Bidirectional fluorescence properties of pyrene-based peroxisome proliferator-activated receptor (PPAR) α/δ dual agonist

Based on X-ray crystallographic analysis of a peroxisome proliferator-activated receptor (PPAR) α/δ dual agonist complexed with human PPARs ligand binding domain (LBD), we previously reported the design and synthesis of a pyrene-based fluorescent PPARα/δ co-agonist 2. Here, we found that the fluorescence intensity of 2 increased upon binding to hPPARα-LBD, in a manner dependent upon the concentration of the LBD. But, surprisingly, the fluorescence intensity of 2 decreased concentration-dependently upon binding to hPPRδ-LBD. Site-directed mutagenesis of the two hPPAR subtypes clearly indicated that Trp264 of hPPARδ-LBD, located between H2' helix and H3 helix (omega loop), is critical for the concentration-dependent decrease in fluorescence intensity, which is suggested to be due to fluorescence resonance energy transfer (FRET) from the pyrene moiety of bound 2 to the nearby side-chain indole moiety of Trp264 in the hPPARδ-LBD.     

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.     

A natural propenoic acid derivative activates peroxisome proliferator-activated receptor-β/δ (PPARβ/δ)

AimsPrevious studies showed that natural prenyloxyphenylpropanoid derivatives have potent biological properties in vivo. Given the structural similarities between these compounds and known peroxisome proliferator-activated receptor (PPAR) agonists, the present study examined the hypothesis that propenoic acid derivatives activate PPARs.Main methodsChimeric reporter assays were performed to identify propenoic acid derivates that could activate PPARs. Quantitative polymerase chain reaction (qPCR) analysis of wild-type and Pparβ/δ-null mouse primary keratinocytes was performed to determine if a test compound could specifically activate PPARβ/δ. A human epithelial carcinoma cell line and primary mouse keratinocytes were used to determine the effect of the compound on cell proliferation.Key findingsThree of the propenoic acid derivatives activated PPARs, with the greatest efficacy being observed with prenyloxycinnamic acid derivatives 4′-geranyloxyferulic acid (compound 1) for PPARβ/δ. Compound 1 increased expression of a known PPARβ/δ target gene through a mechanism that requires PPARβ/δ. Inhibition of cell proliferation by compound 1 was found in a human epithelial carcinoma cell line.SignificanceResults from these studies demonstrate that compound 1 can activate PPARβ/δ and inhibit cell proliferation of a human skin cancer cell line, suggesting that the biological effects of 4′-geranyloxyferulic acid may be mediated in part by activating this PPAR isoform.     

Activation of peroxisome proliferator-activated receptor-β/-δ (PPARβ/δ) prevents endothelial dysfunction in type 1 diabetic rats

Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease. Herein, we have analyzed if the peroxisome proliferator-activated receptor-β/-δ (PPARβ/δ) agonist GW0742 exerts protective effects on endothelial function in type 1 diabetic rats. The rats were divided into 4 groups: control, control-treated (GW0742, 5mgkg(-1)day(-1) for 5 weeks), diabetic (streptozotocin injection), and diabetic-treated. GW0742 administration in diabetic rats did not alter plasma glucose, systolic blood pressure, or heart rate, but reduced plasma triglyceride levels. The vasodilatation induced by acetylcholine was decreased in aortas from diabetic rats. GW0742 restored endothelial function, increasing eNOS phosphorylation. Superoxide production, NADPH oxidase activity, and mRNA expression of prepro endothelin-1, p22(phox), p47(phox), and NOX-1 were significantly higher in diabetic aortas, and GW0742 treatment prevented these changes. In addition, GW0742 prevented the endothelial dysfunction and the upregulation of prepro endothelin-1and p47(phox) after the in vitro incubation of aortic rings with high glucose and these effects were prevented by the PPARβ/δ antagonist GSK0660. PPARβ/δ activation restores endothelial function in type 1 diabetic rats. This effect seems to be related to an increase in nitric oxide bioavailability as a result of reduced NADPH oxidase-driven superoxide production and downregulation of prepro endothelin-1.     

Skin-targeted inhibition of PPAR β/δ by selective antagonists to treat PPAR β/δ-mediated psoriasis-like skin disease in vivo

We have previously shown that peroxisome proliferator activating receptor ?/δ (PPAR β/δ is overexpressed in psoriasis. PPAR β/δ is not present in adult epidermis of mice. Targeted expression of PPAR β/δ and activation by a selective synthetic agonist is sufficient to induce an inflammatory skin disease resembling psoriasis. Several signalling pathways dysregulated in psoriasis are replicated in this model, suggesting that PPAR β/δ activation contributes to psoriasis pathogenesis. Thus, inhibition of PPAR β/δ might harbour therapeutical potential. Since PPAR β/δ has pleiotropic functions in metabolism, skin-targeted inhibition offer the potential of reducing systemic adverse effects. Here, we report that three selective PPAR β/δ antagonists, GSK0660, compound 3 h, and GSK3787 can be formulated for topical application to the skin and that their skin concentration can be accurately quantified using ultra-high performance liquid chromatography (UPLC)/mass spectrometry. These antagonists show efficacy in our transgenic mouse model in reducing psoriasis-like changes triggered by activation of PPAR β/δ. PPAR β/δ antagonists GSK0660 and compound 3 do not exhibit systemic drug accumulation after prolonged application to the skin, nor do they induce inflammatory or irritant changes. Significantly, the irreversible PPAR β/δ antagonist (GSK3787) retains efficacy when applied topically only three times per week which could be of practical clinical usefulness. Our data suggest that topical inhibition of PPAR β/δ to treat psoriasis may warrant further exploration.     

Nutritional regulation and role of peroxisome proliferator-activated receptor δ in fatty acid catabolism in skeletal muscle

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors primarily involved in lipid homeostasis. PPARδ displays strong expression in tissues with high lipid metabolism, such as adipose, intestine and muscle. Its role in skeletal muscle remains largely unknown. After a 24-h starvation period, PPARδ mRNA levels are dramatically up-regulated in gastrocnemius muscle of mice and restored to control level upon refeeding. The rise of PPARδ is accompanied by parallel up-regulations of fatty acid translocase/CD36 (FAT/CD36) and heart fatty acid binding protein (H-FABP), while refeeding promotes down-regulation of both genes. To directly access the role of PPARδ in muscle cells, we forced its expression and that of a dominant-negative PPARδ mutant in C2C12 myogenic cells. Differentiated C2C12 cells responds to 2-bromopalmitate or synthetic PPARδ agonist by induction of genes involved in lipid metabolism and increment of fatty acid oxidation. Overexpression of PPARδ enhanced these cellular responses, whereas expression of the dominant-negative mutant exerts opposite effects. These data strongly support a role for PPARδ in the regulation of fatty acid oxidation in skeletal muscle and in adaptive response of this tissue to lipid catabolism.     

Virtual identification of novel peroxisome proliferator-activated receptor (PPAR) α/δ dual antagonist by 3D-QSAR,molecule docking,and molecule dynamics simulation

Abstract

The therapeutic potential of PPARs antagonists extends beyond diabetes. PPARs antagonists represent a new drug class that holds promise as a broadly applicable therapeutic approach for cancer treatment. Thus, there is a strong need to develop a rational design strategy for creating PPARs antagonists. In this study, three-dimensional quantitative structure-activity relationship (3D-QSAR) models of PPARα receptor (CoMFA-1, q ² = 0.636, r ² = 0.953; CoMSIA-1, q ² = 0.779, r ² = 0.999) and PPARδ receptor (CoMFA-2, q ² = 0.624, r ² = 0.906; CoMSIA-2, q ² = 0.627, r ² = 0.959) were successfully constructed using 35 triazolone ring derivatives. Contour map analysis revealed that the electrostatic and hydrophobic fields played vital roles in the bioactivity of dual antagonists. Molecular docking studies suggested that the hydrogen bonding, electrostatic and hydrophobic interactions all influenced the binding of receptor-ligand complex. Based on the information obtained above, we designed a series of compounds. The docking results were mutually validated with 3D-QSAR results. Three-dimensional-QSAR and absorption, distribution, metabolism, excretion and toxicity (ADMET) predictions indicated that 19 newly designed compounds possessed excellent biological activity and physicochemical properties. In summary, this research could provide theoretical guidance for the structural optimization of novel PPARα and δ dual antagonists.

Communicated by Ramaswamy H. Sarma     

Agonism activities of lyso-phosphatidylcholines (LPC) Ligands binding to peroxisome proliferator-activated receptor gamma (PPARγ)

Abstract

PPARγ is an isoform of peroxisome proliferator-activated receptor (PPAR) belonging to a super family of nuclear receptors and is a primary target of the effective drug to treat the type II diabetes. The experiments found that Lyso-phosphatidylcholines (LPC) could bind to PPARγ, but the binding modes remain unknown. We used the Molecular Docking and Molecular Dynamic (MD) simulations to study the binding of four LPC ligands (LPC16:0, LPC18:0, LPC18:1-1 and LPC18:1-2) to PPARγ. The two-step MD simulations were employed to determine the final binding modes. The 20?ns MD simulations for four final LPC-PPARγ complexes were performed to analyze their structures, the binding key residues, and agonism activities. The results reveal that three LPC ligands (LPC16:0, LPC18:0 and LPC18:1-1) bind to Arm II and III regions of the Ligand Binding Domain (LBD) pocket, whereas they do not interact with Tyr473 of Helix 12 (H12). In contrast, LPC18:1-2 can form the hydrogen bonds with Tyr473 and bind into Arm I and II regions. Comparing with the paradigm systems of the full agonist (Rosiglitazone–PPARγ) and the partial agonist (MRL24–PPARγ), our results indicate that LPC16:0, LPC18:0 and LPC18:1-1 could be the potential partial agonists and LPC18:1-2 could be a full agonist. The in-depth analysis of the residue fluctuations and structure alignment confirm the present prediction of the LPC agonism activities.

Communicated by Ramaswamy H. Sarma     

Roles for peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivators 1α and 1β in regulating response of white and brown adipocytes to hypoxia

Obese white adipose tissue is hypoxic but is incapable of inducing compensatory angiogenesis. Brown adipose tissue is highly vascularized, facilitating delivery of nutrients to brown adipocytes for heat production. In this study, we investigated the mechanisms by which white and brown adipocytes respond to hypoxia. Brown adipocytes produced lower amounts of hypoxia-inducible factor 1α (HIF-1α) than white adipocytes in response to low O(2) but induced higher levels of hypoxia-associated genes. The response of white adipocytes to hypoxia required HIF-1α, but its presence alone was incapable of inducing target gene expression under normoxic conditions. In addition to the HIF-1α targets, hypoxia also induced many inflammatory genes. Exposure of white adipocytes to a peroxisome proliferator-activated receptor γ (PPARγ) ligand (troglitazone) attenuated induction of these genes but enhanced expression of the HIF-1α targets. Knockdown of PPARγ in mature white adipocytes prevented the usual robust induction of HIF-1α targets in response to hypoxia. Similarly, knockdown of PPARγ coactivator (PGC) 1β in PGC-1α-deficient brown adipocytes eliminated their response to hypoxia. These data demonstrate that the response of white adipocytes requires HIF-1α but also depends on PPARγ in white cells and the PPARγ cofactors PGC-1α and PGC-1β in brown cells.     

Novel highly selective peroxisome proliferator-activated receptor δ (PPARδ) modulators with pharmacokinetic properties suitable for once-daily oral dosing

Optimization of benzamide PPARδ modulator 1 led to (E)-6-(2-((4-(furan-2-yl)-N-methylbenzamido)methyl)phenoxy)-4-methylhex-4-enoic acid (18), a potent selective PPARδ modulator with significantly improved exposure in multiple species following oral administration.     

Angiotensinogen and angiotensin-converting enzyme mRNA decrease and AT1 receptor mRNA and protein increase in epididymal fat tissue accompany age-induced elevation of adiposity and reductions in expression of GLUT4 and peroxisome proliferator-activated receptor (PPARγ)

Elevated adiposity is one of the accompanying features of increased age in humans and animals. Angiotensin II (Ang II) is considered as growth promoting peptide to be involved in hypertrophic enlargement of adipose tissue. However, systemic renin-angiotensin system (RAS) seems to decrease with increased age of rats. Local adipose tissue RAS might be independent of the systemic one. Therefore we performed a comprehensive study using rats with increased age from 9 to 26 weeks and evaluated angiotensinogen, angiotensin-converting enzyme (ACE) and AT(1) receptor mRNA in epididymal adipose tissue by RT-PCR. In addition, we determined AT(1) receptor protein by Western blotting and Ang II binding. These RAS parameters were correlated with expression of selected adiposity-dependent proteins such as leptin, adiponectin, insulin-dependent glucose transporter (GLUT4) and PPARgamma. Angiotensinogen and ACE expression decreased with increased age and adiposity. On the contrary, AT(1) receptor mRNA and protein was significantly elevated in 26-week-old rats though the Ang II binding was not different between 9 and 26-week-old animals. These results suggest dynamic adaptation of local adipose tissue RAS components to increased age and adiposity most likely by decreasing local Ang II formation which is thereafter compensated by increased expression of AT(1) receptor. However, this increase in AT(1) receptor mRNA and protein is not reflected in increased receptor binding. We believe that this complex regulation of adipose tissue RAS slows down the negative age and adiposity related changes in adipose tissue leptin, adiponectin, GLUT4 and PPARgamma.