gw-7647 and pirinixic-acid

Peroxisome proliferator-activated receptors (PPARs) are attractive targets for the treatment of the metabolic syndrome. Especially a combination of PPARα and PPARγ agonistic activity seems worthwhile to be pursued. Herein we present the design and synthesis of a series of pirinixic acid derivatives as potent PPARα particularly dual PPARα/γ agonists with 2-((4-chloro-6-((4-(phenylamino)phenyl)amino)pyrimidin-2-yl)thio)octanoicacid having the highest potential. Our investigations based on molecular docking and structure-activity relationship (SAR) studies elucidated structural determinants affecting the potency at PPARα. A diphenylamine-scaffold seems to play a key role. Careful in silico analysis revealed an essential role for a hydrogen bond between the diphenylamine and a water cluster. We confirmed this hypothesis using a mutated PPARα LBD in our transactivation assay to disrupt the water cluster and to validate the proposed interaction.

Topics: Dose-Response Relationship, Drug; Humans; Hydrogen Bonding; Molecular Docking Simulation; Molecular Structure; Mutagenesis, Site-Directed; PPAR alpha; Pyrimidines; Structure-Activity Relationship

Peroxisome proliferator-activated receptor gamma (PPARgamma) is involved in glucose and lipid homeostasis. PPARgamma agonists are in clinical use for the treatment of type 2 diabetes. Lately, a new class of selective PPARgamma modulators (SPPARgammaMs) was developed, which are believed to show less side effects than full PPARgamma agonists. We have previously shown that alpha-substitution of pirinixic acid, a moderate agonist of PPARalpha and PPARgamma, leads to low micromolar active balanced dual agonists of PPARalpha and PPARgamma. Herein we present modifications of pirinixic acid leading to subtype-selective PPARgamma agonists and furthermore the development of a selective PPARgamma modulator guided by molecular docking studies.

Topics: Drug Design; Models, Molecular; PPAR gamma; Pyrimidines

PPAR alpha is a ligand-activated transcription factor belonging to the nuclear receptor superfamily. PPAR alpha is involved in the regulation of in vivo triglyceride levels, presumably through its effects on fatty acid and lipoprotein metabolism. Some nuclear receptors have been involved in rapid effects mediated by non-genomic mechanisms. In this paper, we report the rapid non-genomic effects of PPAR alpha ligands on the intracellular calcium concentration ([Ca2+]i), mitochondrial function, reactive oxygen species (ROS) generation, and secretion of insulin in freshly isolated mouse islets of Langerhans. The hypolipidemic fibrate PPAR alpha agonist WY-14 643 decreased the glucose-induced calcium oscillations in intact islets. This effect was mimicked by the synthetic agonist GW7647 and the endogenous agonist oleylethanolamide. The WY-14 643 action was rapid in onset (5 min) and was still produced in the presence of protein and mRNA synthesis inhibitors, cycloheximide, and actinomycin-d. This suggests that it is independent of gene transcription. In addition, WY-14 623 impaired mitochondrial function, increased ROS formation and decreased insulin release. PPAR alpha is present in beta-cells, mainly in the cytosol and nucleus, with a small subpopulation localized in the plasma membrane. However, the presence of the PPAR alpha ligand effects in mice bearing a disrupted Ppar alpha gene raises the possibility that the rapid effects of the agonists in pancreatic beta-cells are independent of the receptor. We conclude that PPAR alpha agonists produce a decrease in glucose-induced [Ca2+]i signals and insulin secretion in beta-cells through a rapid, non-genomic mechanism.

Topics: Animals; Anticholesteremic Agents; Butyrates; Calcium; Insulin; Insulin Secretion; Insulin-Secreting Cells; Male; Mediator Complex Subunit 1; Mice; Mitochondria; Oleic Acids; Phenylurea Compounds; Pyrimidines; Reactive Oxygen Species; Transcription Factors

Increasing HDL cholesterol concentrations by stimulating de-novo apolipoprotein A-I (apoA-I) production in the liver and/or in the small intestine is a potential strategy to reduce coronary heart disease risk. Although there is quite some knowledge concerning regulatory effects in the liver, less is known concerning potential agents that could elevate de-novo apoA-I production in the small intestine.. Therefore, we compared side-by-side effects of various peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma, retinoid-X-receptor alpha, and farnesoid-X-receptor agonists on de-novo apoA-I production in differentiated CaCo-2 and HepG2 cells.. For PPARa agonists, we showed that GW7647 elevated apoA-I concentrations in the medium of both cell models, whereas WY14643 elevated only de-novo apoA-I concentrations in differentiated CaCo-2 cells. Unexpectedly, fenofibric acid lowered apoA-I medium concentrations in both cell lines, which could not be explained by a lack of PPAR transactivation or a lack of retinoid-X-receptor a activation. For farnesoid-X-receptor agonists, chenodeoxycholic acid strongly reduced apoA-I concentrations both in differentiated CaCo-2 and HepG2 cells, whereas GW4064 and taurocholate only lowered apoA-I in CaCo-2 cells (GW4064) or in HepG2 cells (taurocholate). However, overall effects of all individual components on apoA-I production in differentiated CaCo-2 and HepG2 cells were highly correlated (r = 0.68; P = 0.037; N=9).. We conclude that differentiated CaCo-2 cells are suitable models to study de-novo small intestinal apoA-I production in vitro enabling the possibility to screen for potential bioactive dietary components. This cell model may also determine small-intestinal-specific effects, as some discrepancy was found between both cell models.

Topics: Anticholesteremic Agents; Apolipoprotein A-I; Butyrates; Caco-2 Cells; Cell Differentiation; Drug Evaluation, Preclinical; Hep G2 Cells; Humans; Intestine, Small; Isoxazoles; Models, Biological; Peroxisome Proliferators; Phenylurea Compounds; Pyrimidines; Receptors, Cytoplasmic and Nuclear

Peroxisome proliferator-activated receptors (PPARalpha, -beta and -gamma) are nuclear receptors involved in transcriptional regulation of lipid and energy metabolism. Since the energy demand increases when cardiac progenitor cells are developing rhythmic contractile activity, PPAR activation may play a critical role during cardiomyogenesis of embryonic stem (ES) cells. It is shown that ES cells express PPARalpha, -beta, and -gamma mRNA during differentiation of ES cells towards cardiac cells. Treatment with PPARalpha agonists (WY14643, GW7647, and ciprofibrate) significantly increased cardiomyogenesis and expression of the cardiac genes MLC2a, ANP, MHC-beta, MLC2v, and cardiac alpha-actin. Furthermore, WY14643 increased PPARalpha gene expression and the expression of the cardiogenic transcription factors GATA-4, Nkx2.5, DTEF-1, and MEF 2C. In contrast, the PPARalpha antagonist MK886 decreased cardiomyogenesis, whereas the PPARbeta agonist L-165,041 as well as the PPARgamma agonist GW1929 were without effects. Treatment with PPARalpha, but not PPARbeta, and PPARgamma agonists and MK886, resulted in generation of reactive oxygen species (ROS), which was inhibited in the presence of the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin and the free radical scavengers vitamin E and N-(2-mercapto-propionyl)-glycine (NMPG), whereas the mitochondrial complex I inhibitor rotenone was without effects. The effect of PPARalpha agonists on cardiomyogenesis of ES cells was abolished upon preincubation with free radical scavengers and NADPH oxidase inhibitors, indicating involvement of ROS in PPARalpha, mediated cardiac differentiation. In summary, our data indicate that stimulation of PPARalpha but not PPARbeta and -gamma enhances cardiomyogenesis in ES cells using a pathway that involves ROS and NADPH oxidase activity.

Topics: Animals; Butyrates; Cell Differentiation; Cells, Cultured; Clofibric Acid; Embryonic Stem Cells; Enzyme Inhibitors; Fibric Acids; Free Radical Scavengers; Gene Expression; Gene Expression Regulation, Developmental; Heart; Immunohistochemistry; Indoles; Mice; Myocytes, Cardiac; NADPH Oxidases; Peroxisome Proliferators; Phenylurea Compounds; PPAR alpha; PPAR gamma; PPAR-beta; Pyrimidines; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Oleic acid synthesized by astrocytes behaves as a neurotrophic factor for neurons, up-regulating the molecular markers of axonal and dendritic outgrowth, growth-associated protein 43 and microtubule-associated protein 2. In this work, the nature of the receptor involved in this neurotrophic effect was investigated. As oleic acid has been reported to be a ligand and activator of the peroxisome proliferator-activated receptor (PPAR), we focus on this family of receptors. Our results show that PPARalpha, beta/delta, and gamma are expressed in neurons in culture. However, only the agonists of PPARalpha, Wy14643, GW7647 and oleoylethanolamide, promoted neuronal differentiation, while PPAR beta/delta and gamma agonists did not modify neuronal differentiation. Consequently, we investigated the involvement of PPARalpha (Nr1c1) in oleic acid-induced neuronal differentiation. Our results indicate that oleic acid activates PPARalpha in neurons. In addition, the effect of oleic acid on neuronal morphology, growth-associated protein 43 and microtubule-associated protein 2 expression decreases in neurons after PPARalpha has been silenced by small interfering RNA. Taken together, our results suggest that PPARalpha could be the receptor for oleic acid in neurons, further broadening the range of functions attributed to this family of transcription factors. Although several works have reported that PPARalpha could be involved in neuroprotection, the present work provides the first evidence suggesting a role of PPARalpha in neuronal differentiation.

Topics: Animals; Butyrates; Cell Differentiation; Cell Shape; Cells, Cultured; Central Nervous System; GAP-43 Protein; Growth Cones; Microtubule-Associated Proteins; Nerve Growth Factors; Neurites; Neurons; Oleic Acid; Phenylurea Compounds; PPAR alpha; Pyrimidines; Rats; Rats, Wistar; RNA, Small Interfering

The peroxisome proliferator-activated receptor-alpha (PPARalpha) plays important roles in lipid metabolism, inflammation, and atherosclerosis. PPARalpha ligands have been shown to reduce cardiovascular events in high-risk subjects. PPARalpha expression by arterial cells, including macrophages, may exert local antiatherogenic effects independent of plasma lipid changes.. To examine the contribution of PPARalpha expression by bone marrow-derived cells in atherosclerosis, male and female low-density lipoprotein receptor-deficient (LDLR(-/-)) mice were reconstituted with bone marrow from PPARalpha(-/-) or PPARalpha(+/+) mice and challenged with a high-fat diet. Although serum lipids and lipoprotein profiles did not differ between the groups, the size of atherosclerotic lesions in the distal aorta of male and female PPARalpha(-/-) --> LDLR(-/-) mice was significantly increased (44% and 46%, respectively) compared with controls. Male PPARalpha(-/-) --> LDLR(-/-) mice also had larger (44%) atherosclerotic lesions in the proximal aorta than male PPARalpha(+/+) --> LDLR(-/-) mice. Peritoneal macrophages from PPARalpha(-/-) mice had increased uptake of oxidized LDL and decreased cholesterol efflux. PPARalpha(-/-) macrophages had lower levels of scavenger receptor B type I and ABCA1 protein expression and an accelerated response of nuclear factor-kappaB-regulated inflammatory genes. A laser capture microdissection analysis verified suppressed scavenger receptor B type I and increased nuclear factor-kappaB gene expression levels in vivo in atherosclerotic lesions of PPARalpha(-/-) --> LDLR(-/-) mice compared with the lesions of control PPARalpha(+/+) --> LDLR(-/-) mice.. These data demonstrate that PPARalpha expression by macrophages has antiatherogenic effects via modulation of cell cholesterol trafficking and inflammatory activity.

Topics: Animals; Aortic Diseases; Atherosclerosis; Biological Transport; Bone Marrow Transplantation; Butyrates; Cells, Cultured; Female; Gene Expression Regulation; Inflammation; Lipid Metabolism; Lipoproteins, LDL; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenylurea Compounds; PPAR alpha; Pyrimidines; Radiation Chimera; Receptors, LDL

Nitric oxide (NO) production through the inducible nitric oxide synthase (iNOS) pathway is increased in response to pro-inflammatory cytokines and bacterial products. In inflammation, NO has pro-inflammatory and regulatory effects. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear steroid receptor superfamily, regulate not only metabolic but also inflammatory processes. The aim of the present study was to investigate the role of PPARalpha in the regulation of NO production and iNOS expression in activated macrophages.. The effects of PPARalpha agonists were investigated on iNOS mRNA and protein expression, on NO production and on the activation of transcription factors NF-kappaB and STAT1 in J774 murine macrophages exposed to bacterial lipopolysaccharide (LPS).. PPARalpha agonists GW7647 and WY14643 reduced LPS-induced NO production in a dose-dependent manner as measured by the accumulation of nitrite into the culture medium. However, PPARalpha agonists did not alter LPS-induced iNOS mRNA expression or activation of NF-kappaB or STAT1 which are important transcription factors for iNOS. Nevertheless, iNOS protein levels were reduced by PPARalpha agonists in a time-dependent manner. The reduction was markedly greater after 24 h incubation than after 8 h incubation. Treatment with the proteasome inhibitors, lactacystin or MG132, reversed the decrease in iNOS protein levels caused by PPARalpha agonists.. The results suggest that PPARalpha agonists reduce LPS-induced iNOS expression and NO production in macrophages by enhancing iNOS protein degradation through the proteasome pathway. The results offer an additional mechanism underlying the anti-inflammatory effects of PPARalpha agonists.

Topics: Acetylcysteine; Animals; Butyrates; Cells, Cultured; Dose-Response Relationship, Drug; Leupeptins; Lipopolysaccharides; Macrophages; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Phenylurea Compounds; PPAR alpha; Proteasome Endopeptidase Complex; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; STAT1 Transcription Factor

The fatty-acid ethanolamide, oleoylethanolamide (OEA), is a naturally occurring lipid that regulates feeding and body weight [Rodriguez de Fonseca, F., Navarro, M., Gomez, R., Escuredo, L., Nava, F., Fu, J., Murillo-Rodriguez, E., Giuffrida, A., LoVerme, J., Gaetani, S., Kathuria, S., Gall, C., Piomelli, D., 2001. An anorexic lipid mediator regulated by feeding. Nature 414, 209-212], and serves as an endogenous agonist of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) [Fu, J., Gaetani, S., Oveisi, F., Lo Verme, J., Serrano, A., Rodriguez De Fonseca, F., Rosengarth., A., Luecke, H., Di Giacomo, B., Tarzia, G., Piomelli, D., 2003. Oleoylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-alpha. Nature 425, 90-93], a ligand-activated transcription factor that regulates several aspects of lipid metabolism [. Peroxisome proliferator-activated receptors: nuclear control of metabolism. Endocr. Rev. 20, 649-688]). OEA reduces food intake in wild-type mice, but not in mice deficient in PPAR-alpha (PPAR-alpha(-/-)), an effect that is also observed with the PPAR-alpha agonists Wy-14643 and GW7647 [Brown, P.J., Chapman, J.M., Oplinger, J.A., Stuart, L.W., Willson, T.M. and Wu, Z., 2000. Chemical compounds as selective activators of PPAR-alpha. PCT Int. Appl., 32; . The PPARs: from orphan receptors to drug discovery. J. Med. Chem. 43, 527-550]. By contrast, specific agonists of PPAR-delta/beta (GW501516) or PPAR-gamma (ciglitazone) have no such effect. In obese Zucker rats, which lack functional leptin receptors, OEA reduces food intake and lowers body-weight gain along with plasma lipid levels. Similar effects are seen in diet-induced obese rats and mice. In the present study, we report that subchronic OEA treatment (5mgkg(-1), intraperitoneally, i.p., once daily for two weeks) in Zucker rats initiates transcription of PPAR-alpha and other PPAR-alpha target genes, including fatty-acid translocase (FAT/CD36), liver fatty-acid binding protein (L-FABP), and uncoupling protein-2 (UCP-2). Moreover, OEA decreases neutral lipid content in hepatocytes, as assessed by Oil red O staining, as well as serum cholesterol and triglyceride levels. The results suggest that OEA regulates lipid metabolism and that this effect may contribute to its anti-obesity properties.

Topics: Animals; Body Weight; Butyrates; CD36 Antigens; Cholesterol; Coenzyme A Ligases; Eating; Endocannabinoids; Fatty Acid-Binding Proteins; Hepatocytes; Hyperlipidemias; Ion Channels; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondrial Proteins; Obesity; Oleic Acids; Phenylurea Compounds; PPAR alpha; Pyrimidines; Rats; Rats, Inbred WF; Rats, Zucker; RNA, Messenger; Thiazoles; Thiazolidinediones; Triglycerides; Uncoupling Protein 2