Design, synthesis and molecular characterization of selective inverse agonists and antagonists of the Peroxisome Proliferator-Activated Receptor (PPAR) beta/delta

过氧化物酶体激活受体PPARbeta/delta通过与特定DNA元件结合来调节其基因。虽然其在脂肪酸代谢和能量代谢中的作用已被充分理解，但其在细胞分化、增殖和炎症过程中的作用仍有争议。人类肌成纤维细胞的全基因组分析表明，抑制是PPARbeta/delta介导的转录调控的主要模式。开发具有不同活性特征（激动剂、拮抗剂或反向激动剂）的亚型特异性、高亲和力、生物可利用的PPARbeta/delta抑制剂对于阐明PPARbeta/delta介导的转录的复杂机制至关重要。在我们的初步研究中，我们分别成功地设计了第一个选择性PPARbeta/delta反向激动剂和拮抗剂。值得注意的是，化合物对PPARbeta/delta配体结合结构域的亲和力与其在体外募集辅阻遏肽的能力没有直接相关性。我们可以进一步证明，人乳腺癌细胞对三维基质胶基质的侵袭受到反向PPARbeta/delta激动剂的抑制。进一步的实验将ANGPTL 4鉴定为这些细胞中的主要转录PPARbeta/delta靶基因。为了阐明复杂的PPARbeta/δ反向激动剂介导的靶基因的抑制，关键是鉴定如果反向激动剂结合PPARbeta/δ-LBD则募集的所有相关蛋白和蛋白复合物。与体外充分描述的PIC组分的顺序募集相反，有强有力的证据表明，在体内事件的顺序可能会改变，此外，可能需要额外的辅因子，我们将通过ChIP测定和其他生物化学和/或遗传学方法详细研究，以鉴定对PPARbeta/delta介导的靶基因抑制至关重要的所有蛋白质。此外，为了理解配体的化学结构的微小变化如何使其活性谱从纯反向激动性转变为纯拮抗性，这些配体的结合模式的测定是不可接受的。

The Peroxisome Proliferator-Activated Receptor PPARbeta/delta regulates its genes through binding to specific DNA elements. While its role in fatty acid catabolism and energy metabolism is quite well understood, its role in cell differentiation, proliferation, and inflammatory processes is controversially discussed. Genome-wide analyses of human myofibroblasts indicate that repression is the major mode of PPARbeta/delta-mediated transcriptional regulation. The development of subtype-specific, high-affinity, bioavailable PPARbeta/delta inhibitors with a distinct activity profile (agonist, antagonist, or inverse agonist) is of utmost importance to elucidate the complex mechanism of the PPARbeta/delta-mediated transcription. In our preliminary studies we succeeded in designing the first selective PPARbeta/delta inverse agonists and antagonists, respectively. Noteworthy, there is no direct correlation of the affinity of a compound towards the PPARbeta/delta ligand-binding domain and its ability to recruit a corepressor peptide in vitro. We could furthermore demonstrate that invasion of a three-dimensional matrigel matrix by human breast adenocarcinoma cells is inhibited by inverse PPARbeta/delta agonists. Further experiments identified ANGPTL4 as the major transcriptional PPARbeta/delta target gene in these cells. To elucidate the complex PPARbeta/delta inverse agonist-mediated repression of target genes, it is crucial to identify all relevant proteins and protein complexes which are recruited if an inverse agonist binds to the PPARbeta/delta-LBD. In contrast to the well-described sequential recruitment of components of the PIC in vitro, there is strong evidence that in vivo the sequence of events might be altered and, moreover, additional co-factors might be required, which we will investigate in detail by ChIP assays and additional biochemical and/or genetic methods to identify all proteins being essential for the PPARbeta/delta-mediated repression of target genes. Furthermore, to understand how small changes in the chemical structure of a ligand can shift its activity profile from pure inverse agonistic towards pure antagonistic, the determination of the binding mode of these ligands is irremissible.