新型双功能抗骨质疏松分子调控骨重建的靶标发现与分子机制研究

Bone remodeling is an important mechanism to rebuild the mature bone tissue, mainly includes bone formation and bone resorption. Osteoporosis caused by bone resorption exceeding bone formation is one of the major diseases of aging society. Currently antiosteoporotic treatments have largely depended on antiresorptive, the only clinically available one is recombinant human parathyroid hormone (PTH), and no drug exerts a dual effect that increases bone formation and reduces bone resorption simultaneously. With a long-term effort, we discovered a novel molecule, QOA-8a that possesses a dual effect that inhibits bone resorption and stimulates bone formation both in vitro and in vivo. During the target research with QOA-8a probe, an unnamed protein (tentatively named as NJx-1) was discovered from osteoblasts of mice. The organ distribution analysis with NJx-1 monoclonal antibody revealed that the possible tetramer of NJx-1 exists only in osteoblasts, and preliminary research confirmed that NJx-1 does associate with the bone formation. Therefore, the current project aims at clone, purification and stereostructure exploration of NJx-1 protein; elucidation of the biological functions and position of the protein during the bone remodeling process, and the key interactive molecular signals with NJx-1. Finally, the project systematically analyzes the interaction between QOA-8a and NJx-1, and reveals its interaction sites and molecular mechanism. The project could not only discover a novel signal pathway during bone remodeling process, but also provide a theoretical basis for the anti-osteoporosis drug target discovery and design of regulators of NJx-1.

骨重建是成熟骨组织的一种重要替换机制，包括骨形成和骨吸收。因骨吸收大于骨形成造成的骨质疏松症是老龄化社会的重大疾病之一。目前骨质疏松治疗药以抑制骨吸收为主，促进骨形成的仅有甲状旁腺素，还没有两者兼具的双重作用药物。申请人经长期研究发现了一个在体内外均显示抑制骨吸收和促进骨形成双重作用的新型小分子QOA-8a。以QOA-8a探针为工具的靶标研究从小鼠成骨细胞中发现了一个未命名蛋白（暂命名为NJx-1）。利用NJx-1单抗的组织分布分析发现该蛋白的四聚体仅在成骨细胞中存在；初步功能分析该蛋白与骨形成相关。本项目旨在克隆、纯化NJx-1蛋白，探究其立体结构；阐明其在骨重建过程中的生物功能、地位，及与其相互作用的关键分子信息；最后系统分析QOA-8a与靶蛋白的相互作用，揭示其作用位点和分子机制。本项目不仅可以发现骨重建过程中的崭新信号通路，而且能为抗骨质疏松药物靶标发现及调控分子设计提供理论依据。

重建是成熟骨组织的一种重要替换机制，包括骨形成和骨吸收。因骨吸收大于骨形成造成的骨质疏松症是老龄化社会的重大疾病之一。项目前期研究发现了一个在体内外均显示抑制骨吸收和促进骨形成双重作用的新型小分子QOA-8a，以该化合物制备的探针从成骨细胞中得到的一个暂命名为NJx-1的unnamed蛋白。本项以NJx-1蛋白为中心展开，旨在探索骨重建过程中的崭新信号通路。.生物信息学研究证实NJx-1的基因是一个非内含子嵌套基因(为一个新基因)，未发现NJx-1具有同源蛋白（这使得本项目研究尤其困难）。我们成功表达和鉴定了NJx-1蛋白，并确定其定位于线粒体。NJx-1在成骨细胞中的过表达和敲低影响成骨细胞中多种标记物水平。在此基础上，利用knockin策略成功地制备了NJx-1基因突变小鼠并确认NJx-1的缺失确实影响骨生长，引起骨质疏松症；更有意义的是还发现引起了狭颅症。理论和实验结合的机理研究发现NJx-1与ATP合酶相互作用，影响ATP合酶的稳态，导致能量失衡，引起细胞早衰。对QOA-8a的进一步结构优化发现了抑制骨质疏松作用更好的化合物，并且还可以抑制代谢相关脂肪性肝病和肌肉萎缩。.通过本项目我们发现了一条暂新的与骨重建相关的信号通路，NJx-1蛋白，取得了原创性发现；初步阐明了其和能量调控紧密相关；同时发现了活性更好的化合物并阐述了活性机理。发表了SCI论文7篇；申请专利一项。本研究对研发具有自主知识产权的和骨质疏松及狭颅症相关的新药靶点或疾病标记物具有重要意义，为进一步展开骨、脂肪和肌肉之间的crosstalk研究打下了良好的基础。