Development and Applications of Bioorthogonal Chemistry

生物正交化学的发展与应用

基本信息

批准号：
9309042
负责人：
Qing Lin
金额：
$ 29.81万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-01 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9309042
关键词：
Agonist Alkenes Alkynes Animals Bacteriophages Biophysics 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 Hydroquinones In Situ Kinetics Label Lasers Ligands Lysine Measures Mediating Metabolic Microscope Molecular Conformation 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 arrestin B azobenzene base biomaterial compatibility biophysical analysis chemical function chemical synthesis conformational conversion cycloaddition extracellular fluorophore glucagon-like peptide 1 insight irradiation member mutant next generation novel phenylalanine analog phenylhydrazine public health relevance receptor receptor function tool two-photon unnatural amino acids

项目摘要

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的一个主要障碍，特别是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定点引入这两个域。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，以实现对这两个域的生物物理研究。在我们之前的研究中，我们优化了生物正交的光诱导四唑-烯烃环加成反应（“光点击化学”），并开发了用于选择性的钯介导的交叉偶联反应。基于这些结果，我们计划在该应用中整合生命系统中的蛋白质标记以及用于进化序列特异性生物正交反应的噬菌体辅助反应策略。生物正交化学工具具有独特化学功能的遗传编码，可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，并研究它们的构象转变和光调节具体目标如下： 1）应用光点击化学在GCGR/GLP-1R上原位生成环境敏感荧光探针，并探测活细胞A中配体诱导的构象变化。螺[2,3]hex-1-ene或富马酸衍生的赖氨酸将被位点特异性地掺入GCGR/GLP-1R的细胞外环3区域以指导光点击化学，以及由此产生的荧光标记的GCGR/GLP-1R将用于特定配体诱导的构象转变的研究；2）开发GCGR/GLP-1R双标记的二元生物正交化学来探测活细胞中 FRET 引起的配体诱导构象变化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，以实现在近膜域同时引入两个荧光团。和 N 端胞外结构域，将通过 FRET 分别监测活细胞中特定肽配体灌注后这两个互连结构域的动态运动。 3）开发基于偶氮苯的光化学遗传工具，用于活细胞中GLP-1R激活的光学调节，将开发基于苯肼与氟喹诺酮缩合的生物相容性逆偶氮偶联反应，其与遗传编码一起。氟喹啉丙氨酸，将使我们能够将偶氮苯位点特异性地引入 GLP-1R 的两个调节区域。将使用 b-arrestin-mCherry 作为受体激活的报告基因来评估 GLP-1R 活性的可逆光开关作用，这些研究将为 GLP-1R/GCGR 激活机制提供关键见解。对于开发 GLP-1R/GCGR 双激动剂作为治疗糖尿病和肥胖症的治疗剂至关重要。