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针对与病毒复制相关的病毒组分。这些药物必须能够渗透到细胞中，并具有足够的效力来阻止已经活跃的感染。最近，病毒进入的步骤已经开始被用于药物发现。病毒进入是建立病毒感染的第一步，也是关键的一步。干扰进入的化合物可以影响病毒与受体的结合、病毒摄取到内体中以及最终病毒和内体膜的膜融合。这些步骤中的每一个都可以被不需要渗透细胞膜的药物靶向，因为每个步骤都发生在细胞表面或包含从细胞外部取样的流体的内体内。除了那些针对艾滋病毒和甲型流感的药物外，很少有其他抗病毒药物被确定为目标进入。在这个项目中，我们的目标是识别和表征抑制丝状病毒和沙粒病毒进入的化合物。每个病毒家族都会引起出血热，并被归类为NIH 1类生物威胁和新兴传染因子。我们将在NCGC使用qHTS来鉴定抑制马尔堡（一种与埃博拉病毒相关的丝状病毒）和拉沙热病毒（一种沙粒病毒）感染的化合物。对于这两个家庭知之甚少的进入机制，没有有用的药物可用于治疗。我们已经开发了一个强大的检测平台，用于鉴定病毒进入抑制剂。该系统基于病毒伪型，因此可以在BSL 2上使用。筛选可以在BSL 2安全地进行，并且该测定已经在NCGC优化以在1536孔格式中给出> 25倍的信噪比和> 0.5的Z '。通过萤火虫荧光素酶进行读数，显示出良好的信号剂量-响应线性。我们还将进行一组二级试验，旨在鉴定病毒进入被阻断的步骤（结合、摄取和膜融合）。我们实验室先前已针对这两种病毒类型发表了这些检测方法。还将测试每种化合物对野生型病毒的效力。然后我们将有机会确定代理特异性或广谱抗病毒化合物。未来的工作将涉及将最好的化合物开发成潜在有用的药物，并将其用作探针，以更好地了解每种病毒的病毒进入机制以及感染细胞的途径。 公共卫生相关性：目前尚无有效的药物可用于治疗由丝状病毒或沙粒病毒引起的疾病。该提案旨在确定抑制这些病毒进入细胞的新化合物。将在NIH筛选中心对30万种化合物进行全面筛选，并分析每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.