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

• 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
• 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

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.

细胞膜是细胞与周围环境相互作用和交流的界面， 包括附近的细胞、蛋白质和抗体。健康细胞的膜在患病状态下经常被修饰， 提供外部手段来区分形态相似的细胞。通常，病变细胞表现出 修饰的碳水化合物和聚糖。酶介导的细胞生物学研究进展 表面工程为标记不同细胞类型中的这些修饰提供了有前景的途径。的方法 称为化学酶聚糖标记结合糖基转移酶和非天然核苷酸糖供体 其配备有化学标签以直接标记生物样品中的特定表面聚糖。最近这款 一步标记方法已成功地用于岩藻糖基转移酶和配备有 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是一种更有效的方法。 直接、更简单的程序，更快的周转周期。它也被证明是普遍适用于鼠 肿瘤模型，因此具有转移到临床环境的高潜力。虽然已发表的报告集中在 在少量GDP-岩藻糖标记上，该方法的高潜力仅受以下因素的限制： 与可接受的糖基转移酶配对的核苷酸糖标记。为了加速医学进步， 在细胞相互作用方面，迫切需要扩大核苷酸糖标记和转移酶的可用性 可以通过这种方法使用。 我们第一阶段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， 标记它们选择的细胞膜上的聚糖。实现这一目标将扩大 糖工程试剂和方法超出了已发表的研究。在目标1中，我们将创造12个新的核苷酸糖， 标签衍生品衍生物将在GDP-岩藻糖研究成功的基础上，扩展到CMP-唾液酸 酸核苷酸糖将通过点击化学与荧光染料，生物素，或光探针进行修饰 label.在目标2中，我们将筛选和优化能够将标记的核苷酸糖连接到 聚糖底物和细胞表面。我们将推进10个套件，联合收割机结合最有效的组合标签 和转移酶，沿着标准操作程序和终端用户可获得的条件。用户 然后可以用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探针标记膜 用于交联。数据将支持第二阶段的工作，将这种方法与现有技术进行比较， 特定细胞类型。