Development of Location-specific Sialidase Inhibitors

项目概要/摘要 唾液酸酶（也称为神经氨酸酶）是负责去除唾液酸（Sia）的糖苷酶 来自糖缀合物的聚糖部分的残基（去唾液酸化）。通过去唾液酸化，唾液酸酶能够 调节含Sia分子的功能性和稳定性，并参与两者 生理和病理途径。以前和我们最近的研究表明，溶酶体Neu 1 唾液酸酶可以在LPS刺激后重新定位到巨噬细胞的细胞表面， TLR 4受体的去唾液酸化，导致TLR 4活化和随后的促炎性细胞因子的产生。 细胞因子LPS对TLR 4激活的失调是导致慢性和急性炎症的原因。 这些疾病通常会导致危险的疾病，如败血症， 治疗本申请的第一个目的是定量分析位置特异性表达 Neu 1唾液酸酶，对TLR 4活化及其随后的信号转导至关重要。唾液酸 抑制剂是用于研究唾液酸酶功能和生物活性的相关机制的有用工具。 途径。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 由唾液酸酶引起的途径，如TLR 4激活失调。我们最近的研究表明， 目前可用的PAN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唾液酸酶活性， 唾液酸酶有效。已经报道了几种哺乳动物唾液酸酶抑制剂。但目前的 唾液酸酶抑制剂的设计通常集中在活性位点的结合，而忽略了亚细胞定位 因此，它们在体内不太有效，甚至可能是有毒的，因为它们会影响其他生物。 细胞内的唾液酸酶本申请的第二个目的是开发位置特异性抑制剂 并确定Neu 1唾液酸酶参与LPS/TLR 4信号通路。的 本项目的目标将通过三个具体目标来实现：（1）分析Neu 1唾液酸酶表达 LPS刺激后巨噬细胞表面的迁移;（2）开发溶酶体靶向Neu 1 唾液酸酶抑制剂，有效调节LPS/TLR 4信号通路中的去唾液酸作用;（3）开发细胞 有效调节LPS/TLR 4信号传导中去唾液酸化的表面靶向Neu 1唾液酸酶抑制剂 通路这项研究是创新的，因为它使用了一种新颖的方法，克服了目前的局限性 在（a）分析唾液酸酶表达和重新定位和（B）在亚细胞位置抑制唾液酸酶中。的 拟议的项目是重要的，因为它将（i）揭示特定的去唾液酸化，这是至关重要的 LPS/TLR 4信号通路和（ii）开发新的唾液酸酶抑制剂用于有效调节去唾液酸化 LPS/TLR 4信号通路中。最后，这项建议将加强研究的基础设施， 教育在克利夫兰州立大学，让本科生学习广泛的 现代科学研究中使用的实验技术、数据分析和演示技巧。

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.