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: 10543541
• 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/10543541
• 关键词: Acceleration; Address; Affinity; Area; Automation; Binding; Biochemical; Biological; Biological Assay; Biological Markers; Blood capillaries; Capillary Electrophoresis; Cell surface; Cells; Chemicals; Chromatography; Complex; Complex Mixtures; Consumption; Coupled; Coupling; Derivation procedure; 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; biomaterial compatibility; burden of illness; cold temperature; combat; cost; gel electrophoresis; glycosylation; high throughput screening; improved; inhibitor; innovation; insight; instrument; ion mobility; ionization; mechanical energy; miniaturize; nanopattern; promote resilience; receptor; receptor binding; 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.

唾液酸化聚糖参与复杂的调节和信号传导，并在疾病中发挥关键作用。 在病毒生命周期中，受体结合和唾液酸裂解以促进释放可以竞争和调节。 因此是微妙的平衡。虽然与单个唾液酸化配体的单价结合较弱（KD ~mM）时，血凝素形成三聚体，这使得能够进行多价结合。多价结合涉及 多于1个配体导致强结合（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宿主， 通过仅切割一些唾液酸化的配体，可以进行内化、复制。神经氨酸酶抑制剂 流感疫苗（达菲、瑞乐沙和Rapivab）在某些感染中阻断神经氨酸酶裂解。 阻断血凝素与唾液酸化小分子结合的治疗策略的发展 抑制剂是有限的。分析唾液酸和唾液酸化的几个分析障碍 化合物对这一领域的研究提出了挑战。该项目的长期目标是 通过两个关键的创新来实现技术的差距。一种新的酶筛选方法 并且受体通过使用热可逆纳米凝胶引入。有了这个新策略， 与酶或受体相互作用的唾液酸结构通过毛细血管 电泳这项工作是基于快速在线外切糖苷酶反应促进模式化 纳米凝胶一种基于声机械耦合的新型毛细管质谱接口 引入能量以使得能够耦合两种技术而不关心电压或流速。目的 1创造了一种新的酶抑制功能筛选工具， 以及评估神经氨酸酶制剂的时间。生物相容性、自动化和低成本 目标2利用试剂和样品要求建立定量筛选工具， 选择和评价与受体强烈相互作用的唾液酸化结构的酶抑制 血凝素蛋白的结合域。无标记结构鉴定的全部功能 目标3中概述的毛细管电泳与质谱法的接口利用了 电辅助振动锐边电离信号的前所未有的增益，克服了 分析唾液酸化聚糖的当前分析技术的障碍。拟议活动 因为基于分离的微量测定的低成本、速度和自动化 产生以前无法获得的关于唾液酸化的信息，唾液酸化是减轻病毒感染的基础。 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对抗 病毒性疾病和促进人类健康。