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.

唾液酸化的多糖参与了复杂的调控和信号传递，在疾病中起着关键作用。 在病毒生命周期中，受体结合和唾液酸裂解促进释放可以竞争和 因此达到了微妙的平衡。而与单个唾液酸化配体的单价结合很弱(Kd ~mm)，血凝素形成一个三聚体，使多价结合成为可能。多价结合涉及 一个以上的配体导致强结合(Kd~nM)。这种多价和单价之间的转换 结合允许血凝素以高亲和力结合到宿主上，这种结合很容易被逆转 内化，复制，仅通过切割一些唾液酸化的配体。神经氨酸酶抑制剂 对于流感(达菲、瑞乐沙和Rapivab)，在某些感染中阻止神经氨酸酶的裂解。 唾液酸化小分子阻断血凝素结合的治疗策略研究进展 抑制剂一直是有限的。唾液酸及唾液酸化产物分析中的几个分析障碍 化合物对这一领域的研究提出了挑战。这个项目的长期目标是架起 这与通过两项关键创新实现技术的差距。一种新的酶筛选方法 受体是通过使用热可逆的纳米凝胶引入的。有了这一新战略， 通过毛细管鉴定与酶或受体相互作用的唾液酸结构。 电泳法。这项工作是基于快速内联外糖苷酶反应，通过模式化促进 纳米凝胶。一种基于声机械的新型毛细管电泳质谱接口 引入能量以实现两种技术的耦合，而无需考虑电压或流量。目标 1创造了一种新的酶抑制功能筛选工具，并减少了酶的数量 以及评估神经氨酸酶制剂的时间。生物兼容性、自动化和低成本 在目标2中利用试剂和样本要求来建立定量筛查工具 选择和评估与受体强相互作用的唾液酸化结构的酶抑制 血凝素蛋白的结合域。无标签结构识别的全部威力 与AIM 3中概述的质谱学接口的毛细管电泳会利用 通过电助振动锐边电离，信号获得前所未有的收益，克服了 目前唾液酸聚糖分析技术的障碍。拟开展的活动 具有重要意义，因为基于分离的微量分析的低成本、快速和自动化 提供以前无法获得的关于唾液酸化的信息，这些信息对于减轻病毒感染至关重要。 这些新工具解决了与唾液酸化结构的化学分析相关的挑战 利用唾液酸化在病毒感染中的作用；从而为研究人员提供了对抗 病毒性疾病和促进人类健康。