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）：流感对全球发病率和死亡率做出了重大贡献。流感病毒引起了所有人类大流行病，并每年发生流感病毒感染，并且在近年来，它们占流感总流感病毒感染的50％，疗养院，医院，寄宿学校和监狱的感染率很高。流感是精选代理。流感A病毒的M2离子通道蛋白是抗病毒药物的靶标，而BM2离子通道蛋白是抗病毒药物的潜在靶标，因为每种蛋白的离子通道活性对于复制病毒是必不可少的。需要针对A/M2离子通道开发更有效的抗病毒药物，因为服用该药物的患者出现了逃生突变体，并且目前尚无针对BM2离子通道的抗病毒药。为了筛选抗病毒活性的化合物，社区需要高通量方法，这些方法可以可靠地检测到离子通道活性的降低。我们建议为此目的开发和验证两个高通量筛选测定法。这些测定基于这两种离子通道的质子传导率高，并涉及测量表达离子通道的细胞内pH变化，或者在重构以包含重组离子通道蛋白的脂质体中的pH变化或pH变化。现在，这两种测定法通常在我们的实验室中用于这些离子通道的机械研究，但两种测定都没有用于高通量筛选。我们建议开发使用两种方法在596个井板中执行测定法的机器人方法。我们的目标将是（1）确定实验变量会导致从井到孔中读取的变化，并最大程度地减少这种变化，以使变异系数小于10％和（2），以降低Amantadine在amantadine存在下的基线活性至少于2％的控制值。开发的方法将以三种方式传播给其他方法。首先，我们将在同行评审的科学文献中发布结果。其次，我们将在实验室网站上发布结果和详细方法；第三，我们将提供一个小型道路，将向社区成员开放，并提供进行测定的理论和实际方面，从而减少结果的可变性和解释。这些测定将使针对M2离子通道进行有效的抗病毒药物进行搜索。