Acquisition of a flash chromatography and HPLC preparative system

购置快速色谱和 HPLC 制备系统

• 批准号: 10794678
• 负责人: XUE-LONG SUN
• 金额: $ 7.66万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-15 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10794678
• 关键词: Active Sites; Acute; Affect; Binding Sites; Biological; Biological Process; Cell membrane; Cell surface; Cells; Chromatography; Chronic; Dangerousness; Data Analyses; Disease; Education; Enzymes; Excision; Family; Glycolipids; Glycoproteins; Glycoside Hydrolases; Goals; High Pressure Liquid Chromatography; Immune; Inflammatory; Interleukin-6; Investigation; Learning; Location; Lysosomes; Macrophage; Measures; Mediating; Membrane; Methods; Modernization; Molecular; Neuraminidase; Pathologic; Pathway interactions; Pharmaceutical Preparations; Pharmacological Treatment; Physiological; Polysaccharides; Production; Receptor Activation; Receptor Signaling; Regulation; Reporting; Research; Research Infrastructure; Sepsis; Sialic Acids; Signal Pathway; Substrate Specificity; System; TLR4 gene; TNF gene; Techniques; Testing; Toll-like receptors; Universities; Vertebrates; Western Blotting; analog; cell type; cytokine; design; enzyme activity; in vivo; inhibitor; innovation; microbial; mitochondrial membrane; neglect; novel; novel strategies; novel therapeutic intervention; parent grant; receptor; skills; tool; undergraduate student

Project Summary/Abstract The goal of the parent grant is to develop effective sialidase inhibitors. Sialidases (also called neuraminidases) are glycosidases responsible for the removal of sialic acid (Sia) residues (desialylation) from glycan portions of glycocojugates. By desialylation, sialidases are able to modulate the functionality and stability of the Sia-containing molecules and are involved in both physiological and pathological pathways. Previous and our recent study indicate that lysosomal Neu1 sialidase could relocate to the cell surface of macrophages upon LPS stimulation, where it causes desialylation of TLR4 receptor, leading to TLR4 activation and subsequent production of pro-inflammatory cytokines. Dysregulation of TLR4 activation by LPS is responsible for chronic and acute inflammatory disorders that often causes dangerous disease like sepsis that still lacks specific pharmacological treatment. Sialidase inhibitors are useful tools for studying sialidase function and related mechanisms of the biological pathways. More importantly, effective sialidase inhibitors can be used as drugs to regulate the pathological pathways caused by sialidase, such as dysregulated TLR4 activation. Our recent study indicates that currently available pan sialidase inhibitor and microbial sialidase inhibitors could not inhibit mammalian sialidase effectively. Several mammalian sialidase inhibitors have been reported. However, current sialidase inhibitor design has usually focused on active-site binding, neglecting the subcellular localization of the active enzyme, therefore, they are less effective in vivo or may be even toxic as they will affect other sialidases inside of the cells. Therefore, the objective of this application is to develop location-specific inhibitor for Neu1 sialidase and define the Neu1 sialidase’s involvement in LPS/TLR4 signaling pathway, which will be accomplished by three specific aims: (1) Profile Neu1 sialidase expression and cell surface relocation in macrophages upon LPS stimulation; (2) Develop lysosome-targeting Neu1 sialidase inhibitors for effective regulating desialylation in LPS/TLR4 signaling pathway; (3) Develop cell surface-targeting Neu1 sialidase inhibitors for effective regulating desialylation in LPS/TLR4 signaling pathway. This study is innovative because it uses a novel approach that overcomes the current limitations in (a) profiling sialidase expression and relocation and (b) inhibiting sialidase at subcellular location. The proposed project is significant because it will (i) uncover specific desialylation that is critical to the LPS/TLR4 signal pathway and (ii) develop novel sialidase inhibitors for effective regulation of desialylation in LPS/TLR4 signaling pathway. Finally, this proposal will enhance the infrastructure of research and education at Cleveland State University, allowing undergraduate students to learn a broad spectrum of experimental techniques, data analysis and presentation skills used in modern scientific investigations.

项目概要/摘要 母基金的目标是开发有效的唾液酸酶抑制剂。唾液酸酶（也称为 神经氨酸酶）是负责去除唾液酸 (Sia) 残基（脱唾液酸化）的糖苷酶 来自糖复合物的聚糖部分。通过去唾液酸化，唾液酸酶能够调节功能 和含 Sia 分子的稳定性，并参与生理和病理 途径。之前和我们最近的研究表明，溶酶体 Neu1 唾液酸酶可以重新定位到细胞中 LPS 刺激巨噬细胞表面，导致 TLR4 受体去唾液酸化，从而导致 TLR4 激活并随后产生促炎细胞因子。 TLR4 失调 LPS 的激活导致慢性和急性炎症性疾病，通常会导致危险 像脓毒症这样的疾病仍然缺乏特定的药物治疗。唾液酸酶抑制剂是有用的工具 用于研究唾液酸酶功能和生物途径的相关机制。更重要的是， 有效的唾液酸酶抑制剂可作为药物来调节由唾液酸酶引起的病理途径 唾液酸酶，例如 TLR4 激活失调。我们最近的研究表明，目前可用的平底锅 唾液酸酶抑制剂和微生物唾液酸酶抑制剂不能有效抑制哺乳动物唾液酸酶。 已报道了几种哺乳动物唾液酸酶抑制剂。然而，目前的唾液酸酶抑制剂设计 通常关注活性位点结合，忽略活性酶的亚细胞定位， 因此，它们在体内的效果较差，甚至可能有毒，因为它们会影响体内的其他唾液酸酶 细胞。因此，本申请的目的是开发Neu1的位置特异性抑制剂 唾液酸酶并定义 Neu1 唾液酸酶参与 LPS/TLR4 信号通路，这将是 通过三个具体目标来实现：(1) 分析 Neu1 唾液酸酶表达和细胞表面重定位 LPS 刺激下的巨噬细胞； (2) 开发靶向溶酶体的 Neu1 唾液酸酶抑制剂以有效 调节LPS/TLR4信号通路的去唾液酸化； (3) 开发细胞表面靶向Neu1唾液酸酶 有效调节 LPS/TLR4 信号通路去唾液酸化的抑制剂。这项研究具有创新性 因为它使用了一种新颖的方法，克服了当前在 (a) 分析唾液酸酶表达方面的局限性 和重新定位以及(b)抑制亚细胞位置的唾液酸酶。拟建项目意义重大 因为它将 (i) 揭示对 LPS/TLR4 信号通路至关重要的特定去唾液酸化作用，以及 (ii) 开发新型唾液酸酶抑制剂，有效调节 LPS/TLR4 信号通路中的去唾液酸化。 最后，该提案将加强克利夫兰州立大学的研究和教育基础设施 大学，让本科生学习广泛的实验技术、数据 现代科学研究中使用的分析和表达技巧。