HTS for Filo- and Arena-virus Entry Mechanisms

描述(由申请人提供)：目前临床上的大多数抗病毒药物都针对与病毒复制有关的病毒成分。这些药物必须能够渗透到细胞内，并具有足够的效力来阻止已经活跃的感染。最近，病毒进入的步骤开始被用于药物开发。病毒进入是确定病毒感染的第一步，也是决定性的一步。干扰进入的化合物会影响病毒与受体的结合(S)，影响病毒进入内小体，最终影响病毒和内体膜的膜融合。这些步骤中的每一步都可以被不需要渗透细胞膜的药物作为靶点，因为每一步都发生在细胞表面或包含从细胞外采样的液体的内体内。除了那些针对艾滋病毒和甲型流感的药物外，几乎没有其他抗病毒药物被确定为进入市场的目标。在这个项目中，我们的目标是识别和表征抑制丝状病毒和虫媒病毒进入的化合物。每个病毒家族都会引起出血热，并被归类为美国国立卫生研究院第1类生物病毒和新出现的传染病病原体。我们将在NCGC使用qHTS来识别抑制马尔堡病毒(一种与埃博拉病毒有关的丝状病毒)和拉沙热病毒(一种阿拉伯病毒)感染的化合物。对于这两个家庭来说，对进入机制知之甚少，也没有有用的药物可供治疗。我们已经开发了一个强大的检测平台来鉴定这两种病毒进入抑制剂。该系统基于病毒伪型，因此可以在BSL2使用。在BSL2可以安全地进行筛查，NCGC已经对分析进行了优化，在1536井格式中提供了25倍和Z‘of&gt；0.5的信噪比。用萤火虫荧光素酶读出，显示出良好的信号剂量-反应线性。我们还将进行一系列二次化验，以确定阻止病毒进入的步骤(结合、摄取和膜融合)。我们的实验室之前已经发表了这两种病毒的检测结果。每种化合物还将被测试对野生型病毒的有效性。然后，我们将有机会鉴定特定试剂或广谱抗病毒化合物。未来的工作将包括将最好的化合物开发成潜在有用的药物，并将每种化合物用作探针，以更好地了解每种病毒进入病毒的机制以及感染细胞的途径。 公共卫生相关性：目前还没有有效的药物来治疗丝状病毒或阿雷纳病毒引起的疾病。这项提议寻求确定抑制这些病毒进入细胞的新化合物。美国国立卫生研究院的筛选中心将对30万种化合物进行全面筛选，并分析每一种化合物的作用机制。

DESCRIPTION (provided by applicant): Most antiviral drugs in the clinic today target virus components related to viral replication. These drugs must be able to penetrate into the cell and be of sufficient potency to stop an already active infection. Recently, the step of virus entry has begun to be exploited for drug discovery. Virus entry is the first and committal step toward establishing a virus infection. Compounds that interfere with entry can affect binding of virus to receptor(s), uptake of virus into endosomes and finally membrane fusion of virus and endosomal membranes. Each of these steps can be targeted by drugs that do not need to permeate the cell membrane as each occurs at the cell surface or within endosomes that contain fluid sampled from outside of the cell. Aside from those against HIV and Influenza A, few other antiviral drugs have been identified that target entry. In this project we aim to identify and characterize compounds that inhibit the entry of filoviruses and arenaviruses. Each family of viruses cause hemorrhagic fever and are classified as NIH category 1 biothreat and emerging infectious agents. We will use qHTS at NCGC to identify compounds that inhibit infection of Marburg (a filovirus related to Ebola virus) and Lassa Fever virus (an arenavirus). For both families little is known about the entry mechanism and no useful drugs are available for therapy. We have developed a robust assay platform for identification of virus entry inhibitors for both. The system is based on virus pseudotypes and so can be used at BSL2. Screens can be safely performed at BSL2 and the assay has been optimized at NCGC to give signal-to-noise ratios >25-fold and Z' of >0.5 in the 1536-well format. Readout is by firefly luciferase and shows good dose-response linearity of signal. We will also perform a set of secondary assays designed to identify the step (binding, uptake and membrane fusion) at which virus entry was blocked. These assays have been previously published by our lab for both virus types. Each compound will also be tested for efficacy against wild type virus. We will then have the opportunity to identify agent- specific or broad-spectrum antiviral compounds. Future work will involve development of the best compounds into potentially useful drugs and use of each as probes to better understand the mechanism of virus entry for each virus and the pathways taken to infect the cell. PUBLIC HEALTH RELEVANCE: No effective drugs are available for treatment of disease caused by Filoviruses or Arenaviruses. This proposal seeks to identify new compounds that inhibit the entry of these viruses into cells. A comprehensive screen of 300,000 compounds will be performed at an NIH screening center and the mechanism of action of each hit will be analyzed.