Full-length LRH-1 structural regulation

全长LRH-1结构调整

基本信息

批准号：
10034145
负责人：
Raymond Daniel Blind
金额：
$ 34.6万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10034145
关键词：
Address Affect Affinity Agonist Amino Acids Architecture Benzophenones Bile Acids Binding Biophysics Chemicals Cholesterol Cholesterol Homeostasis Clinic Clinical Communication Computer Models Cryoelectron Microscopy Crystallography DNA DNA Binding Domain Data Development Drug Design Drug Targeting Event Gene Expression Profiling Gene Expression Regulation Genes Genetic Transcription Glucose Hepatocyte Human Hydrophobicity Individual Knockout Mice Length Ligand Binding Ligand Binding Domain Ligands Liver Metabolic Modeling Molecular Mus Mutation NR5A2 gene Non-Insulin-Dependent Diabetes Mellitus Nuclear Nuclear Receptors Oranges Patients Peptides Pharmaceutical Preparations Pharmacology Physiological Physiology Positioning Attribute Post-Translational Protein Processing Regulation Resolution Structural Models Structure Structure-Activity Relationship System Testing Therapeutic Treatment Efficacy base biophysical analysis biophysical techniques blood glucose regulation cohesion crosslink cysteinylcysteine drug development experience flexibility insight liver metabolism mouse model mutant nanomolar non-alcoholic fatty liver disease nonalcoholic steatohepatitis optimism pre-clinical prevent receptor function recruit response simulation small molecule structural biology tool

项目摘要

LRH-1 (NR5A2) is a monomeric nuclear receptor involved in many aspects of liver physiology, including bile acid, cholesterol and glucose homeostasis. LRH-1 activation has beneficial effects on liver metabolism in pre-clinical mouse models. As nuclear receptors like LRH-1 have a very druggable ligand-binding pocket, LRH-1 has been targeted by many drug development efforts with great recent progress, however an LRH-1 agonist is still not available in the clinic. Like most other nuclear receptors, LRH-1 is composed of a DNA-binding domain and a ligand-binding domain, which are connected by a large unstructured Hinge domain. Classic nuclear receptor drug design has focused on the isolated ligand-binding domain, as the regulatory mechanism of this isolated domain is very well understood at the molecular level: binding of a hydrophobic small molecule allosterically alters ligand-binding domain recruitment of a transcriptional coregulator, which regulates nuclear receptor function. However, several lines of evidence suggest inter-domain communication exists between LRH-1 domains, regulating function. Understanding the structural biology behind this inter-domain communication might help LRH-1 drug design efforts, however technical challenges in applying crystallography or cryo-EM has prevented progress, despite great effort from several groups. We used an integrated structural approach to develop a low-resolution, but high confidence model of the intact, full-length LRH-1, using exclusively solution-based biophysical analyses and computational modeling (HDX, SAXS, chemical crosslinking, artificial amino acid benzophenone cross linking, Cys-Cys interdomain crosslinking, Rosetta and MD simulations). The model explains human patient mutations and structure-based mutations predicted to reside in the interface between the domains, which we show alter full length LRH-1 structure and function. Here, we propose to take advantage of this solution-based approach to address several long-standing questions in the field: Aim 1 determines how various ligands change full length LRH-1 interdomain communication. Aim 2 resolves how the SUMO module post-translational modification alters LRH-1 interdomain communication. Aim 3 identifies the genes effected by structure-based LRH-1 mutations in mouse liver and primary hepatocytes. Our current understanding of how LRH-1 structure is regulated is limited to studies of the individual domains. Without understanding how full-length LRH-1 is regulated, we cannot know if our current drug design efforts are taking full advantage of the entire therapeutic capacity of LRH-1.

LRH-1 (NR5A2) 是一种单体核受体，参与肝脏生理学的许多方面，包括胆汁酸、胆固醇和葡萄糖稳态。 LRH-1 激活对临床前小鼠模型的肝脏代谢具有有益影响。由于像LRH-1这样的核受体具有非常可成药的配体结合口袋，LRH-1已成为许多药物开发工作的目标，并且最近取得了巨大进展，然而LRH-1激动剂仍然无法在临床上使用。与大多数其他核受体一样，LRH-1 由 DNA 结合结构域和配体结合结构域组成，它们通过大型非结构化铰链结构域连接。经典的核受体药物设计重点关注分离的配体结合结构域，因为这种分离结构域的调节机制在分子水平上已得到很好的理解：疏水性小分子的结合变构地改变了转录共调节因子的配体结合结构域的招募，调节核受体功能。然而，一些证据表明 LRH-1 域之间存在域间通信，调节功能。了解这种域间通信背后的结构生物学可能有助于 LRH-1 药物设计工作，然而，尽管多个小组付出了巨大努力，但应用晶体学或冷冻电镜方面的技术挑战阻碍了进展。我们使用集成结构方法开发了完整、全长 LRH-1 的低分辨率但高置信度模型，仅使用基于溶液的生物物理分析和计算模型（HDX、SAXS、化学交联、人工氨基酸二苯甲酮）交联、Cys-Cys 域间交联、Rosetta 和 MD 模拟）。该模型解释了人类患者突变和基于结构的突变，这些突变预计存在于结构域之间的界面中，我们表明这些突变会改变全长 LRH-1 的结构和功能。在这里，我们建议利用这种基于解决方案的方法来解决该领域的几个长期存在的问题：目标 1 确定各种配体如何改变全长 LRH-1 域间通信。目标 2 解决 SUMO 模块翻译后修饰如何改变 LRH-1 域间通信。目标 3 鉴定小鼠肝脏和原代肝细胞中受基于结构的 LRH-1 突变影响的基因。我们目前对 LRH-1 结构如何调控的理解仅限于对各个域的研究。如果不了解全长 LRH-1 是如何调节的，我们就无法知道我们当前的药物设计工作是否充分利用了 LRH-1 的全部治疗能力。