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

描述（由申请人提供）：流感是造成全球发病率和死亡率的主要原因。甲型流感病毒是所有人类大流行病的罪魁祸首，乙型流感病毒感染每年都会发生，最近几年，乙型流感病毒感染占流感病毒感染总数的50%，在疗养院、医院、寄宿学校和监狱的感染率很高。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病毒的复制至关重要。有必要开发针对a /M2离子通道的更有效的抗病毒药物，因为在服用该药物的患者中会出现逃逸突变体，而且目前还没有已知的针对BM2离子通道的抗病毒药物。为了筛选具有抗病毒活性的化合物，需要能够可靠地检测离子通道活性降低的高通量方法。我们建议为此目的开发和验证两种高通量筛选试验。这些检测是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内pH变化或脂质体中含有重组离子通道蛋白的pH变化。这两种检测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，但这两种检测方法都没有用于高通量筛选。我们建议开发机器人方法，使用这两种方法在596孔板中进行分析。我们的目标是(1)确定导致井与井之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)在金刚烷胺存在的情况下，将A/M2的基线活度降低到控制值的2%以下。拟定的方法将以三种方式传播给其他国家。首先，我们将在同行评议的科学文献中发表我们的研究结果；第二，我们将在实验室网站上公布结果和详细方法；第三，我们将提供一个向社区成员开放的迷你课程，并将从理论和实践两方面进行分析，减少结果的可变性和解释。这些试验将使针对M2离子通道的抗病毒药物的有效搜索成为可能。

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.