Name: high-throughput screening for broad-spectrum chemical inhibitors of rna viruses
Uploaded: 2014-05-05T16:15:00
Description: Watch this Scientific Journal Video about High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses at JoVE.com

我们感谢伊夫·亚宁博士的卓有成效的意见和建议。我们要感谢HH迦得对植/仁和C Combredet她的技术支持。巴斯德弊端Infectieuses（计划福星到POV和HM- - 这项工作是由巴斯德研究所，中心法国国家科学研究（CNRS），研究所国家德拉桑特逸德拉RECHERCHEMédicale（INSERM），研究所卡诺支持L）时，国民新电倒拉RECHERCHE（ANR-RPIB，计划STING 2.0至POV），和“行政法院区域D&#39;法兰西岛大区法国”（化学库项目，赠款N°我06-222 / R和I 09-1739 / R以HM-L）。对CHIKV /任这项工作是由项目ArbOAS支持（ANR授予2010-INTB-1601-02）。

96孔子板1。准备与化合物<ol><li>将包含化合物的DMSO中的10mM储备溶液在-20℃至室温的96孔母板上。稀释5倍的化合物在DMSO在2毫米，以获得中间96孔稀释板。稀释吹打5微升到20微升的DMSO和混合来实现。 </li><li>吸取1微升的稀释板成白色，条形码组织培养96孔板干井。这第一组子板（D1）将被用来评估化合物以20μM的终浓度的毒性。店子板在-20℃备用。 </li><li>吹打从稀释板4微升到36微升的DMSO和混合再稀...

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening-based+identification+of+paramyxovirus+inhibitors.">High-throughput screening-based identification of paramyxovirus inhibitors.</a> J Biomol Screen. 13, 591-608 (2008).</li><li>Mar&#233;chal, E., Roy, S., Lafanech&#232;re, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemogenomics and Chemical Genetics: A User's Introduction for Biologists, Chemists and Informaticians. , (2011).</li><li>Gentzsch, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+C+virus+complete+life+cycle+screen+for+identification+of+small+molecules+with+pro-+or+antiviral+activity.">Hepatitis C virus complete life cycle screen for identification of small molecules with pro- or antiviral activity.</a> Antiviral Res. 89, 136-148 (2011).</li><li>Caignard, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+V+protein+of+Tioman+virus+is+incapable+of+blocking+type+I+interferon+signaling+in+human+cells.">The V protein of Tioman virus is incapable of blocking type I interferon signaling in human cells.</a> PLoS One. 8, (2013).</li></ol>

这里描述的筛选管线的目的是选择的化合物与合适的配置文件作为RNA病毒的广谱抑制剂。当10000的化合物库中筛选与此 ​​协议，上述过滤条件被应用， 即抑制MV复制优于75％，40个化合物（0.4％）的得分阳性。此外，约一半的人表现出一定的毒性在荧光素酶为基础的可行性分析，并无视这个原因。最后，一​​应俱全的命中打在用量较大再次接受测试。这个小套很容易被重新测试两个细胞...

RNA病毒负责了大量的各种人类感染，并同时拥有在公共健康和经济成本方面对全世界的人口产生巨大的影响。有效的疫苗已经开发了针对几个人RNA病毒，并且被广泛地用作预防性治疗。然而，仍然有严重缺乏的治疗药物抗RNA病毒的感染。事实上，有效的疫苗不提供对主要的人类病原体，如登革热病毒，丙型肝炎病毒或人类呼吸道合胞病毒（hRSV的）。此外，RNA病毒是负责大部分新出现的疾病，它已经在频率，因为全球性的交流和对生态系统人为的影响，增加的。针对代表RNA病毒这一威胁，我们的治疗阿森纳是极其有限的，相对低效的1-3。目前的治疗方法基本上都是基于重组I型干扰素（IFN-α/β），以刺激先天性免疫，或利巴韦林的管理。虽然这核苷类似物的作用方式是有争议的，可能依赖于各种机制，细胞IMPDH（次黄嘌呤核苷酸脱氢酶），这耗尽细胞内GTP池的抑制作用，显然是必要的4。利巴韦林，与聚乙二醇化干扰素-α结合，是对丙型肝炎病毒的主要治疗手段。然而，IFN-α/β和利巴韦林治疗的疗效是比较差在体内对大多数RNA病毒的，因为它们通过毒力表达的有效钝的IFN-α/β信号因子5和经常逃避利巴韦林3。这增加了一个事实，即利巴韦林治疗是提高重要的毒性问题，尽管它最近被批准对严重hRSV的疾病，有争议的好处6。最近，一些病毒特异性治疗方法已被销售，特别是对流感病毒与发展Õf神经氨酸酶抑制剂3。但是，大的多样性和RNA病毒的出现永久排除了对他们在一个相对紧密的未来每一个具体的治疗方法的发展。总之，这强调需要有效的战略，以确定并开发强大的抗病毒分子在不久的将来。 这是微不足道的说，一个广谱抑制剂有效对抗RNA病毒大面板会解决这个问题。虽然这样的分子仍然是一个病毒学家的梦想，我们更好地了解细胞防御机制和先天免疫系统表明，一些可能性存在7,8。几个学术和工业实验室正在寻求刺激的细胞防御机制或代谢途径钝病毒复制的特定方面的分子。虽然这种化合物可能会显示出显著的副作用，对急性病毒感染的治疗会辖编为一相对短的时间，使得即使在长期的一些潜在毒性他们可以接受的。各种策略已经被开发，以识别这样的广谱抗病毒药分子。一些研究项目旨在寻找针对特定抗辩或代谢途径的分子。这包括，例如，病原体识别受体诱发的抗病毒基因的表达和9激活抗病毒因子如引入RNaseL 10，自噬机械，以促进病毒降解11，核苷合成途径12,13，或凋亡级联反应以沉淀病毒感染细胞的死亡14。其他小组已经开发出不是目标为基础13,15-17表型的屏幕。在这种情况下，抗病毒分子通过它们的能力简单地识别，以阻止病毒复制的一个给定的蜂窝式系统中。一般的假设是，抑制2-3无关RNA病毒的化合物，将有一个合适的模式为广大-SPectrum抗病毒分子。有这样的经验方法选择的命中化合物的作用方式只是在第二次确定的，最终，可能导致新的细胞目标的识别抗病毒药物。有趣的是，1999年和2008年间获得美国食品药品监督管理局新药的回顾性分析表明，在一般情况下，这种表型筛查往往表现比目标为基础的方法更好地发现先入类小分子药物18 。 在高通量的基于细胞的测定病毒的复制通常由病毒致细胞病变效应测定。细胞被感染并培养在96 - 或384 - 孔板中测试的化合物的存在。之后的几天，细胞层被固定和染色的染料如结晶紫。最后，吸光度用酶标仪和化合物抑制病毒复制是由它们的能力确定为往复保存细胞层确定米病毒诱导的细胞病变效应。另外，病毒介导的细胞病变效应正在使用标准的可行性分析，如MTS减少评估。此类测定法是高度易处理的和具有成本效益的，但三个主要的限制受到影响。首先，它们需要一个病毒-细胞组合，其中病毒的复制是细胞病变中只有几天，但情况并非总是可能的，从而要求替代方法19。其次，他们都缺乏定量的，因为它们是基于对病毒复制的间接测量。最后，有毒化合物可以评价为阳性命中，并且因此必须与一个计数器屏幕测定细胞活力来消除。要克服一些障碍，重组病毒或复制子已设计通过反向遗传学表达报告蛋白，如绿色荧光蛋白或荧光素酶，由一个附加的转录单位或以帧为与病毒蛋白的基因（几个例子是20-23）。当这些病毒的复制，记者公关oteins与病毒蛋白本身产生在一起。这提供了一个非常定量测定来测量病毒的复制和评估候选分子的抑制活性。这是用于重组病毒表达荧光素酶（或生物发光能力的其他酶），因为此报告系统显示出一个很宽的动态范围内具有高灵敏度，几乎没有背景，尤其如此。此外，不存在激发光源，从而防止干扰化合物的荧光24。 在这里，我们详细地高通量协议来筛选的RNA病毒的广谱抑制剂，化学文库。化合物在感染了重组麻疹病毒（MV）表达萤火虫荧光素酶25（rMV2/Luc， 如图1所示 ，主要画面）人体细胞先进行测试。 MV属于Mononegavirales秩序，往往被视为负链RNA病毒的原型成员ES。因此，MV的基因组作为模板，通过病毒聚合酶合成mRNA分子对病毒蛋白质进行编码。在重组MV株称为rMV2/Luc，萤光素酶表达是由P和M基因（ 图2A）之间插入一个附加的转录单位表达。平行地，化合物使用的是商业荧光素酶为基础的试剂，用于评估，由ATP定量测试它们的毒性对人体细胞，代谢活性细胞中培养的数量（ 图1，主屏幕）。整个化学文库可以很容易地筛选出与这两个试验，以便选择是没有毒性的，有效地阻止MV复制的化合物。然后，点击是重新测试剂量-反应抑制的MV复制，缺乏毒性以及其对损害基孔肯雅病毒（CHIKV）复制（ 图1，二次屏）的能力。 CHIKV是披膜病毒科家族的成员，其基因组是apositive单链RNA分子。因此，它是完全无关的麻疹和化合物抑制两个MV和CHIKV站在一个很好的机会，以抑制RNA病毒大面板。 CHIKV非结构蛋白是直接从病毒基因组的转化，而结构蛋白是由转录和一个亚基因组mRNA分子的翻译编码。对CHIKV我们的体外复制的测定是基于重组菌株称为CHIKV /韧，它通过NSP3和NSP4序列26（ 图2B）之间的报告基因的插入表达的Renilla荧光素酶作为非结构性多蛋白的裂解的部分。海肾荧光素酶活性的度量允许病毒复制的监视在CHIKV生命周期的早期阶段。 这种高通量的协议被用于快速识别化合物在10000 molec商业库一个合适的模式为广谱抗病毒药物ULES丰富的化学多样性。化合物基本上按照五个平斯基的规则，分子量范围从250至600道尔顿，和日志D值低于5。大多数这些分子分别在其他商业库无法使用新的化学实体。

<table border="0" cellpadding="0" cellspacing="0" width="756">
	<colgroup>
		<col />
		<col /> 
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Freedom EVO platform</td>
			<td style="width:105px;">TECAN</td>
			<td style="width:119px;"> </td>
			<td style="width:316px;">Robotic platform</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-Well Polystyrene Cell Culture Microplates, White</td>
			<td style="width:105px;">Greiner Bio One</td>
			<td style="width:119px;">655083</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CellTiter-Glo Luminescent Cell Viability Assay</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">G7570</td>
			<td>Luciferase-based viability assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bright-Glo Luciferase Assay System</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">E2610</td>
			<td>Reagent containing firefly luciferase substrate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Renilla-Glo Luciferase Assay System</td>
			<td style="width:105px;">Promega</td>
			<td style="width:119px;">E2710</td>
			<td>Reagent containing Renilla luciferase substrate</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Britelite plus Reporter Gene Assay System</td>
			<td style="width:105px;">Perkin-Elmer</td>
			<td style="width:119px;">6016761</td>
			<td style="width:316px;">Reagent containing firefly luciferase substrate. Can be used as an alternative to Brigh-Glo reagent to determine luciferase activity</td>
		</tr>
	</tbody>
</table>

high-throughput screening for broad-spectrum chemical inhibitors of rna viruses

RNA病毒是负责重大人类疾病，如感冒，支气管炎，登革热，丙型肝炎或麻疹。他们还表示，因为增加了世界各地的交流与人群渗透越来越多的自然生态系统的一个新威胁。这样的情况出现的一个很好的例子是2005-2006年在印度洋基孔肯雅病毒的流行。在我们的细胞途径控制病毒复制的了解最新进展表明，靶向宿主细胞的功能的化合物，而不是病毒本身，能抑制RNA病毒的大面板。一些广谱抗病毒化合物被确定，与主机针对性的分析。然而，测量病毒复制的细胞培养物的抑制使用减少的细胞病变效应的读出还是代表一个最重要的筛选策略。这种功能的屏幕已经由重组病毒表达记者的发展有了很大的提高酶CA帕布尔生物发光如荧光素酶。在本报告中，我们详细介绍了高通量筛选的管道，它结合了重组麻疹和基孔肯雅病毒与细胞活力检测，以确定化合物具有广谱抗病毒配置文件。

这种筛选流水线首先依赖于抑制MV复制化合物的选择和不显示任何显著的细胞毒性（ 图1，主屏幕）。它利用荧光素酶为基础的检测，以确定细胞毒性和病毒复制的抑制。所有吸移步骤和数据采集可以在高通量环境来执行与机器人平台。 化合物的细胞毒性是使用计算结果的基础上，ATP的存在，它对应于代谢活性细胞的定量在培养的活细胞的数目的荧光素酶为基础?...

Watch this Scientific Journal Video about High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses at JoVE.com

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

在体外测定法来测量病毒的复制已经由重组RNA病毒表达荧光素酶或能生物发光的其它酶的开发大大提高。这里我们详细介绍，结合麻疹和基孔肯雅病毒，从化学文库分离出广谱抗病毒药物，例如重组菌株的高通量筛选的管道。

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Research

JoVE Journal

Immunology and Infection

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

科研

免疫与感染

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

用于适应高通量/高含量筛查的细胞内分枝杆菌定量的微观表型检测

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

细菌人工染色体：对于正链RNA病毒研究的功能基因组学工具

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

在参与宿主因素的高通量筛选基于显微镜的检测方法<em&gt;布鲁氏菌</em&gt; Hela细胞感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

基于荧光的淋巴细胞检测适用于小分子的高通量筛选

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...