Wnt信号通路Frizzled蛋白机制研究和特异性调控分子开发

As a conserved signaling pathway, Wnt pathway plays an indispensable role in embryogenesis and adult tissue regeneration through regulating the cell fate in higher organisms. In most case, dysfunction of Wnt pathway will cause severe problems. Thus, developing specific Wnt pathway modulators is of great significance as they can be used in both stem cell and cancer research as well as clinical trials and disease therapy. Although Wnt pathway has been attracting tremendous attention, the exact underlying molecular mechanism, especially how the Frizzled receptor transduct the outside Wnt binding signal into the intracellular side, remains unclear. In addition, the extreme difficulty in purifying the Wnt protein and the promiscuous interaction between Wnt and Frizzled has always precluded the development of specific agonist or effective antagonist against Wnt pathway. Here we propose to generate synthetic antibody libraries and apply them to screen specific binders for Frizzled and LRP5/6 protein. These identified conformation selective antibodies, on one hand, will facilitate the crystallization of Frizzled protein; on the other hand, will lock the Frizzled in different states. By solving their crystal structures, the Frizzled activation mechanism upon Wnt association could be further deciphered. At the same time, based on the screened Fab, multivalent antibodies in different formats, such as diabody and biospecific IgG, will be constructed with the aim of getting agonistic or strong antagonistic agents for Wnt pathway. In conjunction with the cellular assays, the activity of these multivalent antibodies will be characterized. In sum, this proposal will provide insight into the molecular mechanism underlying Wnt pathway as well as antibody drug development.

Wnt通路在高等生物胚胎发育及成体组织再生中发挥着不可替代的作用，其异常往往会导致严重的后果。开发针对Wnt通路调控分子意义重大，不仅有助于干细胞和肿瘤基础研究，还可用于药物研发。虽然Wnt通路一直广受关注，但目前其确切的分子机制，特别是其受体Frizzled蛋白如何将胞外Wnt结合信号传递至胞内还不清楚。此外，由于Wnt蛋白极难纯化以及缺少Frizzled作用特异性，开发Wnt通路特异性激活剂和高效抑制剂也一直受阻。本项目将设计抗体文库，筛选Frizzled和LRP5/6的特异性抗体。利用所获抗体将Frizzled稳定在不同的构象状态，通过测定其晶体结构，阐明Frizzled发挥功能的分子机制。与此同时，进一步制备不同形式的多价抗体，筛选出可以特异性开启Wnt通路的激活剂以及能够高效抑制Wnt通路的超级抑制剂。总而言之，本项目将为理解Wnt通路分子机制以及抗体药物开发提供新的思路和方向。

Wnt通路是哺乳生物中一种保守的信号通路，在胚胎发育和组织再生中发挥着重要作用。Wnt通路的异常往往和肿瘤或者再生疾病密切相关。因此，靶向Wnt通路的调控分子不仅可以作为研究工具用于基础研究，也有着重要的临床转化价值。Wnt通路的主要受体为frizzled (FZD)，在人体中，FZD受体有10种。由于这10种FZD受体序列高度类似，开发特异性FZD调控分子难度一直很大。据此，本项目选择若干重要FZD受体作为研究对象，通过结合定向合成文库以及“反向”抗原的筛选方法，成功获取了相应的FZD特异性抗体。具体而言，本项目的进展如下：1)构建了靶向FZD10的定向抗体文库并用其筛选获取了3种FZD10特异性抗体（F10_A9、F10_E10、F10_H7），利用结构生物学的手段，解析了F10_A9结合FZD10 CRD的晶体结构，揭示了F10_A9识别FZD10的关键功能表位（site I区域），利用生化手段，证明了F10_E10和F10_H7同样识别FZD10的site I区域；2)构建了靶向FZD1/2/7的定向抗体文库并用其筛选获取了1种FZD1/2/7交叉识别抗体pF7_A5，解析了pF7_A5结合FZD2的晶体结构，揭示了pF7_A5识别FZD1/2/7的关键功能表位（site II区域）；3)基于pF7_A5/FZD2晶体结构，进一步构建了二代文库并用其筛选获取了一种高亲和力FZD1/2/7交叉识别抗体pF7_B6，其结合FZD1/2/7能力均在纳摩尔级别；4)更重要的是，本项目设计并利用反向筛选策略，获得了1种FZD2选择性抗体sF2_C11以及1种FZD7偏好性抗体sF7_A2；5)细胞水平功能验证揭示所获取的所有抗体以及四价的Wnt通路激活性抗体（Flags）均展示了预期的Wnt通路调控功能。项目执行期间，已发表标注资助号的高水平SCI论文2篇，分别为以通讯作者发表在 Nature Communications和PNAS杂志上，尚在审稿阶段论文1篇；培养2名即将毕业博士研究生。如上所述，我们按照计划，圆满地完成项目的研究目标。