Novel selective PPARdelta agonists: optimization of activity by modification of alkynylallylic moiety

Y-shaped molecules bearing alkynylallylic moieties were found to be potent and selective PPARdelta activators. The alkynylallylic moiety was synthesized from alkyn-1-ols by hydroalumination followed by a cross-coupling reaction. Series of active compounds 6 were obtained by stepwise changing the structure of the known PPARpan agonist 5 into Y-shaped compounds. The most active and selective compound, 6f, had a PPARdelta potency of 0.13 microM, which is 50-fold more potent than compound 5.     

Discovery of highly potent and selective benzyloxybenzyl-based peroxisome proliferator-activator receptor (PPAR) delta agonists

A series of 1,4-benzyloxybenzylsulfanylaryl carboxylic acids were prepared and their activities for PPAR receptor subtypes (alpha, delta, and gamma) with potential indications for the treatment of dyslipidemia were investigated. Analog 13a displayed the greatest binding affinity (IC(50)=10nM) and selectivity (120-fold) for PPARdelta over PPARalpha. Many of the analogs investigated were found to be highly selective for PPARdelta and were dependent on the point of attachment of the substituent. In the 1,4-series, analog 28e was found to be the most potent (IC(50)=1.7 nM) and selective (>1000-fold) compound for PPARdelta. None of the compounds tested showed appreciable binding affinity for PPARgamma.     

Phenylpropanoic acid derivatives bearing a benzothiazole ring as PPARdelta-selective agonists

To find novel PPARdelta-selective agonists, we designed and synthesized phenylpropanoic acid derivatives bearing 6-substituted benzothiazoles. Optimization of this series led to the identification of a potent and selective PPARdelta agonist 17. Molecular modeling suggested that compound 17 occupies the Y-shaped pocket of PPARdelta appropriately.     

Discovery of para-alkylthiophenoxyacetic acids as a novel series of potent and selective PPARdelta agonists

A novel series of potent and selective PPARdelta agonists, para-alkylthiophenoxyacetic acids, was identified. The synthesis and structure-activity relationships are described.     

3,4,5-Trisubstituted isoxazoles as novel PPARdelta agonists. Part 2

A series of PPARdelta-selective agonists was investigated and optimized for a favorable in vivo pharmacokinetic profile. Isoxazole LCI765 (17d) was found to be a potent and selective PPARdelta agonist with good in vivo PK properties in mouse (C(max)=5.1 microM, t(1/2)=3.1 h). LCI765 regulated expression of genes involved in energy homeostasis in relevant tissues when dosed orally in C57BL6 mice. A co-crystal structure of compound LCI765 and the LBD of PPARdelta is discussed.     

Synthesis and structure-activity relationships of thiadiazole-derivatives as potent and orally active peroxisome proliferator-activated receptors alpha/delta dual agonists

Replacement of the methyl-thiazole moiety of GW501516 (a PPARdelta selective agonist) with [1,2,4]thiadiazole gave compound 21 which unexpectedly displayed submicromolar potency as a partial agonist at PPARalpha in addition to the high potency at PPARdelta. A structure-activity relationships study of 21 resulted in the identification of 40 as a potent and selective PPARalpha/delta dual agonist. Compound 40 and its close analogs represent a new series of PPARalpha/delta dual agonists. The high potency, high selectivity, significant gene induction, excellent PK profiles, low P450 inhibition or induction, and good in vivo efficacy in four animal models support 40 being selected as a pre-clinical study candidate, and may render 40 as a valuable pharmacological tool in elucidating the complex roles of PPARalpha/delta dual agonists, and the potential usage for the treatment of metabolic syndrome.     

Design, synthesis, and evaluation of potent, structurally novel peroxisome proliferator-activated receptor (PPAR) delta-selective agonists

A series of 3-(4-alkoxyphenyl)propanoic acid derivatives was prepared as candidate peroxisome proliferator-activated receptor (PPAR) delta-selective agonists, based on our previously discovered potent human PPARalpha/delta dual agonist TIPP-401 as a lead compound. Structure-activity relationship studies clearly indicated the importance of the chain length of the alkoxy group at the 4-position, and the n-butoxy compound exhibited the most potent PPARdelta transactivation activity and highest PPARdelta selectivity. The (S)-enantiomer of a representative compound exhibited extremely potent PPARdelta transactivation activity, comparable with or somewhat superior to that of the known PPARdelta-selective agonist, GW-501516. The representative compound regulated the expression of genes involved in lipid and glucose homeostasis, and should be useful not only as a chemical tool to study PPARdelta function, but also as a candidate drug for the treatment of metabolic syndrome.     

Discovery of a novel class of PPARdelta partial agonists

Anthranilic acid GW9371 was identified as a novel class of PPARdelta partial agonist through high-throughput screening. The design and synthesis of SAR analogues is described. GSK1115 and GSK7227 show potent partial agonism of the PPARdelta target genes CPT1a and PDK4 in skeletal muscle cells.     

The peroxisome proliferator activated receptor δ is required for the differentiation of THP-1 monocytic cells by phorbol ester

BACKGROUND: PPARdelta (NR1C2) promotes lipid accumulation in human macrophages in vitro and has been implicated in the response of macrophages to vLDL. We have investigated the role of PPARdelta in PMA-stimulated macrophage differentiation.The THP-1 monocytic cell line which displays macrophage like differentiation in response to phorbol esters was used as a model system. We manipulated the response to PMA using a potent synthetic agonist of PPARdelta, compound F. THP-1 sub-lines that either over-expressed PPARdelta protein, or expressed PPARdelta anti-sense RNA were generated. We then explored the effects of these genetic modulations on the differentiation process. RESULTS: The PPARdelta agonist, compound F, stimulated differentiation in the presence of sub-nanomolar concentrations of phorbol ester. Several markers of differentiation were induced by compound F in a synergistic fashion with phorbol ester, including CD68 and IL8. Over-expression of PPARdelta also sensitised THP-1 cells to phorbol ester and correspondingly, inhibition of PPARdelta by anti-sense RNA completely abolished this response. CONCLUSIONS: These data collectively demonstrate that PPARdelta plays a fundamental role in mediating a subset of cellular effects of phorbol ester and supports observations from mouse knockout models that PPARdelta is involved in macrophage-mediated inflammatory responses.     

Synthesis and SAR of selective benzothiophene, benzofuran, and indole-based peroxisome proliferator-activated receptor delta agonists

Recent literature has suggested the benefit of selective PPARdelta agonists for the treatment of atherosclerosis and other disease states associated with the metabolic syndrome. Herein we report the synthesis and structure-activity relationships of a series of novel and selective PPARdelta agonists. Our search began with identification of a novel benzothiophene template which was modified by the addition of various thiazolyl, isoxazolyl, and benzyloxy-benzyl moieties. Further elucidation of the SAR led to the identification of benzofuran and indole based templates. During the course of our research, we discovered three new chemical templates with varying degrees of affinity and potency for PPARdelta versus the PPARalpha and PPARgamma subtypes.     

Regulation of Wnt/beta-catenin pathway by cPLA2alpha and PPARdelta

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is a rate-limiting key enzyme that releases arachidonic acid (AA) from membrane phospholipid for the production of biologically active lipid mediators including prostaglandins, leukotrienes and platelet-activating factor. cPLA(2)alpha is translocated to nuclear envelope in response to intracellular calcium increase and the enzyme is also present inside the cell nucleus; however, the biological function of cPLA(2)alpha in the nucleus remains unknown. Here we show a novel role of cPLA(2)alpha for activation of peroxisome proliferator-activated receptor-delta (PPARdelta) and beta-catenin in the nuclei. Overexpression of cPLA(2)alpha in human cholangiocarcinoma cells induced the binding of PPARdelta to beta-catenin and increased their association with the TCF/LEF response element. These effects are inhibited by the cPLA(2)alpha siRNA and inhibitors as well as by siRNA knockdown of PPARdelta. Overexpression of PPARdelta or treatment with the selective PPARdelta ligand, GW501516, also increased beta-catenin binding to TCF/LEF response element and increased its reporter activity. Addition of AA and GW501516 to nuclear extracts induced a comparable degree of beta-catenin binding to TCF/LEF response element. Furthermore, cPLA(2)alpha protein is present in the PPARdelta and beta-catenin binding complex. Thus the close proximity between cPLA(2)alpha and PPARdelta provides a unique advantage for their efficient functional coupling in the nucleus, where AA produced by cPLA(2)alpha becomes immediately available for PPARdelta binding and subsequent beta-catenin activation. These results depict a novel interaction linking cPLA(2)alpha, PPARdelta and Wnt/beta-catenin signaling pathways and provide insight for further understanding the roles of these key molecules in human cells and diseases.     

Roles of peroxisome proliferator-activated receptors delta and gamma in myoblast transdifferentiation

Dietary long chain fatty acids and thiazolidinediones act as potent activators of adipogenesis in established preadipose cell lines. High concentrations of thiazolidinediones have also been shown to induce terminal differentiation of non-preadipose cells, such as fibroblasts and myoblasts, into adipose-like cells. This transdifferentiation was observed in both rodent and human myoblasts. In this report, we show that PPARdelta mediates some of the effects exerted by long chain fatty acids on myogenesis and adipogenesis. Activation of PPARdelta by long chain fatty acids impairs the expression of the determination factor MyoD1 and alpha-actin, abolishes the development of multinucleated myotubes, and in parallel induces the expression of PPARgamma gene, a master regulator of adipogenesis. Ectopic expression of PPARdelta in C2C12 myoblasts potentiated the fatty acid-induced expression of adipogenic markers, while expression of a dominant negative PPARdelta mutant exerted opposite effects. Furthermore, a sequential activation of first PPARdelta with long chain fatty acids and then PPARgamma with thiazolidinediones is required for adipogenesis in C2C12 myoblasts. This study demonstrates that PPARdelta, at least in part, is responsible for the dual effects of long chain fatty acids as inhibitors of myogenesis and inducers of transdifferentiation into preadipose-like cells.     

Differential responses of PPARalpha, PPARdelta, and PPARgamma reporter cell lines to selective PPAR synthetic ligands

To characterize the specificity of synthetic compounds for peroxisome proliferator-activated receptors (PPARs), three stable cell lines expressing the ligand binding domain (LBD) of human PPARalpha, PPARdelta, or PPARgamma fused to the yeast GAL4 DNA binding domain (DBD) were developed. These reporter cell lines were generated by a two-step transfection procedure. First, a stable cell line, HG5LN, expressing the reporter gene was developed. These cells were then transfected with the different receptor genes. With the help of the three PPAR reporter cell lines, we assessed the selectivity and activity of PPAR agonists GW7647, WY-14-643, L-165041, GW501516, BRL49653, ciglitazone, and pioglitazone. GW7647, L-165041, and BRL49653 were the most potent and selective agonists for hPPARalpha, hPPARdelta, and hPPARgamma, respectively. Two PPAR antagonists, GW9662 and BADGE, were also tested. GW9662 was a selective PPARgamma antagonist, whereas BADGE was a low-affinity PPAR ligand. Furthermore, GW9662 was a full antagonist on PPARgamma and PPARdelta, whereas it showed partial agonism on PPARalpha. We conclude that our stable models allow specific and sensitive measurement of PPAR ligand activities and are a high-throughput, cell-based screening tool for identifying and characterizing PPAR ligands.     

Novel peroxisome proliferator-activated receptor (PPAR) gamma and PPARdelta ligands produce distinct biological effects

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARalpha, PPARdelta, and PPARgamma. PPARgamma has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPARgamma and PPARdelta that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPARgamma and PPARdelta directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARgamma agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabetic db/db mice all PPARgamma agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selective in vivo activation of PPARdelta did not significantly affect these parameters. In vivo PPARalpha activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPARgamma and PPARdelta; 2) ligand-dependent activation of PPARdelta involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARgamma activation (but not PPARdelta or PPARalpha activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARgamma agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARalpha activation is sufficient to affect triglyceride metabolism, PPARdelta activation does not appear to modulate glucose or triglyceride levels.     

The PPARdelta agonist, GW501516, promotes fatty acid oxidation but has no direct effect on glucose utilisation or insulin sensitivity in rat L6 skeletal muscle cells

Peroxisome proliferator-activated receptor-delta (PPARdelta) activation enhances skeletal muscle fatty acid oxidation and improves whole body glucose homeostasis and insulin sensitivity. Recently, GW501516, a selective PPARdelta agonist, was reported to increase glucose uptake in human skeletal myotubes by an AMPK-dependent mechanism that may contribute to the improved glucose tolerance. Here, we demonstrate that whilst GW501516 increases expression of PGC-1alpha and CPT-1 and stimulates fatty-acid oxidation in L6 myotubes, it fails to enhance insulin sensitivity, AMPK activity or glucose uptake and storage. Our findings exclude sarcolemmal glucose transport as a potential target for the therapeutic action of PPARdelta agonists in skeletal muscle.     

Design, synthesis, and evaluation of a new class of noncyclic 1,3-dicarbonyl compounds as PPARalpha selective activators

Lipid accumulation in nonadipose tissues is increasingly linked to the development of type 2 diabetes in obese individuals. We report here the design, synthesis, and evaluation of a series of novel PPARalpha selective activators containing 1,3-dicarbonyl moieties. Structure-activity relationship studies led to the identification of PPARalpha selective activators (compounds 10, 14, 17, 18, and 21) with stronger potency and efficacy to activate PPARalpha over PPARgamma and PPARdelta. Experiments in vivo showed that compounds 10, 14, and 17 had blood glucose lowering effect in diabetic db/db mouse model after two weeks oral dosing. The data strongly support further testing of these lead compounds in other relevant disease animal models to evaluate their potential therapeutic benefits.