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

基本信息

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
项目类别：
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

项目摘要

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，受体结合和促进释放的唾液酸裂解可以竞争并因此是微妙的平衡。虽然与单个唾液酸化配体的单价结合很弱（KD ~mM)，血凝素形成三聚体，从而实现多价结合。多价结合涉及超过 1 个配体会导致强结合 (KD ~nM)。多价和单价之间的转换结合允许血凝素以高亲和力与宿主结合，并且在以下情况下很容易逆转通过仅切割一些唾液酸化配体进行内化、复制。神经氨酸酶抑制剂治疗流感的药物（达菲、瑞乐沙和拉皮瓦）可阻断某些感染中的神经氨酸酶裂解。阻断血凝素与小分子唾液酸化结合的治疗策略的开发抑制剂受到限制。唾液酸和唾液酸化物分析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架起桥梁通过两项关键创新来弥补这一差距。酶的新筛选方法受体是通过使用热可逆纳米凝胶引入的。凭借这一新战略，通过毛细管识别与酶或受体相互作用的唾液酸结构电泳。这项工作基于通过图案化促进的快速在线外切糖苷酶反应纳米凝胶。一种基于声机械的新型毛细管电泳-质谱联用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。目的 1 创造了一种新的酶抑制功能筛选工具，同时减少了酶的用量以及评估神经氨酸酶制剂的时间。生物相容性、自动化、低目标 2 利用试剂和样品要求建立定量筛选工具选择并评估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。无标签结构识别的全部功能目标 3 中概述的毛细管电泳与质谱联用利用了通过电辅助振动锐边电离获得前所未有的信号增益，克服了当前分析唾液酸化聚糖的分析技术的障碍。拟议的活动意义重大，因为基于分离的微量检测成本低、速度快且自动化产生以前无法获得的有关唾液酸化的信息，这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的手段病毒性疾病并促进人类健康。