Besides its established functions in intermediary metabolism and developmental processes, the nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) has a less defined role in tumorigenesis. In the present study, we have identified a function for PPARβ/δ in cancer cell invasion. We show that two structurally divergent inhibitory ligands for PPARβ/δ, the inverse agonists ST247 and DG172, strongly inhibit the serum- and transforming growth factor β (TGFβ)-induced invasion of MDA-MB-231 human breast cancer cells into a three-dimensional matrigel matrix. To elucidate the molecular basis of this finding, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) and microarray analyses, which identified the gene encoding angiopoietin-like 4 (ANGPTL4) as the major transcriptional PPARβ/δ target in MDA-MB-231 cells, previously implicated in TGFβ-mediated tumor progression and metastatic dissemination. We show that the induction of ANGPTL4 by TGFβ and other oncogenic signals is strongly repressed by ST247 and DG172 in a PPARβ/δ-dependent fashion, resulting in the inhibition of ANGPTL4 secretion. This effect is attributable to these ligands' ability to induce a dominant transcriptional repressor complex at the site of transcription initiation that blocks preinitiation complex formation through an histone deacetylase-independent, non-canonical mechanism. Repression of ANGPTL4 transcription by inverse PPARβ/δ agonists is functionally linked to the inhibition of cancer cell invasion into a three-dimensional matrix, as (i) invasion of MDA-MB-231 cells is critically dependent on ANGPTL4 expression, (ii) recombinant ANGPTL4 stimulates invasion, and (iii) reverses the inhibitory effect of ST247 and DG172. These findings indicate that a PPARβ/δ–ANGPTL4 pathway is involved in the regulation of tumor cell invasion and that its pharmacological manipulation by inverse PPARβ/δ agonists is feasible.

Insights into the thermodynamic and kinetic signature of the transient opening of a protein-binding pocket resulting from accommodation of suitable substituents attached to a given parent ligand scaffold are presented. As a target, we selected human aldose reductase, an enzyme involved in the development of late-stage diabetic complications. To recognize a large scope of substrate molecules, this reductase opens a transient specificity pocket. The pocket-opening step was studied by X-ray crystallography, microcalorimetry, and surface plasmon resonance using a narrow series of 2-carbamoyl-phenoxy-acetic acid derivatives. Molecular dynamics simulations suggest that pocket opening occurs only once an appropriate substituent is attached to the parent scaffold. Transient pocket opening of the uncomplexed protein is hardly recorded. Hydration-site analysis suggests that up to five water molecules entering the opened pocket cannot stabilize this state. Sole substitution with a benzyl group stabilizes the opened state, and the energetic barrier for opening is estimated to be ∼5 kJ/mol. Additional decoration of the pocket-opening benzyl substituent with a nitro group results in a huge enthalpy-driven potency increase; on the other hand, an isosteric carboxylic acid group reduces the potency 1000-fold, and binding occurs without pocket opening. We suggest a ligand induced-fit mechanism for the pocket-opening step, which, however, does not represent the rate-determining step in binding kinetics.

The ligand-regulated nuclear receptor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a potential pharmacological target due to its role in disease-related biological processes. We used TR-FRET-based competitive ligand binding and coregulator interaction assays to screen 2693 compounds of the Open Chemical Repository of the NCI/NIH Developmental Therapeutics Program for inhibitory PPARβ/δ ligands. One compound, (Z)-3-(4-dimethylamino-phenyl)-2-phenyl-acrylonitrile, was used for a systematic SAR study. This led to the design of derivative 37, (Z)-2-(2-bromophenyl)-3-{[4-(1-methyl-piperazine)amino]phenyl}acrylonitrile (DG172), a novel PPARβ/δ-selective ligand showing high binding affinity (IC(50) = 27 nM) and potent inverse agonistic properties. 37 selectively inhibited the agonist-induced activity of PPARβ/δ, enhanced transcriptional corepressor recruitment, and down-regulated transcription of the PPARβ/δ target gene Angptl4 in mouse myoblasts (IC(50) = 9.5 nM). Importantly, 37 was bioavailable after oral application to mice with peak plasma levels in the concentration range of its maximal inhibitory potency, suggesting that 37 will be an invaluable tool to elucidate the functions and therapeutic potential of PPARβ/δ.

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)β/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 PPARβ/δ in physiological and pathophysiological processes.

The stilbene derivative (Z)-2-(2-bromophenyl)-3-{[4-(1-methylpiperazine) amino]phenyl}acrylonitrile (DG172) was developed as a highly selective inhibitory peroxisome proliferator-activated receptor (PPAR)b/d ligand. Here, we describe a novel PPARb/d-independent, yet highly specific, effect of DG172 on the differentiation of bone marrow cells (BMCs). DG172 strongly augmented granulocytemacrophage-colony-stimulating factor (GM-CSF)-induced differentiation of primary BMCs from Ppard null mice into two specific populations, characterized as mature (CD11c Ly6B1 cells. In agreement with these findings, transcriptome analyses showed a strong DG172-mediated repression of genes encoding neutrophilic markers in both differentiating wild-type and Ppard null cells, while macrophage/DC marker genes were up-regulated. DG172 also inhibited the expression of transcription factors driving granulocytic differentiation (Cebpe, Gfi1, and Klf5), and increased the levels of transcription factors promoting macrophage/DC differentiation (Irf4, Irf8, Spib, and Spic). DG172 exerted these effects only at an early stage of BMC differentiation induced by GM-CSF, did not affect macrophage-colony-stimulating factortriggered differentiation to macrophages and had no detectable PPARb/d-independent effect on other cell types tested. Structurefunction analyses demonstrated that the 4-methylpiperazine moiety in DG172 is required for its effect on DC differentiation, but is dispensable for PPARb/d binding. Based on these data we developed a new compound, (Z)-2-(4-chlorophenyl)-3-[4-(4-methylpiperazine-1-yl)phenyl]acrylonitrile (DG228), which enhances DC differentiation in the absence of significant PPARb/d binding.