Discovery & Characterization of Filo-. Arena -and Alpha Virus Entry Inhibitors

RNA病毒是NIAID A、B和C类生物防御和新兴病毒名单中的重要成员 传染病病原体。在这项提案中，治疗学正在被识别、表征和发展。 作为我们的目标，我们选择专注于受体的相互作用和进入。这是建立 事实证明，破坏是一种感染，在防止感染和病毒传播方面是有效的。我们有 开发并发布了一系列检测方法，已成为鉴定此类疾病的平台技术 药物，更重要的是，用于作用机制分析。我们的系统现在发展到了这样一个地步 我们可以表演非常高密度的银幕。首先，我们将使用一个包含200,000个化合物的库 与美国国立卫生研究院化学基因组中心合作。活性化合物的优先顺序将基于 结构-活性关系的比较分析跨由多个 剂量。我们的UTMB团队将致力于将HITS开发成有用的药物。我们将系统地比较两个 三类病毒的成员，丝状病毒、埃博拉病毒和马尔堡病毒；阿雷纳病毒、拉萨病毒和朱宁病毒； 还有甲型病毒、委内瑞拉马脑炎和基孔肯雅病毒。比较每种病毒在 一次筛查将是识别针对1)特定病毒类型，2)病毒的潜在活性药物的有效手段 家庭，或3)广谱活动的家庭。所有药物都将接受针对野生型病毒的测试。接下来，我们 将跟进我们的埃博拉病毒研究，在该研究中，我们确定了FDA批准的有效阻断埃博拉病毒的药物 病毒感染。在用小的药物文库和siRNA进行初步筛选时，我们发现 阻断钙离子流入细胞可有效抑制埃博拉病毒感染和细胞进入，从而防止 细胞病变效应。我们将描述每种药物的作用机制。首先，我们将确定最多的 通过我们专门的病毒进入试验和使用野生型病毒，有效的钙通道阻断药物。 其次，我们将通过确定下游信令目标(例如，CAMPE和 CaM激酶)，这反过来又可能成为抗病毒治疗的有用靶点。第三，我们将研究 通过对包膜蛋白-细胞的生化分析发现病毒包膜蛋白在触发这一级联反应中的作用 互动。最后，我们将在动物疾病模型中测试候选药物。我们还将测试其他 丝状病毒，包括马尔堡病毒。我们的发现将有助于开发一种强大的丝状病毒疗法和 了解钙信号在病毒感染和潜在致病机制中的作用。

RNA viruses are prominent members of NIAID's Category A, B, and C lists of biodefense and emerging infectious disease agents. In this proposal therapeutics are being identified, characterized and developed. As our target we have chosen to focus on receptor interaction and entry. This is the first step in establishing an infection, and disruption has proved effective at preventing infection and virus spread. We have developed and published a series of assays that have become a platform technology for identifying such drugs and, importantly, for mechanism of action analysis. Our systems are now developed to the point where we can perform very high-density screens. To begin, we will use a library of 200,000 compounds in collaboration with the NIH Chemical Genomics Center. Active compounds will be prioritized based on comparative analyses of structure-activity relationships across the panel of assays determined from multiple dosages. Our UTMB team will work to develop hits into useful drugs. We will systematically compare two members each of three classes of virus, the filoviruses, Ebola and Marburg; arenaviruses, Lassa and Junin; and the alphaviruses, Venezuelan equine encephalitis and chikungunya viruses. Comparing each virus in one screen will be an effective means to identify potential drugs active against 1) specific virus types, 2) virus families, or 3) those of broad spectrum activity. All drugs will be tested against wild-type viruses. Next, we will follow up on our Ebola virus study, in which we identified FDA-approved drugs effective at blocking Ebola virus infection. In preliminary screens with small drug libraries and siRNA, we discovered that drugs that block calcium flux into cells are effective at inhibiting Ebola virus infection and cell entry, thereby preventing cytopathic effect. We will characterize each drug's mechanism of action. First, we will identify the most potent calcium channel blocking drugs via our specialized virus entry assays and use of wild-type virus. Second, we will address mechanism of action by identifying downstream signaling targets (e.g., CALM and CAM kinases) that may, in turn, be useful targets for anti-viral therapy. Third, we will examine the role of virus envelope protein in triggering this cascade by biochemically analyzing envelope protein-cell interactions. Finally, we will test the candidate drugs in animal disease models. We will also test other filoviruses, including Marburg virus. Our findings will aid in developing a robust filovirus therapy and an understanding of how calcium signaling functions in virus infection and potentially pathogenesis.