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

行政设备补充使技术能够筛选和量化唾液酸化结构的抗病毒酶和受体活性

• 批准号: 10389191
• 负责人: Lisa A Holland
• 金额: $ 20.5万
• 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/10389191
• 关键词: Address; Affinity; Area; Automation; Binding; Biological Assay; Capillary Electrophoresis; Chemicals; Complex; Coupling; Disease; Enzyme Inhibition; Enzymes; Equipment; Exoglycosidases; Health; Hemagglutinin; Human; Infection; Influenza; Label; Life Cycle Stages; Ligands; Mass Spectrum Analysis; Mechanics; NanoGel; Neuraminidase; Neuraminidase inhibitor; Oseltamivir; Pattern; Play; Polysaccharides; Preparation; Proteins; Reaction; Reagent; Regulation; Research; Research Personnel; Role; Sampling; Screening procedure; Sialic Acids; Signal Transduction; Speed; Structure; Techniques; Technology; Time; Viral; Virus; Virus Diseases; Work; base; biomaterial compatibility; combat; cost; inhibitor/antagonist; innovation; ionization; receptor; receptor binding; screening; sialylation; small molecule; therapeutic development; tool; vibration; voltage; zanamivir

Abstract for R01GM140560: 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 replication and release 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 barrier s 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 outl ined in Aim 3 leverages the unprecedented gains in signal with electrically assisted vibrational sharp edge ionization, overcoming barriers of current analytical technologies. 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 virus related diseases and advance human health.

R 01 GM 140560摘要：唾液酸化聚糖参与复杂的调控和信号传导， 在疾病中起着关键作用。在病毒的生命周期中，受体结合和唾液酸裂解促进 释放可以竞争，因此是微妙的平衡。虽然单价结合单个 唾液酸化配体是弱的（KD ~mM），血凝素形成三聚体，这使得能够进行多价结合。 涉及多于1个配体的多价结合导致强结合（KD ~nM）。这种转换 多价和单价结合允许血凝素以高亲和力结合宿主， 在复制后容易逆转，并通过仅切割一些唾液酸化配体释放。 流感的神经氨酸酶抑制剂（达菲、瑞乐沙和Rapivab）阻断神经氨酸酶 在某些感染中出现分裂。阻断血凝素结合的治疗策略的发展 小分子唾液酸化抑制剂的应用受到限制。分析的几个分析障碍 唾液酸和唾液酸化的化合物对这一领域的研究提出了挑战。长期目标 该项目的主要目标是通过两项关键创新，利用使能技术弥合这一差距。一个新 通过使用热可逆的酶和受体的筛选方法被引入 纳米凝胶通过这种新策略，与酶或受体相互作用的唾液酸结构被 通过毛细管电泳鉴定。这项工作是基于快速在线外切糖苷酶 用图案化的纳米凝胶促进反应。一种新的毛细管电泳-质谱法 引入了基于声机械能的接口，以使得能够耦合两种技术， 关注电压或流速。AIM 1创建了一种新的酶抑制功能筛选工具 并且减少了酶的量和评估神经氨酸酶制剂的时间。的 Aim 2利用生物相容性、自动化以及低试剂和样品要求， 建立定量筛选工具以选择和评价唾液酸化结构的酶抑制 与血凝素蛋白的受体结合域强烈相互作用。的全部力量 毛细管电泳-质谱联用的无标记结构鉴定 在目标3中，利用了前所未有的信号增益， 电离，克服了目前分析技术的障碍。拟议的活动是 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 病毒相关疾病，促进人类健康。