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

R01GM140560摘要：唾液酸化的多糖参与复杂的调节和信号转导，以及 在疾病中起着关键作用。在病毒生命周期中受体的结合和唾液酸的裂解 释放可以竞争，因此是微妙的平衡。而单价结合与单一的 唾液酸化的配体很弱(kD~mM)，血凝素形成三聚体，使多价结合成为可能。 多个配体的多价结合导致强结合(Kd~NM)。这种切换在 多价和单价结合允许血凝素以高亲和力与宿主结合 仅通过切割一些唾液酸化的配体，在复制和释放后很容易逆转。 治疗流感的神经氨酸酶抑制剂(达菲、瑞乐沙和雷帕单抗)阻断神经氨酸酶 在某些感染中出现卵裂。阻断血凝素结合的治疗策略的发展 用小分子唾液酸化的抑制剂已经受到限制。S的几个分析障碍分析 唾液酸和唾液酸化化合物对这一领域的研究提出了挑战。长期目标 该项目的关键是通过两项关键创新，通过使能技术来弥合这一差距。一种新的 介绍了利用热可逆性筛选酶和受体的方法。 纳米凝胶。在这种新策略下，与酶或受体相互作用的唾液酸结构是 经毛细管电泳法鉴定。这项工作是基于快速顺式外切糖苷酶 利用图案化的纳米凝胶促进反应。一种新的毛细管电泳质谱 引入了基于声学机械能的接口，使这两种技术无需 关注电压或流量。AIM 1创造了一种新的酶抑制功能筛选工具 并且减少了酶的数量和评估神经氨酸酶制剂的时间。这个 目标2利用了生物兼容性、自动化以及低试剂和样品要求 建立选择和评价唾液酸化结构酶抑制作用的定量筛选工具 它们与血凝素蛋白的受体结合域相互作用很强。的全部力量 毛细管电泳质谱联用的无标记结构鉴定 在Aim 3中，利用电助振动锐边实现了前所未有的信号增益 电离，克服当前分析技术的障碍。建议的活动包括 意义重大，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 病毒相关疾病和促进人类健康。

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.