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:10543732
- 负责人:
- 金额:$ 39.62万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2019
- 资助国家:美国
- 起止时间:2019-01-01 至 2024-04-30
- 项目状态:已结题
- 来源:
- 关键词:AddressAgonistAlkynesAmino AcidsArrestinsBiologicalBiologyBiophysicsBiosensorCellsChemicalsChemistryComplexCouplingCrosslinkerDevelopmentDiabetes MellitusDrug TargetingFluoresceinFluorescenceFluorescence Resonance Energy TransferG-Protein-Coupled ReceptorsGeneticGenetic CodeIn SituLabelLigandsLigationMammalian CellMapsMass Spectrum AnalysisMediatingMolecular Biology TechniquesMolecular ConformationMovementObesityPalladiumPhotoaffinity LabelsProtein ConformationProteinsProteomicsReactionReagentReceptor ActivationReceptor SignalingResearchSignal TransductionSiteTimecyanine dye 5cycloadditiondesignextracellularfluorophoreinsightinterestmembernovelprotein protein interactionreal time monitoringreceptorresponsetargeted treatmenttooltool development
项目摘要
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 tetrazolealkene cycloaddition reaction (‘photoclick’ chemistry), the spiroalkenetetrazine
ligation reaction, the palladium-mediated cross-coupling reactions, and the sequence-specific 2-cyanobenzo-
thiazolecysteine 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 EGFRGrb2 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.
生物正交化学的发展与应用
摘要
这个MIRA应用程序结合了我们实验室的两项研究工作,以解决以下研究中的关键障碍:
B类GPCR生物物理学和活细胞中的信号传导,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的瞬时且高度动态的GPCR相互作用蛋白质
有偏见的对抗。我们长期以来一直致力于开发基于反应性的化学工具,
这是使用常规分子生物学技术难以解决的重大生物学问题。在
在过去的五年里,我们继续在工具开发和这些工具的应用方面取得进展,
解决重要的生物学问题。例如,我们优化了几个生物正交反应,包括
光诱导的四唑-四烯烃环加成反应(“光点击”化学),螺烯烃-四嗪
连接反应、钯介导的交叉偶联反应和序列特异性2-氰基苯并-
噻唑-半胱氨酸连接反应。再加上涉及螺烯氨基的遗传密码扩展
酸,这些反应中的两个(光点击化学和四嗪连接)被利用用于位点特异性的
在GLP-1 R和GCGR的细胞外环3处引入有机荧光团(荧光素、Cy 3和Cy 5),
两个与糖尿病和肥胖有关的B类GPCR成员,用于正在进行的单细胞FRET研究
在活细胞中配体诱导的受体活化过程中的结构域运动。此外,我们还制作了一个
一个偶然的发现,2-芳基-5-羧基四唑(ACT)提供了一个新的邻近依赖的光交叉,
连接子,然后将其用于设计能够原位捕获和
随后鉴定药物靶标以及用于位点特异性的遗传编码的氨基酸,
在哺乳动物细胞中,瞬时EGFR α Grb 2相互作用复合物的掺入和随后的捕获。
在这些结果的基础上,在本申请中,我们计划继续研究正交化学反应性,
化学-生物学接口,并进行以下两个相关的项目。在项目1中,我们将构建
基于FRET的GLP-1 R和GCGR生物传感器通过生物正交标记来探测构象
参与活细胞中受体活化和信号传导的动力学。一套新的荧光“开启”
将设计用于GLP-1 R细胞内环3(ICL 3)生物正交荧光标记的试剂
和GCGR,以允许活细胞中受体构象的单细胞内和分子间FRET分析。
在项目2中,我们将开发一种含有炔基的基因编码ACT光交联剂,
应用该光交联剂按质量绘制时间依赖性GLP-1 R和β-抑制蛋白相互作用组图谱
响应于配体刺激的光谱分析。我们希望这些研究不仅能验证新的
用于实时监测活细胞中蛋白质构象和蛋白质-蛋白质相互作用的化学工具,
还提供了新的见解GLP-1 R和GCGR激活和偏置信号,这是至关重要的
开发用于治疗糖尿病和肥胖症的靶向疗法。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Qing Lin其他文献
Qing Lin的其他文献
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{{ truncateString('Qing Lin', 18)}}的其他基金
Development of Orally Administered Peptide Hormones for Treatment of Diabetes and Obesity
用于治疗糖尿病和肥胖症的口服肽激素的开发
- 批准号:
10323876 - 财政年份:2021
- 资助金额:
$ 39.62万 - 项目类别:
Development and Applications of Bioorthogonal Chemistry: Administrative Supplement for Equipment
生物正交化学的发展与应用:设备管理补充
- 批准号:
10581256 - 财政年份:2019
- 资助金额:
$ 39.62万 - 项目类别:
Development and Applications of Bioorthogonal Chemistry
生物正交化学的发展与应用
- 批准号:
10317075 - 财政年份:2019
- 资助金额:
$ 39.62万 - 项目类别:
Development and Applications of Photoinducible Bioorthogonal Chemistry
光诱导生物正交化学的发展及应用
- 批准号:
8240114 - 财政年份:2009
- 资助金额:
$ 39.62万 - 项目类别:
Development and Applications of Bioorthogonal Chemistry
生物正交化学的发展与应用
- 批准号:
8913203 - 财政年份:2009
- 资助金额:
$ 39.62万 - 项目类别:
Development and Applications of Bioorthogonal Chemistry
生物正交化学的发展与应用
- 批准号:
9309042 - 财政年份:2009
- 资助金额:
$ 39.62万 - 项目类别:
Development and Applications of Photoinducible Bioorthogonal Chemistry
光诱导生物正交化学的发展及应用
- 批准号:
8460102 - 财政年份:2009
- 资助金额:
$ 39.62万 - 项目类别:
Development and Applications of Photoinducible Bioorthogonal Chemistry
光诱导生物正交化学的发展及应用
- 批准号:
7793428 - 财政年份:2009
- 资助金额:
$ 39.62万 - 项目类别:
Development and Applications of Bioorthogonal Chemistry
生物正交化学的发展与应用
- 批准号:
9266090 - 财政年份:2009
- 资助金额:
$ 39.62万 - 项目类别:
Development and Applications of Bioorthogonal Chemistry
生物正交化学的发展与应用
- 批准号:
8759491 - 财政年份:2009
- 资助金额:
$ 39.62万 - 项目类别:
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