Development and Applications of Bioorthogonal Chemistry

生物正交化学的发展及应用 摘要 此Mira应用程序结合了我们实验室的两项研究工作，以解决研究中的关键障碍 B类GPCR生物物理学和活细胞中的信号传递，即缺乏合适的工具来构建功能 GPCR生物传感器以及捕获参与其中的瞬时和高动态GPCR相互作用蛋白 有偏见的激励主义。我们对开发基于反应性的化学工具有着长期的兴趣，以解决 用传统的分子生物学技术难以解决的重大生物学问题。在 在过去的五年中，我们在工具开发和这些工具的应用方面继续取得进展 解决重要的生物问题。例如，我们优化了几个生物正交反应，包括 光诱导的四唑烯烃环加成反应(光点化学)，螺烯四嗪 连接反应，钯介导的交叉偶联反应，以及序列特异性的2-氰基苯并-2 噻唑半胱氨酸连接反应。与涉及螺烯氨基的遗传密码扩展一起 酸，这些反应中的两个(光点击化学和四嗪连接)被利用来定位 在GLP-1R和GCGR的胞外环3引入有机荧光团(荧光素、Cy3和Cy5)， 两名B类GPCRs成员与糖尿病和肥胖症有关，正在进行的单细胞FRET研究 在配体诱导的活细胞受体激活过程中的结构域移动。此外，我们还制作了一个 偶然发现2-芳基-5-羧基四氮唑(ACT)提供了一种新的邻位依赖的光交叉反应. 链接器，然后在设计光亲和标记时使用，从而实现原位捕获和 随后识别药物靶点以及针对特定部位的遗传编码氨基酸 哺乳动物细胞中瞬时EGFRGrb2相互作用复合体的掺入和随后捕获。 在这些结果的基础上，在本申请中，我们计划在以下位置继续我们的正交化学反应研究 化学-生物接口和追求以下两个相关的项目。在项目1中，我们将构建 基于FRET的GLP-1R和GCGR生物传感器的生物正交标记研究 参与活细胞中受体激活和信号传递的动力学。一套新的荧光灯“开启” 将设计用于GLP-1R的细胞内环3(ICL3)的生物正交荧光标记的试剂 和GCGR，允许对活细胞中的受体构象进行单细胞内和分子间FRET分析。 在项目2中，我们将开发一种基因编码的ACT光交联剂，该交联剂含有炔基和 应用这种光交联剂对依赖时间的GLP-1R和-arrestins的相互作用进行质量映射 光谱对配体刺激的反应。我们预计，这些研究不仅将验证新的 实时监测活细胞中蛋白质构象和蛋白质相互作用的化学工具，但 还提供了对GLP-1R和GCGR激活和有偏信号的新见解，这些信号对 开发治疗糖尿病和肥胖症的靶向疗法。

Development and Applications of Bioorthogonal Chemistry ABSTRACT This MIRA application combines two research efforts in our lab in tackling the critical barriers in the study of class B GPCR biophysics and signaling in live cells, i.e., the lack of suitable tools for constructing functional GPCR biosensors as well as capturing the transient and highly dynamic GPCR-interacting proteins involved in biased agonism. We have a long-standing interest in developing the reactivity-based chemical tools to address significant biological problems that are difficult to solve using conventional molecular biology techniques. In the past five years we continued to make progress in both tool development and the applications of these tools to address important biological problems. For instance, we optimized several bioorthogonal reactions, including the photoinduced tetrazolealkene cycloaddition reaction (‘photoclick’ chemistry), the spiroalkenetetrazine ligation reaction, the palladium-mediated cross-coupling reactions, and the sequence-specific 2-cyanobenzo- thiazolecysteine ligation reaction. Together with the genetic code expansion involving a spiroalkene amino acid, two of these reactions (photoclick chemistry and tetrazine ligation) were harnessed for site-specific introduction of organic fluorophore (fluorescein, Cy3 and Cy5) at the extracellular loop 3 of GLP-1R and GCGR, two members of the class B GPCRs implicated in diabetes and obesity, for an ongoing single-cell FRET study of the domain movement during ligand-induced receptor activation in live cells. In addition, we made a serendipitous discovery that 2-aryl-5-carboxytetrazole (ACT) offers a new proximity-dependent photo-cross- linker, which was then used in the design of the photo-affinity labels that enabled in situ capture and subsequent identification of the drug targets as well as a genetically encoded amino acid for site-specific incorporation and subsequent capture of the transient EGFRGrb2 interaction complex in mammalian cells. Built upon these results, in this application we plan to continue our studies of orthogonal chemical reactivity at the chemistry-biology interface and pursue the following two related projects. In Project 1, we will construct the FRET-based biosensors of GLP-1R and GCGR via bioorthogonal labeling to probe the conformational dynamics involved in the receptor activation and signaling in live cells. A new set of fluorescence ‘turn-on’ reagents will be designed for bioorthogonal, fluorescent labeling of the intracellular loop 3 (ICL3) of GLP-1R and GCGR to allow single-cell intra- and intermolecular FRET analysis of receptor conformations in live cells. In Project 2, we will develop a genetically encoded ACT photo-cross-linker containing an alkyne group and apply this photo-cross-linker to map the time-dependent GLP-1R and -arrestins interactomes by mass spectrometry in response to ligand stimulation. We expect that these studies will not only validate new chemical tools for real-time monitoring of protein conformations and protein-protein interactions in live cells but also provide novel insights into the GLP-1R and GCGR activation and biased signaling that are crucial for the development of targeted therapies for the treatment of diabetes and obesity.