Design and Synthesis of Highly Active Peroxisome Proliferator-Activated Receptor (PPAR) ß/δ Inverse Agonists with Prolonged Cellular Activity.

Based on 3-(((4-(hexylamino)-2-methoxyphenyl)amino)sulfonyl)-2-thiophenecarboxylic acid methyl ester (ST247, compound 2), a recently described peroxisome proliferator-activated receptor (PPAR)ß/δ-selective inverse agonist, we designed and synthesized a series of structurally related ligands. The structural modifications presented herein ultimately resulted in a series of ligands that display increased cellular activity relative to 2. Moreover, with methyl 3-(N-(2-(2-ethoxyethoxy)-4-(hexylamino)phenyl)sulfamoyl)thiophene-2-carboxylate (PT-S264, compound 9 u), biologically relevant plasma concentrations in mice were achieved. The compounds presented in this study will provide useful novel tools for future investigations addressing the role of PPARß/δ in physiological and pathophysiological processes.

The transcriptional PPARß/δ network in human macrophages defines a unique agonist-induced activation state.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is a lipid ligand-inducible transcription factor with established metabolic functions, whereas its anti-inflammatory function is poorly understood. To address this issue, we determined the global PPARß/δ-regulated signaling network in human monocyte-derived macrophages. Besides cell type-independent, canonical target genes with metabolic and immune regulatory functions we identified a large number of inflammation-associated NFκB and STAT1 target genes that are repressed by agonists. Accordingly, PPARß/δ agonists inhibited the expression of multiple pro-inflammatory mediators and induced an anti-inflammatory, IL-4-like morphological phenotype. Surprisingly, bioinformatic analyses also identified immune stimulatory effects. Consistent with this prediction, PPARß/δ agonists enhanced macrophage survival under hypoxic stress and stimulated CD8(+) T cell activation, concomitantly with the repression of immune suppressive target genes and their encoded products CD274 (PD-1 ligand), CD32B (inhibitory Fcγ receptor IIB) and indoleamine 2,3-dioxygenase 1 (IDO-1), as well as a diminished release of the immune suppressive IDO-1 metabolite kynurenine. Comparison with published data revealed a significant overlap of the PPARß/δ transcriptome with coexpression modules characteristic of both anti-inflammatory and pro-inflammatory cytokines. Our findings indicate that PPARß/δ agonists induce a unique macrophage activation state with strong anti-inflammatory but also specific immune stimulatory components, pointing to a context-dependent function of PPARß/δ in immune regulation.

Deregulation of PPARß/δ target genes in tumor-associated macrophages by fatty acid ligands in the ovarian cancer microenvironment.

The nuclear receptor peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is a lipid ligand-inducible transcription factor associated with macrophage polarization. However, its function in tumor-associated macrophages (TAMs) has not been investigated to date. Here, we report the PPARß/δ-regulated transcriptome and cistrome for TAMs from ovarian carcinoma patients. Comparison with monocyte-derived macrophages shows that the vast majority of direct PPARß/δ target genes are upregulated in TAMs and largely refractory to synthetic agonists, but repressible by inverse agonists. Besides genes with metabolic functions, these include cell type-selective genes associated with immune regulation and tumor progression, e.g., LRP5, CD300A, MAP3K8 and ANGPTL4. This deregulation is not due to increased expression of PPARß/δ or its enhanced recruitment to target genes. Instead, lipidomic analysis of malignancy-associated ascites revealed high concentrations of polyunsaturated fatty acids, in particular linoleic acid, acting as potent PPARß/δ agonists in macrophages. These fatty acid ligands accumulate in lipid droplets in TAMs, thereby providing a reservoir of PPARß/δ ligands. These observations suggest that the deregulation of PPARß/δ target genes by ligands of the tumor microenvironment contributes to the pro-tumorigenic polarization of ovarian carcinoma TAMs. This conclusion is supported by the association of high ANGPTL4 expression with a shorter relapse-free survival in serous ovarian carcinoma.

Development of improved PPARß/δ inhibitors.

GSK0660 (1) is the first peroxisome proliferator-activated receptor (PPAR) ß/δ-selective inhibitory ligand described in the literature. Based on its structure, we designed and synthesized a series of modified compounds to establish preliminary structure-activity relationships. Most beneficial for increased binding affinity towards the PPARß/δ ligand binding domain was the replacement of the 4'-aminophenyl substituent by medium-length n-alkyl chains, such as n-butyl or iso-pentyl. These compounds show activity down to the one-digit nanomolar range, thus possessing up to a tenfold higher binding affinity compared with GSK0660. Additionally, the subtype-specific inhibition of PPARß/δ was confirmed in a cell-based assay making these compounds invaluable tools for the further exploration of the functions of PPARß/δ.

High-affinity peroxisome proliferator-activated receptor ß/δ-specific ligands with pure antagonistic or inverse agonistic properties.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is a ligand-regulated nuclear receptor with essential functions in metabolism and inflammation. We have synthesized a new derivative [methyl 3-(N-(4-(hexylamino)-2-methoxyphenyl)sulfamoyl)thiophene-2-carboxylate (ST247) structurally related to the published PPARß/δ inhibitory ligand methyl 3-(N-(2-methoxy-4-(phenylamino)phenyl)sulfamoyl)thiophene-2-carboxylate (GSK0660). ST247 has a higher affinity to PPARß/δ than GSK0660, and at equimolar concentrations, it more efficiently 1) induces the interaction with corepressors both in vitro and in vivo, 2) inhibits the agonist-induced transcriptional activity of PPARß/δ, and 3) down-regulates basal level expression of the peroxisome proliferator responsive element-driven PPARß/δ target gene ANGPTL4. Methyl 3-(N-(4-(tert-butylamino)-2-methoxyphenyl)sulfamoyl)thiophene-2-carboxylate (PT-S58), another high-affinity derivative from our series, also efficiently inhibits agonist-induced transcriptional activation, but in contrast to ST247, it does not enhance the interaction of PPARß/δ with corepressors. PT-S58 rather prevents corepressor recruitment triggered by the inverse agonist ST247. These findings classify ST247 as an inverse agonist, whereas PT-S58 is the first pure PPARß/δ antagonist described to date. It is noteworthy that ST247 and PT-S58 are also effective on PPRE-independent functions of PPARß/δ: in monocytic cells, both ligands modulate expression of the activation marker CCL2 in the opposite direction as an established PPARß/δ agonist. The possibility to differentially modulate specific functions of PPARß/δ makes these novel compounds invaluable tools to advance our understanding of PPARß/δ biology.