Structural Genomics of Orphan Nuclear Receptors

描述（由申请人提供）：本项目的长期目标是确定所有孤儿核受体配体结合域（LBD）的结构，将其结构与这些受体的生物学功能相关联，并探索发现靶向这些受体的药物的结构信息。核受体（Nuclear receptors，NRs）是一个与DNA结合的转录因子大家族，参与调节多种基因的表达。与其他转录因子相比，NR的活性受小分子配体的调节，这些配体直接结合NR以诱导活性或抑制性构象。它们通过小分子的调节使NR成为治疗药物最重要和最成功的靶标之一。所有核受体都含有两个高度保守的结构域中的至少一个：位于中心的DNA结合结构域（DBD）和C-末端LBD。LBD是NR的关键结构和功能域。除了配体结合之外，LBD还含有二聚化基序和介导配体调节的共激活子和共抑制子的募集以进行转录调节的保守表面。因此，LBD一直是密集的结构研究的焦点和药物发现的直接目标。所有“经典”内分泌核受体和所有“收养孤儿受体”（受体的配体在受体被鉴定后被鉴定）的晶体结构都已得到解决。这些结构揭示了一个保守的三明治折叠，其中包含一个配体结合口袋和一个C-末端激活螺旋（AF-2），介导配体调节的核受体功能。重要的是，这些结构有助于揭示核受体的配体调节和配体发现的关键机制，如SF-1和COUP-TFII在该资助的第一阶段所示。目前，只有三个亚家族（SHP，GCNF和TLX/PNR）的人核受体的LBD结构仍有待解决。SHP、GCNF、TLX和PNR分别是胆固醇稳态、神经发生、眼发育和干细胞再生的重要调节因子，但对其结构和配体调控知之甚少。结构信息的缺乏已成为理解这些受体介导的生物学和信号通路的关键障碍。基于对其他孤儿核受体的配体鉴定和核受体的共同结构特征，我们假设剩余的孤儿核受体（SHP、GCNF、TLX/PNR）也是配体调节的。在这一更新的应用中，计划将X射线晶体学与生物化学和功能测定相结合，以验证这一假设。实现我们的特定目标将克服上述与这些受体相关的科学进步的关键障碍，为这些受体的配体调节提供全面的结构和分子框架，以及专注于靶向这些受体的药物发现的知识基础。 公共卫生相关性：SHP、TLX/PNR和GCNF是孤儿核受体，在脂质和胆汁酸的代谢或神经和胚胎干细胞的维持和发育中起关键作用。然而，很少有人知道他们的结构和配体调控方面。缺乏这些受体的结构信息已经成为这些受体研究进展的关键障碍。这些受体的晶体结构及其在各自生物学背景下的功能相关性不仅将确定这些孤儿核受体是否为配体调节的受体，而且还将作为靶向这些受体用于癌症、代谢疾病和神经退行性疾病的干细胞治疗的药物设计的合理模板。

DESCRIPTION (provided by applicant): The long-term objective of this project is to determine the structures of all orphan nuclear receptor ligand binding domains (LBDs), to correlate their structures with the biological functions of these receptors, and to explore the structural information for discovery of drugs that target these receptors. Nuclear receptors (NRs) constitute a large family of DNA-binding transcription factors that modulate the expression of genes involved in a broad spectrum of physiology. In contrast to other transcription factors, the activity of NRs is regulated by small-molecule ligands that directly bind NRs to induce active or repressive conformations. Their regulation by small molecules has made NRs one of the most important and successful targets for therapeutic drugs. All nuclear receptors contain at least one of two highly conserved domains: the centrally located DNA-binding domain (DBD) and the C-terminal LBD. The LBD is the key structural and functional domain of a NR. In addition to ligand binding, the LBD contains dimerization motifs and a conserved surface that mediates ligand-regulated recruitment of coactivators and co-repressors for transcriptional regulation. The LBD has thus been the focus of intense structural study and the direct target of pharmaceutical discovery. Crystal structures of all "classical" endocrine nuclear receptors and all "adopted orphan receptors" (receptors for which ligands were identified after the receptor was identified) have been solved. These structures reveal a conserved sandwich fold that harbors a ligand binding pocket and a C-terminal activation helix (AF-2) that mediates the ligand-regulated function of nuclear receptors. Importantly, these structures have been instrumental in revealing key mechanisms of ligand regulation and ligand discovery for nuclear receptors, as demonstrated for SF-1 and COUP-TFII in the first period of this grant. Currently, there are only three subfamilies (SHP, GCNF, and TLX/PNR) of human nuclear receptors for which the LBD structure remains to be solved. SHP, GCNF, TLX, and PNR are essential regulators in cholesterol homeostasis, neurogenesis, eye development, and stem cell regeneration, respectively, but little is known about their structures and ligand regulation. The lack of structural information has become a critical barrier for understanding the biology and signaling pathways mediated by these receptors. Based on ligand identification for other orphan nuclear receptors and the common structural features of nuclear receptors, we hypothesize that the remaining orphan nuclear receptors (SHP, GCNF, TLX/PNR) are also ligand-regulated. In this renewed application, the plan is to use X-ray crystallography in combination with biochemical and functional assays to test this hypothesis. Achieving our specific aims will overcome the above critical barrier to scientific advancements related to these receptors by providing a comprehensive structural and molecular framework for ligand regulation of these receptors, as well as a knowledgeable foundation for drug discovery focused on targeting these receptors. PUBLIC HEALTH RELEVANCE: SHP, TLX/PNR, and GCNF are orphan nuclear receptors that play crucial roles in the metabolism of lipids and bile acid, or maintenance and development of neural and embryonic stem cells. However, little is known about their structures and aspects of ligand regulation. The lack of structural information for these receptors has become critical barriers to progress of these receptors. Crystal structures of these receptors and their functional correlation under their respective biological context will not only establish whether these orphan nuclear receptors are ligand-regulated receptors, but will also serve as a rational template for drug design targeting of these receptors for cancers, metabolic diseases, and stem cell therapy for neurodegenerative diseases.