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.

生物物理研究B类GPCR构象转变的主要障碍，特别是在配体诱导激活过程中两个大结构域的运动，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，以便对活细胞中的多结构域信号蛋白（如B类gpcr）进行生物物理研究。在我们之前的研究中，我们优化了一个生物正交，光诱导的四唑-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，用于生命系统中选择性蛋白质标记，以及噬菌体辅助的反应性策略，用于进化序列特异性生物正交反应。基于这些结果，在本应用中，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结合起来，原位生成化学修饰的GLP-1R/GCGR——与糖尿病和肥胖有关的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)利用光点击化学在GCGR/GLP-1R上原位生成环境敏感荧光探针，探测配体诱导的活细胞构象变化。螺旋[2,3]己-1-烯或富马酸衍生的赖氨酸将在GCGR/GLP-1R的细胞外环3区域特异性结合以指导光点击化学，所得到的荧光标记GCGR/GLP-1R将用于研究由特定配体诱导的构象转变；2)建立双标记GCGR/GLP-1R的二元生物正交化学方法，通过FRET探测配体在活细胞中诱导的构象变化。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，以便分别在近膜结构域和n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时，这两个相互连接的结构域的动态运动将由FRET使用共聚焦显微镜监测；3)开发基于偶氮苯的光化学遗传工具，用于GLP-1R在活细胞中活化的光学调控。基于苯基肼与氟喹诺醇缩合的生物相容性逆偶氮偶联反应将被开发出来，再加上氟喹诺丙氨酸的遗传编码，将使我们能够将偶氮苯位点特异性地引入GLP-1R的两个调控区域。在GLP-1缺失或存在的情况下，可逆光开关对GLP-1R活性的影响将使用b-arrestin-mCherry作为受体激活的报告因子进行评估。这些研究将提供GLP-1R/GCGR激活机制的关键见解，这对于开发GLP-1R/GCGR双重激动剂作为治疗糖尿病和肥胖症的药物至关重要。