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

项目摘要 肝受体同源物-1（LRH-1）是一种核受体（NR），已显示出作为抗糖尿病药物的前景。 在小鼠研究中的治疗作用，并调节胆固醇转运、胆汁酸生物合成、类固醇生成和 葡萄糖稳态，使其成为治疗多种疾病的有吸引力的靶标。NR是变构效应物， 通过选择性募集辅助调节酶传递配体结合状态以改变靶基因表达 修饰染色质并募集转录机器。虽然磷脂是内源性配体， LRH-1，我的实验室已经使用结构-活性关系（SAR）研究来开发合成激动剂， 用于在实验和临床环境中靶向该蛋白。我们采用了结构导向的方法 直接接触结合口袋中的残基，以产生结合和激活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， 通过改变辅助调节因子的偏好仍然有限，我们目前的一系列激动剂的工作已经 增强小分子的亲和力，同时最低限度地改善LRH-1的细胞内折叠活化。如何 LRH-1感觉配体招募辅调节因子知之甚少，正如我们的高亲和力小分子所证明的那样。 不能有效驱动辅调节子结合以增强激活的分子。因此，我将研究 合成激动剂结合和对小分子进行修饰如何影响辅调节因子偏好 这将直接靶向LRH-1激活功能表面（AFS），辅调节因子结合界面。我 假设合成激动剂诱导构象变化，有利于与 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 通过确定如何影响辅调节因子的募集来检查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）结合测定以测试小分子和内源性磷脂如何改变辅调节因子偏好。 然后，我将使用荧光素酶报告基因测定法来确定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助调节因子的表达。在目标2中，我将使用 结构导向的方法来进行直接调节AFS构象动力学的修饰。使用FP 竞争和荧光素酶报告分析，我将确定如何修改影响结合和细胞内激活 LRH-1有前途的小分子将进一步研究与X射线晶体学和氢 氘交换与质谱联用（HDX-MS），以研究这些修饰如何驱动AFS 构象和动力学。我工作的长期目标是提高小分子调节剂的设计 LRH-1的活性，这将有助于探测LRH-1生物学和治疗代谢疾病。

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.