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(申请人提供)：生物正交化学的发展和应用摘要在B类GPCR构象转变的生物物理研究中的一个主要障碍，特别是在配体诱导的激活过程中两个大结构域的移动，是很少有可用的技术允许在不改变受体功能的情况下将特定部位的生物物理探针引入这两个结构域。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，使活细胞中的多结构域信号蛋白(如B类GPCRs)能够进行生物物理研究。在我们之前的研究中，我们优化了生物正交光诱导的四氮唑-烯烃环加成反应(光点击化学)，并开发了钯介导的交叉偶联反应用于生物系统中选择性的蛋白质标记，以及噬菌体辅助的反应策略的询问，以形成序列特异性的生物正交反应。在这些结果的基础上，在这一应用中，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，以原位产生化学修饰的GLP-1R/GCGR�，这是与糖尿病和肥胖症相关的B类GPCR的两个成员，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)应用光点化学法在GCGR/GLP-1R上原位产生环境敏感的荧光探针，并探测配体诱导的活细胞构象变化。将螺环[2，3]己烯或富马酸衍生的赖氨酸定点掺入GCGR/GLP-1R的胞外环3区域以指导光点击化学，得到的荧光标记的GCGR/GLP-1R将用于研究特定配体诱导的构象转变；2)建立双标记GCGR/GLP-1R的生物正交化学，以探测活细胞中FRET诱导的配体诱导的构象变化。光点化学将与序列特异性的钯介导的交叉偶联或半胱氨酸-腈缩合反应一起使用，以使两个荧光团分别在膜旁区域和N-末端胞外区域同时引入。这两个相互连接的结构域在活细胞中灌流特定的多肽配体后的动态移动将通过FRET使用共聚焦显微镜进行监测；3)开发基于偶氮苯的光化学遗传工具，用于光调控活细胞中GLP-1R的激活。我们将开发一种基于苯肼和氟喹酚缩合的生物相容的反向偶氮偶合反应，它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-1R的两个调控区。在没有或存在GLP-1的情况下，可逆光开关对GLP-1R活性的影响将被用b-arrestin-mCherry作为受体激活的报告来评估。这些研究将为GLP-1R/GCGR激活机制提供关键的见解，这对于开发GLP-1R/GCGR双激动剂作为治疗糖尿病和肥胖症的药物至关重要。