RXRα硝基配体鉴定及其RXRα依赖的生物学活性研究

The functions of nuclear receptors are tightly and delicately regulated by their cognate ligands, and discovering and studying novel ligands will reveal unidentified activities of nuclear receptors. Retinoid x receptor alpha (RXRα) is a unique member of nuclear receptor family and plays important and complex roles in the biological and pathological processes. We have reported N-terminally truncated RXRα (tRXRα) is able to bind the PI3K regulatory subunit p85α and promote PI3K/AKT signal activity, which is dramatically inhibited by RXRα ligands sulindac sulfide and its optimized derivative K-80003. Recently our prelimilary studies presented a novel type of RXRα ligand, the nitro-ligands - - Z compounds (Z-10 and Z-12), which bound RXRα and strongly activated RXRα transcriptional activity. Our prelimilary data also indicated that Z compounds negatively regulated TNFα/NFκB signal activity and synergistically induced cancer cell apoptosis with TNFα in a RXRα/tRXRα dependent manner. In this proposed studies, we will apply integrated multidisciplinary approaches to focuse on studying the following questions, 1) what is the mode of Z compounds binding to RXRα? 2) How do Z compounds regulate genomic and non-genomic functions of RXRα? 3) What is the molecular mechanism by which Z compounds inhibit TNFα/NFκB signal in a RXRα/tRXRα dependent manner? 4) How do Z compounds and TNFα synergistically induce cancer cell apoptosis? 5) How to optimize Z compounds in their abilities of binding RXRα and exerting RXRα/tRXRα dependent functions. The results from this research will reveal the unidentified nitro-ligands of RXRα and their unique regulations of RXRα activities, which will modify the concept of RXRα ligands. Our proposed studies will also provide a novel regulation mode of TNFα/NFκB signal mediated by RXRα/tRXRα and a mechanism of tumor development. Furthmore, the anti-cancer functions of Z compounds and their optimized derivatives may provide lead drugs and therapeutic approaches for cancer treatment.

核受体的活性受到其配体的特异性调节，发现和研究新型配体将揭示核受体重要的功能。我们的前期研究首次揭示了核受体RXRα的新型硝基配体 - Z化合物（Z-10和Z-12），并发现Z化合物以RXRα/tRXRα依赖的方式抑制TNFα/NFκB信号并且激活TNFα介导的肿瘤细胞凋亡。据此本课题将在分子、细胞和动物水平，用多学科交叉的方法深入研究Z化合物结合RXRα的独特方式以及其对RXRα的基因型和非基因型活性的调节；同时研究Z化合物以RXRα/tRXRα依赖的方式调节TNFα/NFκB信号以及和TNFα协同诱导肿瘤细胞凋亡的机制；并且研究Z化合物依赖RXRα/tRXRα的结构和活性优化。本课题的开展将首次展示一种新型RXRα硝基配体及其独特活性，修正RXRα配体的概念；同时揭示RXRα/tRXRα与TNFα/NFκB信号的新的相互作用方式，以及一种肿瘤发展的新机制和潜在的肿瘤治疗药物和方案。

核受体RXRα和其N端缺失的突变体tRXRα同多种疾病发生发展密切相关。通过小分子配体对RXRα和tRXRα的生理学和病理学功能进行调控有望产生对上述疾病有效的治疗作用。.我们发现硝基苯乙烯化合物Z-1、Z-10和Z-12具有选择性激活Gal4-DBD-RXRα-LBD融合蛋白转录的功能。Z-10/Z-12的硝基和RXRα配体结合口袋的Cys432对于Z-10/Z-12和RXRα的结合至关重要。Z-10/Z-12抑制RXRα同源二聚体的转录功能，这是由于Z-10和Z-12能够诱导构象特异的RXRα二聚体的产生。.我们发现Z-10/Z-12能够显著性地抑制TNFα/NFκB信号通路。机制研究发现tRXRα通过和TRAF2相互作用从而激活TNFα/NFκB信号。Z-10/Z-12通过和tRXRα结合，从而抑制tRXRα和TRAF2的相互作用。Z-10/Z-12和TNFα能够以tRXRα依赖的方式协同诱导乳腺癌细胞MCF-7的凋亡。.急性早幼粒细胞白血病（APL）主要是PML-RARα融合基因所导致的。研究发现Z-10诱导ATRA敏感和抵抗的APL细胞凋亡。RXRα能够同RARα和PML-RARα结合进而稳定RARα和PML-RARα蛋白。Z-10通过和RXRα结合进而抑制其同PML-RARα的结合，导致PML-RARα易于受到caspase的切割。Z-10不会诱导RARα的降解。这主要在于Z-10对RXRα和RARα的结合并没有抑制作用。我们还得到了两个优化的衍生物。.RXR配体Bexarotene通过激活RXR/LXR and RXR/PPAR异源二聚体，达到清除老年痴呆症（AD）模型小鼠脑中Aβ的功能。我们的研究发现，Z-10邻位甲基和甲氧基取代的衍生物比相应的对位取代衍生物能够更有效地激活RXRα同源和异源二聚体。邻位取代的衍生物具有更强的RXRα结合能力。乙氧基修饰的衍生物Z-36具有最好的RXR/LXR和RXR/PPAR异源二聚体激活能力。Z-10和Z-36均能显著性减少AD小鼠大脑皮层和海马区的Aβ斑块，且Z-36具有更好的作用。.综上所述，本项目揭示了RXRα硝基配体具有独特的结合RXRα模式和RXRα依赖的生物学功能。此类配体通过结合RXRα或tRXRα，产生抗乳腺癌、APL和AD的功效。同时，我们还揭示了RXRα和tRXRα新的生物学功能和作用机制。