Structural Genomics of Orphan Nuclear Receptors

描述(申请人提供)：该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，将它们的结构与这些受体的生物学功能联系起来，并探索针对这些受体的药物的结构信息。核受体(NRs)是一个DNA结合转录因子大家族，可调节广泛的生理学相关基因的表达。与其他转录因子不同，NRs的活性受小分子配体的调控，小分子配体直接与NRs结合，诱导活性或抑制性构象。它们受到小分子的调节，使NRS成为治疗药物最重要和最成功的靶点之一。所有的核受体都至少包含两个高度保守的结构域中的一个：位于中心的DNA结合域(DBD)和C末端的LBD。LBD是NR的关键结构和功能结构域。除了配体结合，LBD还含有二聚化基序和一个保守的表面，它介导了配体调节的辅助激活因子和辅助抑制因子的招募，以进行转录调控。因此，LBD一直是密集的结构研究的焦点和药物发现的直接靶点。所有“经典”内分泌核受体和所有“领养孤儿受体”(受体的配体在受体确定后被确定)的晶体结构已被解决。这些结构揭示了一个保守的三明治折叠，它含有一个配体结合口袋和一个C末端激活螺旋(AF-2)，后者介导核受体的配体调节功能。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在本赠款的第一阶段所展示的那样。目前，人类核受体只有三个亚家族(SHP、GCNF和TLX/PNR)的LBD结构尚待解决。SHP、GCNF、TLX和PNR分别是胆固醇稳态、神经发生、眼睛发育和干细胞再生的重要调节因子，但对它们的结构和配体调节知之甚少。缺乏结构信息已成为了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基鉴定和核受体的共同结构特征，我们假设其余的孤儿核受体(SHP、GCNF、TLX/PNR)也是受配体调节的。在这一新的应用中，计划使用X射线结晶学结合生化和功能分析来验证这一假设。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一个全面的结构和分子框架，并为专注于这些受体的药物开发提供一个知识基础。 公共卫生相关性：SHP、TLX/PNR和GCNF是孤儿核受体，在脂质和胆汁酸的新陈代谢或神经和胚胎干细胞的维持和发育中发挥关键作用。然而，人们对它们的结构和配体调控方面知之甚少。缺乏这些受体的结构信息已经成为这些受体进展的关键障碍。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是配体调节的受体，而且还将成为针对癌症、代谢性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.