Modulating Coregulator Preference of Liver Receptor Homolog-1 with Small Molecules

项目总结 肝脏受体同系物-1(LRH-1)是一种核受体，已被认为是一种有希望的抗糖尿病药物 治疗在小鼠研究和调节胆固醇转运，胆汁酸生物合成，类固醇合成，和 葡萄糖动态平衡，使其成为治疗各种疾病的有吸引力的靶点。NRs是变构效应器， 选择性招募辅调节酶传递配体结合状态改变靶基因表达 来修饰染色质并招募转录机器。虽然磷脂是内源性的配体，但 LRH-1，我的实验室使用结构-活性关系(SAR)研究来开发合成激动剂 对于在实验和临床环境中靶向该蛋白质很有用。我们使用了结构导向的方法 直接接触结合袋中的残基，产生与LRH-1结合并激活LRH-1的化合物 纳摩尔效力。然而，我们对这些小分子如何驱动LRH-1激活的理解 通过改变辅助调节偏好仍然有限，我们目前系列激动剂的工作已经 增强小分子的亲和力，同时最小限度地改善LRH-1的细胞内折叠激活。多么 LRH-1的正义配体招募共调节因子知之甚少，正如我们的高亲和力小鼠所证明的那样 不能有效地驱动协同调节关联以增强激活的分子。因此，我将审查 合成激动剂结合和对小分子的修饰如何影响辅助调节因子的偏好 这将直接靶向LRH-1激活功能表面(AFS)，即辅助调节因子结合界面。我 假设合成激动剂诱导构象变化有利于与之相互作用 辅助激活剂和LRH-1活性可以通过直接改变AFS动力学来调节。在《目标1》中，我将 通过确定辅调节因子招募是如何受到影响的，来研究配体介导的激活机制 通过激动剂结合。我将使用荧光偏振(FP)和生物发光共振能量转移 (BRET)结合试验，以测试小分子和内源性磷脂如何改变辅助调节因子的偏好。 然后，我将使用荧光素酶报告分析来建立观察到的相互作用的功能相关性 配体介导的LRH-1激活是否依赖于这些辅调节因子的表达。在目标2中，我将使用 以结构为导向的方法进行修饰，直接调节AFS构象动力学。使用FP 竞争和荧光素酶报告分析，我将确定修饰如何影响结合和细胞内激活 LRH-1。有希望的小分子将用X射线结晶学和氢气进行进一步的研究 氢交换-质谱仪(HDX-MS)研究这些修饰是如何驱动AFS的 构象和动力学。我工作的长期目标是改进小分子调制器的设计 LRH-1活性的研究将有助于探测LRH-1生物学和治疗代谢性疾病。

PROJECT SUMMARY Liver receptor homolog-1 (LRH-1) is a nuclear receptor (NR) that has shown promise as an anti-diabetic therapeutic in murine studies and regulates cholesterol transport, bile acid biosynthesis, steroidogenesis, and glucose homeostasis, making it an attractive target for treating a variety of diseases. NRs are allosteric effectors, transmitting ligand binding status to alter target gene expression by selectively recruiting coregulator enzymes that modify chromatin and recruit transcriptional machinery. Although phospholipids are endogenous ligands of LRH-1, my lab has used structure-activity relationship (SAR) studies to develop synthetic agonists that are more useful for targeting this protein in experimental and clinical contexts. We have used a structure-guided approach to directly contact residues in the binding pocket to produce compounds that bind and activate LRH-1 with low- nanomolar potency. However, our understanding of how these small molecules drive LRH-1 activation through altered coregulator preference remains limited, and work on our current series of agonists has enhanced affinity of small molecules while minimally improving in-cell fold activation of LRH-1. How LRH-1 senses ligand to recruit coregulators is poorly understood, as evidenced by our high-affinity small molecules that do not efficiently drive coregulator association to enhance activation. Therefore, I will examine how coregulator preference is influenced by synthetic agonist binding and make modifications to small molecules that will directly target the LRH-1 activation function surface (AFS), the coregulator binding interface. I hypothesize that synthetic agonists induce conformational changes favoring interaction with coactivators and that LRH-1 activity can be modulated by directly altering AFS dynamics. In Aim 1, I will examine the mechanism of ligand-mediated activation by determining how coregulator recruitment is influenced by agonist binding. I will use fluorescence polarization (FP) and bioluminescence resonance energy transfer (BRET) binding assays to test how small molecules and endogenous phospholipids alter coregulator preference. I will then establish functional relevance for observed interactions using a luciferase reporter assay to examine whether ligand-mediated activation of LRH-1 relies upon expression of these coregulators. In Aim 2, I will use a structure-guided approach to make modifications that directly modulate AFS conformational dynamics. Using FP competition and luciferase reporter assays, I will identify how modifications impact binding and in-cell activation of LRH-1. Promising small molecules will be studied further with both X-ray crystallography and hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) to examine how these modifications drive AFS conformation and dynamics. The long-term goal of my work is to enhance design of small molecule modulators of LRH-1 activity that will be useful for probing LRH-1 biology and treating metabolic diseases.