Quantification of tagitinin C in Tithonia diversifolia by reversed-phase high-performance liquid chromatography

A simple, rapid and reliable reversed-phase high-performance liquid chromatographic method for the determination of tagitinin C, an anti-plasmodial sesquiterpene lactone isolated from the aerial parts of Tithonia diversifolia, has been developed. The assay has been used to quantify tagitinin C in various extracts of the aerial parts of T. diversifolia.     

2-(2-Bromophenyl)-formononetin and 2-heptyl-formononetin are PPARγ partial agonists and reduce lipid accumulation in 3T3-L1 adipocytes

Isoflavones are bioactive compounds that have been shown to decrease lipid accumulation in vitro. However, the knowledge of the isoflavone formononetin is limited. The aim of the study was to assess the effects of formononetin and its two synthetic analogues, 2-(2-bromophenyl)-formononetin and 2-heptyl-formononetin, on lipid accumulation in 3T3-L1 adipocytes and investigate possible mechanisms. Formononetin and the two analogues were added day 0–8 or day 8–10 of the differentiation period, and lipid accumulation, glycerol release and gene expression were measured. Additionally, competitive peroxisome proliferator-activated receptor (PPAR)-γ binding assay, PPARγ transactivation assay and Western blot for phosphorylated AMP-activated protein kinase (AMPK) were performed. Chronic treatment (day 0–8) with formononetin increased lipid accumulation, whereas the two analogues decreased lipid accumulation partly due to decreased differentiation. The two analogues, but not formononetin, also decreased lipid content in mature adipocytes. 2-Heptyl-formononetin increased glycerol release and lipolytic gene expression and decreased lipogenic gene expression. Formononetin did not bind to or activate PPARγ whereas both analogues bound to the receptor and behaved as PPARγ partial agonists in the transactivation assay. Neither of the compounds affected phosphorylation of AMPK. In conclusion, the analogues of formononetin decreased lipid accumulation possibly in part by acting as PPARγ partial agonists.     

Docosahexaenoic acid suppresses the activity of peroxisome proliferator-activated receptors in a colon tumor cell line

Fatty acids are generally considered as agonists for peroxisome proliferator-activated receptors (PPARs). Fatty acids have been shown to bind to and transactivate PPARs; it is not known whether fatty acids act as generalized agonists for PPARs in different cell types, and thus, stimulate the expression of PPAR-regulated target genes. Here, we investigated the potency of unsaturated fatty acids on transactivation of PPRE, DNA-binding activity of PPARs, and the expression of a PPAR-regulated gene product, CD36. Docosahexaenoic acid (DHA) suppressed the basal and PPAR agonist-induced transactivation of PPRE, and DNA binding of PPARs in colon tumor cells (HCT116). The suppression of PPAR transactivation by DHA leads to reduced expression of CD36 in HCT116 cells and human monocytic cells (THP-1) as determined by promoter reporter gene assay and flow cytometric analysis. Our results demonstrate that DHA and other unsaturated fatty acids act as antagonists instead of agonists for transactivation of PPRE and PPAR-regulated gene expression in the cell lines tested. These results suggest that PPAR-mediated gene expression and cellular responses can be dynamically modulated by different types of dietary fatty acids consumed.     

Oleanane-type triterpene saponins from the bark of Aralia elata and their NF-κB inhibition and PPAR activation signal pathway

Two new oleanane-type triterpene saponins, tarasaponin IV (1) and elatoside L (2), and four known; stipuleanoside R(2) (3), kalopanax-saponin F (4), kalopanax-saponin F methylester (5), and elatoside D (6) were isolated from the bark of Aralia elata. Kalopanax-saponin F methyl ester was isolated from nature for the first time. Their chemical structures were elucidated using the chemical and physical methods as well as good agreement with those of reported in the literature. Oleanane-type triterpene saponins are the main component of A. elata. All compounds were investigated the anti-inflammatory activity. We measured their inhibition of NF-κB and activation of PPARs activities in HepG2 cells using luciferase reporter system. As results, compounds 2 and 4 were found to inhibit NF-κB activation stimulated by TNFα in a dose-dependent manner with IC(50) values of 4.1 and 9.5 μM, respectively, when compared with that of positive control, sulfasalazine (0.9 μM). Compounds 2 and 4 also inhibited TNFα-induced expression of iNOS and COX-2 mRNA. Furthermore, compounds 1-6 were evaluated PPAR activity using PPAR subtype transactivation assays. Among of them, compounds 4-6 significantly increased PPARγ transactivation. However, compounds 4-6 did not activate in any other PPAR subtypes.     

1,3-Diphenyl-1H-pyrazole derivatives as a new series of potent PPARγ partial agonists

A new series of PPARγ partial agonists, 1,3-diphenyl-1H-pyrazole derivatives, were identified using an improved virtual screening scheme combining ligand-centric and receptor-centric methods. An in vitro assay confirmed the nanomolar binding affinity of 1,3-diphenyl-1H-pyrazole derivatives such as SP3415. We also characterized the competitive antagonism of SP3415 against rosiglitazone at micromolar concentrations. They showed a PPARγ partial agonistic activity similar to that of a known PPARγ drug, pioglitazone, in a cell-based transactivation assay. Furthermore, the structure-activity relationships of the pyrazole derivatives were investigated through comparative molecular field analysis and binding mode analysis, which provided new insight concerning their partial agonistic effect on PPARγ.     

Molecular determinants for improved activity at PPARα: Structure–activity relationship of pirinixic acid derivatives,docking study and site-directed mutagenesis of PPARα

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.     

Structure optimization of a new class of PPARγ antagonists

Peroxisome proliferator-activated receptor gamma (PPARγ) modulators have found wide application for the treatment of cancers, metabolic disorders and inflammatory diseases. Contrary to PPARγ agonists, PPARγ antagonists have been much less studied and although they have shown immunomodulatory effects, there is still no therapeutically useful PPARγ antagonist on the market. In contrast to non-competitive, irreversible inhibition caused by 2-chloro-5-nitrobenzanilide (GW9662), the recently described (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl)benzyl)oxy)-benzylidene)-hexanoic acid (MTTB, T-10017) is a promising prototype for a new class of PPARγ antagonists. It exhibits competitive antagonism against rosiglitazone mediated activation of PPARγ ligand binding domain (PPARγLBD) in a transactivation assay in HEK293T cells with an IC₅₀ of 4.3 µM against 1 µM rosiglitazone. The aim of this study was to investigate the structure-activity relationships (SAR) of the MTTB scaffold focusing on improving its physicochemical properties. Through this optimization, 34 new derivatives were prepared and characterized. Two new potent compounds (T-10075 and T-10106) with much improved drug-like properties and promising pharmacokinetic profile were identified.     

Medium chain fatty acids are selective peroxisome proliferator activated receptor (PPAR) γ activators and pan-PPAR partial agonists

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8-C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products.     

Design and synthesis of benzoxazole containing indole analogs as peroxisome proliferator-activated receptor-γ/δ dual agonists

A series of benzoxazole or benzothiazole containing indole analogs, 6-alkoxyindole-2-carboxylic acids and 5-alkoxy-3-indolylacetic acids, were synthesized as novel candidates of PPARγ/δ dual agonists and their ligand activities for PPAR subtypes (α, γ, and δ) were investigated. In transient transactivation assay, several compounds activated PPARγ and δ with little activity of PPARα. Putative binding mode of the compounds 1a and 2a in the active site of PPARγ was similar with that of rosiglitazone and the molecular modeling provides molecular insight to the observed activity.     

Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, independently of PPARγ in human glioma cells

Peroxisome proliferator-activated receptor γ (PPARγ) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPARγ in CGZ-induced cell death was examined. At concentrations of greater than 30 μM, CGZ, a synthetic PPARγ agonist, activated caspase-3 and induced apoptosis in T98G cells. Treatment of T98G cells with less than 30 μM CGZ effectively induced cell death after pretreatment with 30 μM of the PPARγ antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPARγ was down-regulated cells by siRNA, lower concentrations of CGZ (<30 μM) were sufficient to induce cell death, although higher concentrations of CGZ (≥30 μM) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPARγ. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPARγ in glioma cells, by down-regulating Akt activity and inducing MMP collapse.     

巨噬细胞选择性活化的信号通路及其干预措施的研究进展

巨噬细胞在不同环境刺激下分化为经典活化巨噬细胞和选择性活化巨噬细胞,巨噬细胞选择性活化的信号通路包括:JAK/STAT6途径、M2分化成熟的转录调节途径(KLF4的转录调节,PPARs的转录调节)以及Jmjd3表观遗传学调节途径。选择性活化对机体而言是一种保护机制,可以依据上述分子理论予以干预,如:细胞因子、PPARγ完全性激动剂、PPARγ部分性激动剂、微量元素硒以及生活方式等通过IL-4/STAT6/PPARγ途径促进巨噬细胞选择性极化。对巨噬细胞选择性活化的信号通路及其促进措施进行了简述。     

Pseudomonas signal molecule 3-oxo-C12-homoserine lactone interferes with binding of rosiglitazone to human PPARγ

Peroxisome proliferator activated receptor (PPARγ) has been suggested as a target for anti-inflammatory therapy in chronic lung disease, including infection with Pseudomonas aeruginosa. However, the P. aeruginosa signal molecule N-(3-oxo-dodecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) has been reported to inhibit function of PPARs in mammalian cells. This suggests that binding of 3-oxo-C12-HSL to PPARs could increase inflammation during P. aeruginosa infection, particularly if it could compete for binding with other PPAR ligands. We investigated the ability of 3-oxo-C12-HSL to bind to a PPARγ ligand binding domain (LBD) construct, and to compete for binding with the highly active synthetic PPARγ agonist rosiglitazone. We demonstrate that 3-oxo-C12-HSL binds effectively to the PPARγ ligand binding domain, and that concentrations of 3-oxo-C12-HSL as low as 1 nM can effectively interfere with the binding of rosiglitazone to the PPARγ ligand binding domain. Because 3-oxo-C12 HSL has been demonstrated in lungs during P. aeruginosa infection, blockade of PPARγ-dependent signaling by 3-oxo-C12-HSL produced by the infecting P. aeruginosa could exacerbate infection-associated inflammation, and potentially impair the action of PPAR-activating therapy. Thus the proposed use of PPARγ agonists as anti-inflammatory therapy in lung P. aeruginosa infection may depend on their ability to counteract the effects of 3-oxo-C12-HSL.     

Switching subtype-selectivity: Fragment replacement strategy affords novel class of peroxisome proliferator-activated receptor α/δ (PPARα/δ) dual agonists

Peroxisome proliferator-activated receptors (PPARs) are important drug targets for treatment of dyslipidemia, type 2 diabetes, cardiovascular disease, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, and great efforts have been made to develop novel PPAR ligands. However, most existing PPAR ligands contain a carboxylic acid (CA) or thiazolidinedione (TZD) structure (acidic head group) that is essential for activity. We recently discovered non-CA/TZD class PPARα/δ partial agonists, which contain an acetamide moiety and adjacent methyl group, linked to a 1,2,4-oxadiazole ring (“fragment a”). We hypothesized that the acetamide structure might interact with the CA/TZD-binding pocket. To test this idea, we firstly replaced fragment a in one of our compounds with the α-alkoxy-CA structure often found in PPAR agonists. Secondly, we replaced the α-alkoxy-CA head group of several reported PPAR agonists with our acetamide-based fragment a. The agonistic activities of the synthesized hybrid compounds toward PPARs (PPARα, PPARγ and PPARδ) were evaluated by means of cell-based reporter gene assays. All the hybrid molecules showed PPAR-agonistic activities, but replacement of the α-alkoxy-CA head group altered the maximum efficacy and the subtype-specificity. The acetamide-based hybrid molecules showed partial agonism toward PPARα and PPARδ, whereas the α-alkoxy-CA-based molecules were generally selective for PPARα and PPARγ, with relatively high activation efficacies. Thus, the fragment replacement strategy appears promising for the development of novel acetamide-based PPARα/δ dual agonists.