High Throughput Assays for Ion Channel Activities of Influenza A & B Viruses

甲型流感离子通道活性的高通量测定

• 批准号: 7153194
• 负责人: LAWRENCE H PINTO
• 金额: $ 60.59万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7153194
• 关键词: infection; influenza; ions; proteins

DESCRIPTION (provided by applicant): Influenza contributes substantially to worldwide morbidity and mortality. Influenza A virus has caused all human pandemics and influenza B virus infections occur annually and in some recent years they have constituted 50% of total influenza virus infections, with high infection rates in nursing homes, hospitals, boarding schools and prisons. Influenza is a select agent. The M2 ion channel protein of influenza A virus is the target of the antiviral drug amantadine and the BM2 ion channel protein is a potential target for antiviral drugs because the ion channel activity of each protein is essential for replication of the virus. There is a need to develop more effective antiviral drugs against the A/M2 ion channel because escape mutants arise in patients taking the drug and there are no presently known antivirals against the BM2 ion channel. To screen compounds for antiviral activity the community needs high-throughput methods that can reliably detect decreases in ion channel activity. We propose to develop and validate two high-throughput screening assays for this purpose. The assays are based on the high rate of proton conduction of both of these ion channels and involve measurement of either intracellular pH changes in mammalian cells that express the ion channel or pH changes in liposomes reconstituted to contain the recombinant ion channel protein. Both of these assays are now used routinely in our laboratories for mechanistic studies of these ion channels but neither of the assays has been developed for high-throughput screening. We propose to develop robotic methods to perform assays in 596 well plates using both methods. Our goals will be (1) to identify the experimental variables that give rise to variation in the readings from well-to-well and minimize this variation such that the coefficient of variation is less than 10% and (2) to reduce the baseline activity for A/M2 in the presence of amantadine to less than 2% of the control value. The methods developed will be disseminated to others in three ways. First, we will publish our results in the peer-reviewed scientific literature; second, we will post the results and a detailed method on our laboratory web-sites; third, we will offer a minicourse that will be open to members of the community and will give both theoretical and practical aspects of performing the assays, reducing variability and interpretation of results. These assays will make it possible to perform efficient searches for antiviral drugs against the M2 ion channels.

描述（由申请人提供）：流感对全世界的发病率和死亡率有重大影响。甲型流感病毒引起了所有人类大流行，乙型流感病毒感染每年都会发生，近年来，它们已占流感病毒感染总数的50%，在疗养院、医院、寄宿学校和监狱中感染率很高。流感是一种选择性病原体。甲型流感病毒的M2离子通道蛋白是抗病毒药物金刚烷胺的靶点，而BM2离子通道蛋白是抗病毒药物的潜在靶点，因为每种蛋白的离子通道活性对于病毒的复制至关重要。需要开发更有效的针对 A/M2 离子通道的抗病毒药物，因为服用该药物的患者会出现逃逸突变体，并且目前还没有已知的针对 BM2 离子通道的抗病毒药物。为了筛选化合物的抗病毒活性，业界需要能够可靠检测离子通道活性下降的高通量方法。我们建议为此目的开发和验证两种高通量筛选测定法。该测定基于这两种离子通道的高质子传导率，并涉及测量表达离子通道的哺乳动物细胞中的细胞内pH变化或重构以含有重组离子通道蛋白的脂质体中的pH变化。这两种测定方法现在在我们的实验室中常规用于这些离子通道的机制研究，但是这两种测定方法都尚未开发用于高通量筛选。我们建议开发机器人方法，使用这两种方法在 596 孔板中进行测定。我们的目标是 (1) 识别导致孔间读数变化的实验变量，并将这种变化最小化，使变化系数小于 10%；(2) 降低基线活性在金刚烷胺存在下，A/M2 低于对照值的 2%。所开发的方法将通过三种方式传播给其他人。首先，我们将在同行评审的科学文献中发表我们的结果；其次，我们将在实验室网站上发布结果和详细方法；第三，我们将提供一门向社区成员开放的迷你课程，并将提供进行分析、减少变异性和结果解释的理论和实践方面的内容。这些测定将使针对 M2 离子通道有效搜索抗病毒药物成为可能。