Development and Applications of Bioorthogonal Chemistry

生物正交化学的发展与应用

• 批准号: 9266090
• 负责人: Qing Lin
• 金额: $ 10.31万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9266090
• 关键词: Agonist; Alkenes; Alkynes; Animals; Arrestins; Bacteriophages; Biocompatible; Cells; Chemicals; Chemistry; Color; Confocal Microscopy; Coupling; Cysteine; Development; Diabetes Mellitus; Energy Transfer; Enteroglucagon; Environment; Equilibrium; Extracellular Domain; Fluorescein; Fluorescent Probes; Fumarates; Funding; G-Protein-Coupled Receptors; Genetic; Glucagon; Goals; Half-Life; Hand; Health; Hydroquinones; In Situ; Kinetics; Label; Lasers; Life; Ligands; Lysine; Measures; Mediating; Microscope; Monitor; Movement; Mutagenesis; N-terminal; Nitriles; Nucleic Acid Regulatory Sequences; Obesity; Optics; Organism; Palladium; Peptides; Perfusion; Pharmacologic Substance; Physical condensation; Physiologic pulse; Positioning Attribute; Property; Proteins; Reaction; Receptor Activation; Regulation; Reporter; Scanning; Signal Transduction; Signaling Protein; Site; Specificity; Surface; System; Techniques; Tetrazoles; Therapeutic Agents; Time; azobenzene; base; biomaterial compatibility; biophysical analysis; conformational conversion; cycloaddition; extracellular; fluorophore; glucagon-like peptide 1; insight; irradiation; member; mutant; next generation; novel; phenylalanine analog; phenylhydrazine; receptor; receptor function; tool; two-photon; unnatural amino acids

DESCRIPTION (provided by applicant): Development and Applications of Bioorthogonal Chemistry ABSTRACT A major hurdle in biophysical studies of class B GPCR conformational transitions, particularly the movements of the two large domains during ligand-induced activation, is that there are very few techniques available that allow site-specific introduction o biophysical probes into these two domains without altering the receptor function. To overcome this limitation, our long-term goal is to develop bioorthogonal chemistry tools that enable biophysical studies of the multi-domain signaling proteins such as class B GPCRs in living cells. In our previous studies, we have optimized a bioorthogonal, photoinduced tetrazole-alkene cycloaddition reaction ('photoclick chemistry'), and developed the palladium-mediated cross-coupling reactions for selective protein labeling in living systems as well as a phage-assisted interrogation of reactivity strategy for evolving the sequence-specific bioorthogonal reactions. Built upon these results, in this application we plan to integrate the bioorthogonal chemistry tools with the genetic encoding of unique chemical functionalities to generate in situ the chemically modified GLP-1R/GCGR�two members of the class B GPCRs that are implicated in diabetes and obesity, and study their conformational transitions and photo-regulation in living cells. The specific aims are as follows: 1) Apply photoclick chemistry to generate in situ the environment-sensitive fluorescent probes on GCGR/GLP-1R and probe the ligand-induced conformational changes in living cells. A spiro[2,3]hex-1-ene or fumarate-derived lysine will be site-specifically incorporated at the extracellular loop 3 region of GCGR/GLP- 1R to direct the photoclick chemistry, and the resulting fluorescent labeled GCGR/GLP-1R will be used in the studies of the conformational transitions induced by the specific ligands; 2) Develop binary bioorthogonal chemistry for dual-labeling of GCGR/GLP-1R to probe the ligand-induced conformational changes by FRET in living cells. The photoclick chemistry will be used in tandem with the sequence-specific palladium-mediated cross-coupling or the cysteine-nitrile condensation reaction to enable the simultaneous introduction of two fluorophores at the juxtamembrane domain and the N-terminal extracellular domain, respectively. The dynamic movements of these two interconnected domains upon perfusion of the specific peptide ligand in living cells will be monitored by FRET using confocal microscope; 3) Develop the azobenzene-based optochemical genetic tools for optical regulation of GLP-1R activation in living cells. A biocompatible inverse azo-coupling reaction based on the condensation of phenylhydrazines with fluoroquinols will be developed, which together with the genetic encoding of fluoroquinolalanine, will allow us to introduce the azobenzenes site-specifically into the two regulatory regions of GLP-1R. The effect of reversible photoswitching on GLP-1R activity in the absence or presence of GLP-1 will be assessed using b-arrestin-mCherry as a reporter for receptor activation. These studies will provide the key insights into GLP-1R/GCGR activation mechanisms, which are crucial for the development of GLP-1R/GCGR dual agonists as therapeutic agents for the treatment of diabetes and obesity.

描述（由申请人提供）：生物正交化学的开发和应用摘要B类GPCR构象转变的生物物理学研究中的一个主要障碍，特别是在配体诱导的活化过程中两个大结构域的运动，是很少有技术可以在不改变受体功能的情况下将生物物理探针位点特异性地引入这两个结构域。为了克服这一限制，我们的长期目标是开发生物正交化学工具，使生物物理研究的多结构域信号蛋白，如B类GPCR在活细胞中。在我们以前的研究中，我们已经优化了生物正交，光诱导的四唑-烯烃环加成反应（“photoclick化学”），并开发了钯介导的交叉偶联反应，用于在生命系统中选择性的蛋白质标记，以及噬菌体辅助的反应性策略的询问，以发展序列特异性生物正交反应。基于这些结果，在本申请中，我们计划将生物正交化学工具与独特化学功能的遗传编码相结合，以原位生成化学修饰的GLP-1 R/GCGR-两种与糖尿病和肥胖有关的B类GPCR成员，并研究它们在活细胞中的构象转变和光调节。具体目的如下：1）应用光点击化学技术原位制备GCGR/GLP-1 R的环境敏感荧光探针，探测配体诱导的活细胞构象变化。将螺[2，3]己-1-烯或衍生自天冬氨酸的赖氨酸位点特异性地掺入GCGR/GLP-1 R的胞外环3区以指导光点击化学，并且所得荧光标记的GCGR/GLP-1 R将用于研究由特异性配体诱导的构象转变; 2）开发用于GCGR/GLP-1 R双标记的二元生物正交化学，以通过FRET在活细胞中探测配体诱导的构象变化。光点击化学将与序列特异性钯介导的交叉偶联或半胱氨酸-腈缩合反应串联使用，以分别在质膜结构域和N-末端细胞外结构域同时引入两个荧光团。在活细胞中灌注特定肽配体后，这两个相互连接的结构域的动态运动将通过使用共聚焦显微镜的FRET来监测; 3）开发用于活细胞中GLP-1 R活化的光学调控的基于偶氮苯的光化学遗传工具。将开发基于苯肼与氟喹啉缩合的生物相容性反偶氮偶联反应，其与氟喹啉丙氨酸的遗传编码一起，将使我们能够将偶氮苯位点特异性地引入GLP-1 R的两个调控区域。将使用b-抑制蛋白-mCherry作为受体激活的报告物，评估在不存在或存在GLP-1的情况下可逆光开关对GLP-1 R活性的影响。这些研究将为GLP-1 R/GCGR激活机制提供关键见解，这对于开发GLP-1 R/GCGR双重激动剂作为治疗糖尿病和肥胖症的治疗药物至关重要。