Design and synthesis of a next generation glycobiology toolbox for cell surface labeling

用于细胞表面标记的下一代糖生物学工具箱的设计和合成

基本信息

批准号：
10699270
负责人：
Samuel Justin Polizzi
金额：
$ 29.09万
依托单位：
CHEMILY, LLC
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10699270
关键词：
Acceleration Alkynes Antibodies Bacteria Bioinformatics Biological Biological Assay Biotin Biotinylation Carbohydrates Cell Communication Cell membrane Cell surface Cells Chemicals Chemistry Clinical Communication Custom Cytidine Monophosphate N-Acetylneuraminic Acid DNA Primers Data Development Discipline Disease Engineering Enzymes Flow Cytometry Fluorescent Dyes Folic Acid Fucosyltransferase Genetic Glycobiology Glycoengineering Glycoproteins Goals Guanosine Diphosphate Fucose Health Image In Situ Label Mammalian Cell Mass Spectrum Analysis Mediating Medical Membrane Metabolic Metabolism Methods Modeling Modification Morphology Mus Nature Other Genetics Phase Physiology Polysaccharides Procedures Process Proteins Protocols documentation Publishing Reagent Recombinants Reporting Rhamnose Role Route Sampling Selection Criteria Sialyltransferases Signal Transduction Surface Techniques Technology Testing Therapeutic Tissues Transferase Work biochemical tools biomarker discovery cell type chimeric antigen receptor T cells commercialization crosslink design fluorophore glycosyltransferase immunoregulation live cell imaging meetings next generation stem sugar sugar nucleotide targeted delivery tool tumor user-friendly

项目摘要

The cell membrane is the interface by which a cell interacts and communicates with its surroundings, including nearby cells, proteins, and antibodies. Membranes of healthy cells are often modified in diseased states, providing an external means to distinguish between morphologically similar cells. Often, diseased cells display modified carbohydrates and glycans on their cell membranes. Recent developments in enzyme-mediated cell surface engineering provide promising routes for labeling these modifications in different cell types. The approach known as chemoenzymatic glycan labeling combines glycosyltransferases and unnatural nucleotide sugar donors equipped with chemical tags to directly label specific surface glycans within biological samples. Recently, this single step labelling approach has been successfully used with fucosyltransferase and GDP-fucose equipped with biotin tags. Compared with other genetic and metabolic techniques, this method of labeling living cells is a more direct, simpler procedure with a faster turnover cycle. It has also been shown to be generally applicable to murine tumor models, and thus has a high potential for transfer to a clinical setting. While published reports have focused on a low number of GDP-fucose labels, the high potential of this method is limited only by the availability of nucleotide sugar labels paired with acceptable glycosyltransferases. In order to accelerate medical advances based on cellular interactions, there is an urgent need to expand the availability of nucleotide sugar labels and transferases that can be used by this method. The goal of our Phase I project is to meet this need and provide non-expert users with a platform for labeling glycans on the membranes of their choice cells. Meeting this goal will expand the availability of glycoengineering reagents and methods beyond published studies. In Aim 1, we will create 12 new nucleotide sugar derivatives with labels. Derivatives will be based on successful studies of GDP-fucose, and expanded to CMP-sialic acid. Nucleotide sugars will be modified through click-chemistry with a fluorescent dye, biotin, or photoprobe label. In Aim 2, we will screen and optimize glycosyltransferases capable of attaching labeled nucleotide sugars to glycan substrates and cell surfaces. We will advance 10 kits that combine the most effective combinations of label and transferase, along with standard operating procedures and conditions that are accessible to end users. Users may then label membranes with fluorescent tags for cell surface imaging, biotin for cell selection, or photoprobes for crosslinking. Data will support Phase II efforts to compare this method to existing technologies and probe specific cell types.

细胞膜是细胞相互作用并与周围环境通信的界面，包括附近的细胞，蛋白质和抗体。健康细胞的膜经常在患病状态中修饰，提供一种外部手段来区分形态上相似的细胞。通常，病细胞显示其细胞膜上修饰的碳水化合物和聚糖。酶介导的细胞的最新发展表面工程提供了有希望的路线，用于将这些修饰标记为不同的细胞类型。方法被称为化学酶聚糖标记结合了糖基转移酶和不自然的核苷酸供体供体配备化学标签，直接在生物样品中标记特定的表面糖。最近，这个单步标记方法已成功用于配备配备有生物素标签。与其他遗传和代谢技术相比，这种标记活细胞的方法更多直接，更简单的过程具有更快的周转周期。它也已被证明通常适用于鼠肿瘤模型，因此具有很高的转移到临床环境的潜力。虽然已发表的报告集中在低数量的GDP饮用标签上，该方法的高电位仅受可用性的限制核苷酸糖标签与可接受的糖基转移酶配对。为了加速基于医疗进步在细胞相互作用上，迫切需要扩大核苷酸糖标签和转移酶的可用性该方法可以使用。我们阶段I项目的目标是满足这一需求，并为非专家用户提供一个平台在其选择细胞的膜上标记聚糖。实现此目标将扩大可用性超出已发表研究的糖化试剂和方法。在AIM 1中，我们将创建12种新的核苷酸糖带有标签的衍生物。衍生物将基于对GDP-凝胶糖的成功研究，并扩展到CMP-Sialic 酸。核苷酸糖将通过荧光染料，生物素或光探针通过点击化学修饰标签。在AIM 2中，我们将筛选并优化能够将标记的核苷酸糖连接到的糖基转移酶聚糖底物和细胞表面。我们将推进10个套件，以结合标签的最有效组合和转移酶，以及最终用户可访问的标准操作程序和条件。用户然后可以标记具有用于细胞表面成像的荧光标签，用于细胞选择的生物素或光周期的膜进行交联。数据将支持第二阶段的努力，将此方法与现有技术和探测进行比较特定的细胞类型。