamatorios/uso terapéutico , Colitis/tratamiento farmacológico , Isoxazoles/química , Receptor Cannabinoide CB2/agonistas , Animales , Antiinflamatorios/química , Antiinflamatorios/toxicidad , Proliferación Celular/efectos de los fármacos , Colitis/inducido químicamente , Colitis/patología , Sulfato de Dextran/toxicidad , Células HT29 , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Isoxazoles/uso terapéutico , Isoxazoles/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

The relevance of CB(2)-mediated therapeutics is well established in the treatment of pain, neurodegenerative and gastrointestinal tract disorders. Recent works such as the crystallization of class-A G-protein-coupled receptors in a range of active states and the identification of specific anchoring sites for CB(2) agonists challenged us to design a reliable agonist-bound homology model of CB(2) receptor. Docking-scoring enrichment tests of a high-throughput virtual screening of 140 compounds led to 13 hits within the micromolar affinity range. Most of these hits behaved as CB(2) agonists, among which two novel full agonists emerged. Although the main challenge was a high-throughput docking run targeting an agonist-bound state of a CB(2) model, a prior 2D ligand-based Bayesian network was computed to enrich the input commercial library for 3D screening. The exclusive discovery of agonists illustrates the reliability of this agonist-bound state model for the identification of polar and aromatic amino acids as new agonist-modulated CB(2) features to be integrated in the wide activation pathway of G-protein-coupled receptors.

Asunto(s)

Receptor Cannabinoide CB2/agonistas , Teorema de Bayes , Sitios de Unión , Enlace de Hidrógeno , Ligandos , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Curva ROC , Receptor Cannabinoide CB2/metabolismo

RESUMEN

In line of a scaffold hopping strategy of pyrazole structures, especially known as potent CB(2) receptor antagonists, we exploited an original and convergent synthesis of a new class of C4-benzyl pyrazolines and derivatives from readily available hydrazones and enones (two or three steps). Making use of a mixture of resin supported reagents strategy an efficient domino process allowed the easy construction of various dihydropyrazoles in 63-83% yields. The obtained family of pyrazolines featured significant hCB(2)/hCB(1) selectivity in favor of hCB(2) receptors while more than 1000-3000 nM affinity was only measured for hCB(1) receptors. This is closely related to pyrazole SR144528 inverse agonist/antagonist, although a partial agonist behavior in the [(35)S]-GTPγS binding assay was mainly measured in our case pointing out a functional switch in action. Furthermore, this hCB(2) selectivity is unique within the pyrazoline CB ligands although the affinity ranging from 251 to 689 nM remains to be improved which give, however, an opportunity for further structure-activity relationship.

Asunto(s)

Pirazoles/farmacología , Receptor Cannabinoide CB2/antagonistas & inhibidores , Unión Competitiva/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Receptor Cannabinoide CB1/antagonistas & inhibidores , Relación Estructura-Actividad

RESUMEN

Further on to our earlier work on the 4-oxo-1,4-dihydropyridine, we describe herein our strategy to get access to potent selective CB2 receptor agonists. Thus, we designed and synthesized 29 compounds, evaluated on both hCB1 and hCB2 cannabinoid receptors, and assessed 11 of them in the TNBS-induced colitis model in mice. Compound 48 was found to be the most efficient of our series, exhibiting an exquisite protection against experimental colitis, superior to the one observed after treatment with Pentasa.

Asunto(s)

Adamantano/análogos & derivados , Adamantano/síntesis química , Antiinflamatorios no Esteroideos/síntesis química , Colitis/prevención & control , Piridinas/síntesis química , Receptor Cannabinoide CB2/agonistas , Adamantano/química , Adamantano/farmacología , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Unión Competitiva , Proteínas Sanguíneas/metabolismo , Células CHO , Colitis/inducido químicamente , Colitis/patología , Cricetinae , Cricetulus , Humanos , Absorción Intestinal , Ligandos , Ratones , Microsomas Hepáticos/metabolismo , Unión Proteica , Piridinas/química , Piridinas/farmacología , Ensayo de Unión Radioligante , Relación Estructura-Actividad , Ácido Trinitrobencenosulfónico

RESUMEN

INTRODUCTION: The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) plays a major regulatory function of genes involved in energy metabolism and is a therapeutic target for dyslipidemia. The last decade provided a constellation of findings demonstrating that PPARα behaves as a modulator of both acute and chronic inflammation. PPARα became a rational potential therapeutic target for the treatment of inflammatory disorders. AERAS COVERED: The ability of PPARα to control inflammatory signaling pathways via a diversity of molecular mechanisms is discussed. This review is especially focused on the global action of PPARα on inflammation in several tissues from data obtained in numerous cell types and in vivo models exposed to inflammatory stimuli. EXPERT OPINION: Available PPARα agonists currently used in clinic belong to the class of hypolipidemic drugs but were not expected and not designed to act as anti-inflammatory drugs. To date, accumulating preclinical suggest evidence promising benefits when considering PPARα as a drug target to treat inflammatory disorders. However, clinical studies are needed to validate this concept. Drug design should also be directed toward the elaboration of PPARα agonists more specifically active in the control inflammatory signaling.

Asunto(s)

Inflamación/metabolismo , PPAR alfa/metabolismo , Animales , Humanos , Ligandos , Conformación Proteica

RESUMEN

Glucocorticoid receptor alpha (GRalpha) and peroxisome proliferator-activated receptor alpha (PPARalpha) are transcription factors with clinically important immune-modulating properties. Either receptor can inhibit cytokine gene expression, mainly through interference with nuclear factor kappaB (NF-kappaB)-driven gene expression. The present work aimed to investigate a functional cross-talk between PPARalpha- and GRalpha-mediated signaling pathways. Simultaneous activation of PPARalpha and GRalpha dose-dependently enhances transrepression of NF-kappaB-driven gene expression and additively represses cytokine production. In sharp contrast and quite unexpectedly, PPARalpha agonists inhibit the expression of classical glucocorticoid response element (GRE)-driven genes in a PPARalpha-dependent manner, as demonstrated by experiments using PPARalpha wild-type and knockout mice. The underlying mechanism for this transcriptional antagonism relies on a PPARalpha-mediated interference with the recruitment of GRalpha, and concomitantly of RNA polymerase II, to GRE-driven gene promoters. Finally, the biological relevance of this phenomenon is underscored by the observation that treatment with the PPARalpha agonist fenofibrate prevents glucocorticoid-induced hyperinsulinemia of mice fed a high-fat diet. Taken together, PPARalpha negatively interferes with GRE-mediated GRalpha activity while potentiating its antiinflammatory effects, thus providing a rationale for combination therapy in chronic inflammatory disorders.

Asunto(s)

Glucocorticoides/farmacología , FN-kappa B/metabolismo , PPAR alfa/metabolismo , Receptores de Glucocorticoides/metabolismo , Activación Transcripcional , Animales , Línea Celular Tumoral , Grasas de la Dieta/administración & dosificación , Fenofibrato/farmacología , Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hiperinsulinismo/inducido químicamente , Hiperinsulinismo/prevención & control , Ratones , Ratones Noqueados , PPAR alfa/agonistas , PPAR alfa/antagonistas & inhibidores , PPAR alfa/genética , Regiones Promotoras Genéticas , Receptores de Glucocorticoides/agonistas

RESUMEN

Hypertriglyceridemia is an independent risk factor for coronary artery disease. Because apolipoprotein (Apo)A5 regulates plasma triglyceride levels, we investigated the impact of human (h)ApoA5 on atherogenesis. The influence of hApoA5 transgenic expression was studied in the ApoE2 knock-in mouse model of mixed dyslipidemia. Our results demonstrate that hApoA5 lowers plasma triglyceride levels in Western diet-fed ApoE2 knock-in mice. Moreover, atherosclerotic lesion development was significantly decreased in the hApoA5 transgenic mice. Finally, pharmacologic activation of hApoA5 expression by the peroxisome proliferator-activated receptor-alpha agonist fenofibrate resulted in an enhanced atheroprotection. These results identify an atheroprotective role of hApoA5 in a mouse model of mixed dyslipidemia.

Asunto(s)

Apolipoproteínas A/genética , Apolipoproteínas A/metabolismo , Aterosclerosis/fisiopatología , Dislipidemias/fisiopatología , Triglicéridos/sangre , Alimentación Animal , Animales , Apolipoproteína A-V , Apolipoproteína E2/sangre , Apolipoproteína E2/genética , Aterosclerosis/metabolismo , Colesterol/sangre , Grasas de la Dieta/farmacología , Modelos Animales de Enfermedad , Dislipidemias/tratamiento farmacológico , Dislipidemias/metabolismo , Femenino , Fenofibrato/farmacología , Homeostasis/fisiología , Humanos , Hipolipemiantes/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos

RESUMEN

The acute-phase response is characterized by the modulation of liver expression of many proteins involved in a diversity of biological functions. Among them, some are associated with the pathology of atherosclerosis. We previously found that peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists attenuate the IL-6 induction of acute-phase response gene expression in vitro and in vivo. In the current work, we found a PPARalpha-dependent regulation of hepatic acute-phase response stimulated by IL-1. We also found that IL-1-stimulated expression of secondary wave cytokines such as IL-6 is prevented upon PPARalpha activation in liver. Direct involvement of hepatic PPARalpha was demonstrated using a liver-restricted expression of PPARalpha in mice. IL-1- or IL-6-mediated acute-phase response was inhibited by fenofibrate treatment in liver-specific PPARalpha-expressing mice but not in PPARalpha-deficient mice. In addition, we demonstrated that PPARalpha exerts a general control of the acute-phase response by using an inflammation/infection model of lipopolysaccharide. In such a context, liver-specific PPARalpha-expressing mice displayed lower circulating levels of TNF, IL-1, and IL-6 cytokines. We found a distal repercussion of this lowering at the vascular wall level as illustrated by a decreased expression of adhesion molecules in aorta. In conclusion, we demonstrated that through a specific liver action, PPARalpha behaves as a modulator of systemic inflammation and of the associated vascular response.

Asunto(s)

Hígado/fisiología , PPAR alfa/fisiología , Proteínas de Fase Aguda/genética , Animales , Aterosclerosis/etiología , Aterosclerosis/fisiopatología , Núcleo Celular/fisiología , Fenofibrato/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/fisiopatología , Interleucina-1/farmacología , Interleucina-8/genética , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , ARN/genética

RESUMEN

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor. PPARalpha regulates lipid and glucose metabolism and controls the inflammatory response. Recently, we have shown that PPARalpha is a short-lived protein degraded by the ubiquitin-proteasome system. In this study, we have analysed the effects of interaction with RXRalpha, CBP, and N-CoR and also the implication of phosphorylation on ubiquitination and stability of PPARalpha. Our results show that interaction of PPARalpha with RXRalpha or CBP leads to an increase in the turnover of the protein. In contrast, interaction with the corepressor N-CoR, which inhibits its transcriptional activity, leads to a stabilization of the protein. Interestingly, treatment of cells with an inhibitor of Ser/Thr phosphatases known to lead to hyperphosphorylation of PPARalpha induces its transcriptional activity which is accompanied by a stabilization of the protein. These data indicate that heterodimerization, recruitment of cofactors, and post-translational modifications can modulate the stability of PPARalpha.

Asunto(s)

Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Animales , Células COS , Dimerización , Ligandos , Fosforilación , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Receptores X Retinoide , Factores de Transcripción/metabolismo , Ubiquitina/metabolismo

RESUMEN

Peroxisome proliferator-activated receptor (PPAR) alpha is a nuclear receptor implicated in several physiological processes such as lipid and lipoprotein metabolism, glucose homeostasis, and the inflammatory response. PPARalpha is activated by natural fatty acids and synthetic compounds like fibrates. PPARalpha activity has been shown to be modulated by its phosphorylation status. PPARalpha is phosphorylated by kinases such as the MAPKs and cAMP-activated protein kinase A. In this report, we show that protein kinase C (PKC) inhibition impairs ligand-activated PPARalpha transcriptional activity. Furthermore, PKC inhibition decreases PPARalpha ligand-induction of its target genes including PPARalpha itself and carnitine palmitoyltransferase I. By contrast, PKC inhibition enhances PPARalpha transrepression properties as demonstrated using the fibrinogen-beta gene as model system. Finally, PKC inhibition decreases PPARalpha phosphorylation activity of hepatocyte cell extracts. In addition, PPARalpha purified protein is phosphorylated in vitro by recombinant PKCalpha and betaII. The replacement of serines 179 and 230 by alanine residues reduces the phosphorylation of the PPARalpha protein. The PPARalpha S179A-S230A protein displays an impaired ligand-induced transactivation activity and an enhanced trans-repression activity. Altogether, our data indicate that the PKC signaling pathway acts as a molecular switch dissociating the transactivation and transrepression functions of PPARalpha, which involved phosphorylation of serines 179 and 230.

Asunto(s)

PPAR alfa/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Activación Transcripcional , Alanina/genética , Alanina/metabolismo , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Fibrinógeno/genética , Expresión Génica , Humanos , Ligandos , Mutagénesis Sitio-Dirigida/genética , PPAR alfa/química , PPAR alfa/genética , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Serina/genética , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos