Development of Location-specific Sialidase Inhibitors

项目摘要/摘要 唾液酸酶(又称神经氨酸酶)是负责清除唾液酸(Sia)的糖苷酶。 糖偶联物的多糖部分的残留物(去分析)。通过分析，唾液酸酶能够 调节含有SiA的分子的官能度和稳定性，并参与 生理和病理途径。先前和我们最近的研究表明，溶酶体Neu1 唾液酸酶可以在内毒素刺激下重新定位到巨噬细胞的细胞表面，从而导致 TLR4受体缺失，导致TLR4激活并随后产生促炎因子 细胞因子。内毒素对TLR4激活的调节失调是慢性和急性炎症的原因 经常导致危险疾病的疾病，如败血症，但仍缺乏特定的药物 治疗。这个应用程序的第一个目标是定量描述特定位置的表达 Neu1唾液酸酶，它对TLR4的激活和随后的信号转导至关重要。唾液酸酶 抑制剂是研究唾液酸酶功能及其相关生物学机制的有用工具。 小路。更重要的是，有效的唾液酸酶抑制剂可以作为药物来调节病理。 唾液酸酶引起的通路，如TLR4激活失调。我们最近的研究表明 目前可用的泛唾液酸酶抑制剂和微生物唾液酸酶抑制剂不能抑制哺乳动物 有效的唾液酸酶。已有几种哺乳动物唾液酸酶抑制剂的报道。但是，当前 唾液酸酶抑制剂的设计通常侧重于活性部位结合，而忽略了亚细胞定位 因此，它们在体内的效果较差，甚至可能是有毒的，因为它们会影响其他 细胞内的唾液酸酶。本申请第二个目的是开发位置特定的抑制剂 并明确Neu1唾液酸酶在脂多糖/TLR4信号通路中的作用。这个 该项目的目标将通过三个具体目标来实现：(1)表达Neu1唾液酸酶 和巨噬细胞在内毒素刺激下细胞表面的重新定位；(2)发展溶酶体靶向Neu1 唾液酸酶抑制剂可有效调节内毒素/TLR4信号通路的失活；(3)发展细胞 表面靶向Neu1唾液酸酶抑制剂有效调控脂多糖/TLR4信号转导途径的研究 路径。这项研究具有创新性，因为它使用了一种新的方法，克服了目前的局限性 在(A)分析唾液酸酶的表达和重定位以及(B)在亚细胞位置抑制唾液酸酶。这个 拟议的项目意义重大，因为它将(I)揭示对以下方面至关重要的具体愿望 LPs/TLR4信号通路和(Ii)开发新型唾液酸酶抑制剂以有效调节脱氢作用 在内毒素/TLR4信号通路中。最后，这项提议将加强研究的基础设施和 克利夫兰州立大学的教育，允许本科生学习广泛的 现代科学调查中使用的实验技术、数据分析和演示技能。

Project Summary/Abstract 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. The first objective of this application is to quantitatively profile the location-specific expression of Neu1 sialidase that is critical for TLR4 activation and its subsequent signal transduction. 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. The second 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. The objectives of this project 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.