Enabling Technology to Screen and Quantify Sialylated Structures for Activity Against Viral Enzymes and Receptors

筛选和量化唾液酸化结构抗病毒酶和受体活性的技术

• 批准号: 10321682
• 负责人: Lisa A Holland
• 金额: $ 31.16万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10321682
• 关键词: Address; Affinity; Area; Automation; Binding; Biochemical; Biological; Biological Assay; Biological Markers; Blood capillaries; Capillary Electrophoresis; Cell surface; Cells; Chemicals; Complex; Complex Mixtures; Consumption; Coupled; Coupling; Detection; Development; Discrimination; Disease; Economic Burden; Electrophoresis; Enzyme Inhibition; Enzyme Inhibitor Drugs; Enzymes; Exoglycosidases; Functional disorder; Gel; Health; Hemagglutinin; Heterogeneity; Hospitalization; Human; Immune system; Infection; Influenza; Intercept; Isomerism; Label; Lectin; Life Cycle Stages; Ligands; Lipids; Liquid Chromatography; Liquid substance; Mass Spectrum Analysis; Measurement; Methods; NanoGel; Neuraminidase; Neuraminidase inhibitor; Oseltamivir; Pattern; Pharmaceutical Preparations; Physiological; Play; Polysaccharides; Post-Translational Protein Processing; Preparation; Proteins; Reaction; Reagent; Reference Standards; Regulation; Research; Research Personnel; Role; Sampling; Screening procedure; Sensitivity and Specificity; Sialic Acids; Signal Transduction; Source; Specificity; Speed; Structure; Techniques; Technology; Temperature; Therapeutic; Time; United States; Viral; Virus; Virus Diseases; Virus Receptors; Virus Replication; Viscosity; Work; analog; base; biomaterial compatibility; burden of illness; cold temperature; combat; cost; glycosylation; high throughput screening; improved; inhibitor; innovation; insight; instrument; ion mobility; ionization; mechanical energy; miniaturize; receptor; receptor binding; resilience; screening; sialylation; small molecule; tandem mass spectrometry; therapeutic development; tool; uptake; vibration; viron; voltage; zanamivir

Sialylated glycans are involved in complex regulation and signaling and play a critical role in disease. In the virus life cycle, receptor binding and sialic acid cleavage to facilitate release can compete and are therefore delicately balanced. While monovalent binding with a single sialylated ligand is weak (KD ~mM), hemagglutinin forms a trimer, which enables multivalent binding. Multivalent binding involving more than 1 ligand leads to strong binding (KD ~nM). This switch between multivalent and monovalent binding allows the hemagglutinin to bind to the host with high affinity that is easily reversed following internalization, replication, by cleavage of only some of the sialylated ligands. Neuraminidase inhibitors for influenza (Tamiflu, Relenza, and Rapivab) block the neuraminidase cleavage in some infections. The development of therapeutic strategies to block hemagglutinin binding with small molecule sialylated inhibitors has been limited. Several analytical barriers to the analysis of sialic acids and sialylated compounds have challenged research in this area. The long term objective of this project is to bridge this gap with enabling technology through two key innovations. A new screening approach for enzymes and receptors is introduced through the use of thermally reversible nanogels. With this new strategy, the sialic acid structures that interact with enzymes or receptors are identified through capillary electrophoresis. This work is based on rapid in-line exoglycosidase reactions facilitated with patterned nanogels. A new capillary electrophoresis-mass spectrometry interface based on acousto-mechanical energy is introduced to enable coupling both techniques without concern for voltage or flow rate. Aim 1 creates a new functional screening tool for enzyme inhibition and reduces both the amount of enzyme and the time to evaluate a neuraminidase preparation. The biocompatibility, automation, and low reagent and sample requirements are harnessed in Aim 2 to establish a quantitative screening tool to select and evaluate enzyme inhibition of sialylated structures that interact strongly with the receptor binding domain of the hemagglutinin protein. The full power of label-free structural identification of capillary electrophoresis interfaced to mass spectrometry outlined in Aim 3 leverages the unprecedented gains in signal with electrically assisted vibrational sharp edge ionization, overcoming barriers of current analytical technologies for the analysis of sialylated glycans. The proposed activities are significant because the low cost, speed, and automation of the separation-based microscale assays yield previously unattainable information about sialylation fundamental to mitigating viral infections. These new tools address challenges associated with chemical analyses of sialylated structures to leverage the role of sialylation in viral infections; thereby, providing researchers the means to combat viral diseases and advance human health.

囊囊的聚糖参与复杂的调节和信号传导，并在疾病中起关键作用。 在病毒生命周期中，受体结合和唾液酸切割以促进释放可以竞争，并且 因此，保持平衡。与单个siaLated配体的单价结合较弱（kd 〜mm），血凝集素形成三聚体，可实现多价结合。涉及多价结合 超过1个配体会导致较强的结合（Kd〜nm）。多价和单价之间的转换 结合使血凝素蛋白具有高亲和力与宿主结合，很容易逆转以下 内在化，复制，仅通过某些溶解的配体进行切割。神经氨酸酶抑制剂 对于流感（Tamiflu，Relenza和Rapivab），在某些感染中阻止了神经氨酸酶的裂解。 与小分子囊化的治疗策略开发以阻断血凝素结合的策略 抑制剂受到限制。分析唾液酸和溶解的几个分析障碍 化合物挑战了这一领域的研究。该项目的长期目标是桥接 通过两项关键创新来促进技术的差距。酶的新筛查方法 并通过使用热可逆纳米凝胶引入受体。有了这个新策略， 通过毛细管鉴定与酶或受体相互作用的唾液酸结构 电泳。这项工作是基于以图案化促进的快速内外糖苷酶反应 纳米凝胶。基于声学机械的新毛细管电泳质谱界面 引入能量以启用两种技术的耦合，而不必担心电压或流速。目的 1创建了一种用于酶抑制的新功能筛选工具，并减少了酶的量 以及评估神经氨酸酶制剂的时间。生物相容性，自动化和低 在AIM 2中利用试剂和样品要求，以建立定量筛选工具 选择并评估与受体强烈相互作用的硫化结构的酶抑制 血凝素蛋白的结合结构域。无标签结构识别的全部功能 毛细管电泳连接到AIM 3杠杆中概述的质谱法 具有电辅助振动锋利边缘电离的信号中空前的收益，克服 当前分析技术的障碍，用于分析溶解的聚糖。拟议的活动 之所以重要，是因为基于分离的微观测定的低成本，速度和自动化 产生以前无法实现的有关减轻病毒感染基本基础的溶解的信息。 这些新工具解决了与溶解结构的化学分析相关的挑战 利用囊酰化在病毒感染中的作用；因此，为研究人员提供战斗的手段 病毒疾病并改善人类健康。