Wnt信号转导机制的化学生物学研究

The Wnt signaling pathway is highly conserved in organic evolution. It plays crucial role in diverse processes, including cell fate determination, embryonic development and tumorigenesis. To further understand the signaling transduction, it has been an attractive research to screen novel chemical small molecule targeting Wnt signaling and explore its mechanism. NC043 is a novel inhibitor of Wnt/β-catenin signaling pathway we have identified. By pull down with biotin-linked NC043 coupled with MS analysis, we identified that NPHXL is one of protein targets of NC043. Moreover, we found that NPHXL positively regulates Wnt/β-catenin signaling, also controls early embryonic development of zebrafish with Wnt signal. Regarding to mechanism, NPHXL interacts with Dvl(s), which is a crucial component family in Wnt signaling, thus may facilitate the formation of β-catenin/TCF transcription complex. Meanwhile, through structure-activity evaluation of NC043, we figured out the critical groups for maintaining and improving its Wnt inhibitory activity. In the following work, we will expound the mechanism of NPHXL in Wnt signaling. also explore its biological function. On the other hand, there are many hints that NPHXL is not the unique target of NC043, so we are going to dig the MS data for novel protein target(s) of NC043 and investigate their function in Wnt signaling.

Wnt信号通路在生物进化中高度保守，调控细胞命运决定、胚胎发育、肿瘤发生等过程。筛选针对Wnt信号通路的小分子，通过揭示小分子的作用机理以完善对Wnt信号转导过程的理解，已成为Wnt信号转导研究领域的一个热点。NC043是我们筛选到的Wnt信号通路的新的小分子抑制剂。通过蛋白质组学方法，我们鉴定出NPHXL是NC043的一个作用靶点。NPHXL能增强经典Wnt信号通路，并参与调控斑马鱼早期发育。机制上，NPHXL能够在核内结合Dvl，从而可能促进下游转录复合体的形成。同时，我们开展对NC043的构效关系研究，明确了NC043发挥作用的关键部位及增效基团。在接下来的工作中，我们将进一步阐述NPHXL的生物学功能及其作用机理。另一方面，许多结果提示我们NPHXL并非NC043抑制Wnt信号通路的唯一靶点，我们还将从质谱数据中挖掘NC043的新的蛋白质靶点，探讨其在Wnt信号通路中的作用机理。

Wnt信号通路在生物进化中高度保守，调控细胞命运决定、胚胎发育、肿瘤发生等过程。筛选针对Wnt信号通路的小分子,应用化学生物学手段揭示小分子作用的靶点机制对于Wnt信号通路失调相关疾病的治疗以及完善对Wnt信号转导过程的理解都有十分重要的意义。NC043是我们筛选到的Wnt信号通路特异的小分子抑制剂。为了阐明NC043的作用机理，我们对其进行了生物素交联,通过亲和沉淀和质谱分析，我们发现CARF是NC043的一个共价结合蛋白。进一步研究表明，CARF能够在细胞核内与Dvl结合，促进由Dvl介导的多元转录复合体的形成和稳固，进而增加经典Wnt信号通路活性。NC043结合CARF后，多元转录复合体不能正常形成，从而Wnt信号通路受到抑制。为了进一步研究CARF的生物学功能，我们用CRISPR/cas9基因编辑技术构建了CARF突变的斑马鱼。突变鱼具有早期胚胎发育缺陷、造血干细胞形成缺陷以及尾鳍再生延迟等经典Wnt信号通路相关的表型。另一方面，我们还明确了NC043的内酯环结构和迈克尔受体是结合CARF的关键基团。综上所述，我们在自主发现的Wnt信号通路小分子抑制剂NC043的基础上，进一步揭示了NC043的作用机理，并在该过程中发现了Wnt信号通路的一个新的调控分子---CARF。我们的研究结果不仅为NC043的应用提供了理论依据和指导，同时更深化了对经典Wnt信号通路的认识。

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