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

过氧化物酶体增殖物激活受体PPARbeta/Delta通过与特定的DNA元件结合来调节其基因。虽然它在脂肪酸分解代谢和能量代谢中的作用已被很好地了解，但它在细胞分化、增殖和炎症过程中的作用仍存在争议。对人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。开发具有不同活性的亚型特异性、高亲和力、生物可用的PPARbeta/Delta抑制剂(激动剂、拮抗剂或反向激动剂)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，我们成功地设计了第一个选择性的PPARβ/Delta反向激动剂和拮抗剂。值得注意的是，化合物对PPARbeta/Delta配体结合域的亲和力与其在体外招募辅阻遏肽的能力没有直接关系。我们还可以进一步证明，反向PPARbeta/Delta激动剂可以抑制人乳腺腺癌细胞对三维基质的侵袭。进一步的实验证实，ANGPTL4是这些细胞中主要的转录PPARbeta/Delta靶基因。为了阐明复杂的PPARbeta/Delta反向激动剂介导的靶基因抑制，关键是确定当反向激动剂与PPARbeta/Delta-LBD结合时招募的所有相关蛋白质和蛋白质复合体。与PIC组分在体外的顺序募集形成鲜明对比的是，有强有力的证据表明，在体内，事件的序列可能会被改变，而且可能需要额外的辅助因子，我们将通过芯片分析和额外的生化和/或遗传方法进行详细研究，以确定所有对PPARbeta/Delta介导的靶基因抑制至关重要的蛋白质。此外，为了了解配体化学结构的微小变化如何将其活性从纯反向激动剂转变为纯拮抗剂，确定这些配体的结合模式是不允许的。

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.