Benzofurazan sulfide fluorogenic agents for live cell subcellular thiol imaging

用于活细胞亚细胞硫醇成像的苯并呋喃硫化物荧光剂

• 批准号: 8688581
• 负责人: XIANGMING GUAN
• 金额: $ 32.75万
• 依托单位: SOUTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8688581
• 关键词: Affect; Aging; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Analytical Chemistry; Biochemical; Biochemical Process; Biological Assay; Biological Process; Cell Line; Cell Nucleus; Cell physiology; Cells; Confocal Microscopy; Cytoplasm; Cytosol; Development; Disease; Endoplasmic Reticulum; Environment; Environmental Risk Factor; Event; Extracellular Space; Fluorescence; Fluorescence Microscopy; Fluorescent Dyes; Functional disorder; Goals; Green Fluorescent Proteins; Human; Image; Imagery; Knowledge; Label; Life; Link; Location; Lysosomes; Malignant neoplasm of lung; Methods; Microscopy; Mitochondria; Molecular; Monitor; Monkeys; Morphologic artifacts; Neurons; Nuclear; Organelles; Organism; Oxidation-Reduction; Parkinson Disease; Photons; Physiological; Play; Preparation; Property; Proteins; Rattus; Reaction; Reporting; Research; Role; Sampling; Series; Staging; Sulfhydryl Compounds; Sulfides; System; Techniques; Time; Tissues; Work; analytical method; base; cell fixing; design; fluorophore; improved; interest; kidney cell; multi-photon; novel; peroxisome; prevent; public health relevance; targeted delivery; therapy development; tool; two-photon

DESCRIPTION: Thiol groups play significant roles in various normal and abnormal cellular functions. Thiol status is compartmentalized at the subcellular level with the most reducing to the most oxidizing in the order of mitochondria > nuclei > cytoplasm > endoplasmic reticulum > extracellular space. Thiol status is affected by various physiological, pathological and environmental factors. A change in thiol status in mitochondria/nucleus has been associated with aging and various disease states such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Current understanding of the impacts of thiol changes on these diseases is mostly based on research through thiol concentration determination using isolated mitochondria/nuclei. Many details and specifics remain unknown on thiols' role in the pathophysiology of these diseases. This application is aimed to develop thiol specific fluorogenic agents that can be used to image and quantify thiols in mitochondria/nuclei in real-time in live cells through fluorescence microscopy such as confocal microscopy and multi- photon (mostly two-photon) microscopy. These agents can provide detailed, specific, dynamic, and potentially molecular level information on thiols in mitochondria/nuclei in its intact and native physiological environment compared with most other analytical methods that provide information only on thiol concentration. The specific information can help us better understand thiols' role in the pathophysiology of the diseases and help prevent and find better treatment of the diseases. Currently, there is no agent that can be used to image and quantify thiols in mitochondria/nuclei in live cells through fluorescence microscopy. Our proposed work is based on our recent finding of a novel thiol specific sulfide-thiol exchange reaction. Based on this reaction, we have developed a class of benzofurazan sulfides that convert thiols specifically, rapidly, and completely to fluorescent molecules. One of the benzofurazan sulfides was selected to demonstrate the ability to image and quantify thiols in live cells through fluorescence microscopy. The work was reported recently in Analytical Chemistry. In this proposed work, we will take advantage of this novel finding and extend it to the development of agents that can image and quantify thiols in mitochondria/nuclei. We will link a benzofurazan sulfide with a well-established mitochondria or nucleus targeting molecule. These designed molecules will be synthesized. Their one-photon and two- photon fluorescence properties will be determined, and their ability to image and quantify thiols in mitochondria and nuclei will be evaluated in three different cell lines. The long term goal of this project is to develop agents that can image and quantify thiols in other subcellular organelles by linking a benzofurazan sulfide to a subcellular targeting molecule. These agents will be valuable tools in investigating thiols' roles in various disease states at the subcellular levels.

描述：巯基在各种正常和异常的细胞功能中发挥重要作用。巯基状态在亚细胞水平上被区室化，从最还原到最氧化的顺序为线粒体>细胞核>细胞质>内质网>细胞外空间。巯基状态受各种生理、病理和环境因素的影响。线粒体/细胞核中巯基状态的变化与衰老和各种疾病状态如阿尔茨海默病（AD）、帕金森病（PD）和肌萎缩侧索硬化（ALS）相关。目前对巯基变化对这些疾病的影响的理解主要是基于使用分离的线粒体/细胞核通过巯基浓度测定的研究。关于硫醇在这些疾病的病理生理学中的作用的许多细节和细节仍然未知。本申请旨在开发硫醇特异性荧光剂，其可用于通过荧光显微镜如共聚焦显微镜和多光子（主要是双光子）显微镜在活细胞中实时成像和定量线粒体/细胞核中的硫醇。与仅提供硫醇浓度信息的大多数其他分析方法相比，这些试剂可以提供关于线粒体/细胞核中硫醇在其完整和天然生理环境中的详细、特异性、动态和潜在分子水平信息。这些具体的信息可以帮助我们更好地了解硫醇在疾病的病理生理学中的作用，并有助于预防和找到更好的治疗方法。目前，没有试剂可以用于通过荧光显微镜成像和定量活细胞中线粒体/细胞核中的硫醇。 我们提出的工作是基于我们最近发现的一种新的硫醇特异性硫化物-硫醇交换反应。基于该反应，我们开发了一类苯并呋咱硫化物，其将硫醇特异性地、快速地且完全地转化为荧光分子。选择一种苯并呋咱硫化物来证明通过荧光显微镜成像和定量活细胞中硫醇的能力。这项工作最近发表在《分析化学》上。 在这项拟议的工作中，我们将利用这一新的发现，并将其扩展到开发可以成像和量化线粒体/细胞核中硫醇的试剂。我们将把苯并呋咱硫化物与成熟的线粒体或细胞核靶向分子连接起来。这些设计的分子将被合成。将测定它们的单光子和双光子荧光性质，并将在三种不同的细胞系中评价它们对线粒体和细胞核中的硫醇进行成像和定量的能力。 该项目的长期目标是开发能够通过将苯并呋咱硫化物连接到亚细胞靶向分子来成像和定量其他亚细胞器中的硫醇的试剂。这些试剂将是在亚细胞水平上研究硫醇在各种疾病状态中的作用的有价值的工具。

项目成果

Editorial of Virtual Special Issue on Progress in Medicinal Chemistry.

关于药物化学进展的虚拟特刊的社论。

• DOI: 10.1016/j.apsb.2016.08.002
• 发表时间: 2016
• 期刊: Acta pharmaceutica Sinica. B
• 作者: Guan,Xiangming

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.

使用新型苯并呋喃硫化物三苯基磷荧光化合物对活细胞中的非蛋白质硫醇成像和定量。

• DOI: 10.1007/s00216-017-0285-y
• 发表时间: 2017
• 期刊: Analytical and bioanalytical chemistry
• 影响因子: 4.3
• 作者: Yang,Yang;Guan,Xiangming
• 通讯作者: Guan,Xiangming

In Vitro and In Vivo Antimetastatic Effect of Glutathione Disulfide Liposomes.

• DOI: 10.1177/1179064417695255
• 发表时间: 2017
• 期刊: Cancer growth and metastasis
• 作者: Sadhu SS;Wang S;Dachineni R;Averineni RK;Seefeldt T;Xie J;Tummala H;Bhat GJ;Guan X
• 通讯作者: Guan X

Glutathione Disulfide Liposomes - a Research Tool for the Study of Glutathione Disulfide Associated Functions and Dysfunctions.

• DOI: 10.1016/j.bbrep.2016.06.017
• 发表时间: 2016-09
• 期刊: Biochemistry and biophysics reports
• 影响因子: 2.7
• 作者: Sadhu SS;Xie J;Zhang H;Perumal O;Guan X
• 通讯作者: Guan X

Cancer metastases: challenges and opportunities.

• DOI: 10.1016/j.apsb.2015.07.005
• 发表时间: 2015-09
• 期刊: Acta pharmaceutica Sinica. B
• 作者: Guan X