A cell based high-throughput screening approach for the discovery of new inhibitors of respiratory syncytial virus.
A cell based high-throughput screening approach for the discovery of new inhibitors of respiratory syncytial virus.
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DOI：
10.1186/1743-422x-10-19
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发表时间：
2013-01-10
期刊：
影响因子：
4.8
通讯作者：
Severson WE
中科院分区：
文献类型：
--
作者：
Chung DH;Moore BP;Matharu DS;Golden JE;Maddox C;Rasmussen L;Sosa MI;Ananthan S;White EL;Jia F;Jonsson CB;Severson WE
人类呼吸道合胞病毒(HRSV)是一种高度传染性的病原体，是一岁以下婴幼儿毛细支气管炎和肺炎的最常见原因。在世界范围内，超过3300万5岁以下儿童受到HRSV的影响，导致300万人住院，20万人死亡。然而，严重的下呼吸道疾病可能发生在任何年龄，特别是老年人或那些心脏、肺部或免疫系统受损的人。目前还没有商业上可用的疫苗。现有的治疗急性感染的方法是利巴韦林和预防性人源化单抗(MedImmune的Synagis®)，仅限于用于高危儿科患者。因此，新的HRSV抑制剂的发现将是有益的临床。我们已经开发并验证了一种基于384孔细胞的高通量检测方法，该方法使用基于发光的信号终点检测系统(细胞滴度Glo®)来测量HRSV(株Long)对Hep-2细胞的细胞病变效应。该方法灵敏、可靠，Z因子大于0.8，背景信号大于35，信号噪声大于24。利用这一方法，从分子文库小分子库中筛选出313,816个化合物，在10μM下进行筛选，初步鉴定了7,583个化合物，它们对细胞病变的抑制率超过22%。选择前2,500个化合物进行确认性筛选，其中409个化合物对CPE的抑制率至少为50%，被认为具有活性。根据效力、选择性和化学可操作性，我们选择了51种化合物进行进一步的剂量反应评估和二次检测，其中几种化合物的SI50值大于3，而最具活性的化合物的SI50值为58.9。已开发、优化和验证了一种基于自动发光的高通量屏幕，该屏幕测量HRSV诱导的Hep-2细胞病变效应的抑制，用于从大型化合物库中快速识别潜在的抑制剂。屏幕上识别的活性化合物代表不同类别的分子，包括以前未被鉴定为具有抗HRSV活性的芳基磺酰基吡咯烷。
Human respiratory syncytial virus (hRSV) is a highly contagious pathogen and is the most common cause of bronchiolitis and pneumonia for infants and children under one year of age. Worldwide, greater than 33 million children under five years of age are affected by hRSV resulting in three million hospitalizations and 200,000 deaths. However, severe lower respiratory tract disease may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. There is no vaccine commercially available. Existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis® from MedImmune) that is limited to use in high risk pediatric patients. Thus, the discovery of new inhibitors for hRSV would be clinically beneficial. We have developed and validated a 384-well cell-based, high-throughput assay that measures the cytopathic effect of hRSV (strain Long) in HEp-2 cells using a luminescent-based detection system for signal endpoint (Cell Titer Glo®). The assay is sensitive and robust, with Z factors greater than 0.8, signal to background greater than 35, and signal to noise greater than 24. Utilizing this assay, 313,816 compounds from the Molecular Libraries Small Molecule Repository were screened at 10 μM. We identified 7,583 compounds that showed greater than 22% CPE inhibition in the primary screen. The top 2,500 compounds were selected for confirmation screening and 409 compounds showed at least 50% inhibition of CPE and were considered active. We selected fifty-one compounds, based on potency, selectivity and chemical tractability, for further evaluation in dose response and secondary assays Several compounds had SI50 values greater than 3, while the most active compound displayed an SI50 value of 58.9. A robust automated luminescent-based high throughput screen that measures the inhibition of hRSV-induced cytopathic effect in HEp-2 cells for the rapid identification of potential inhibitors from large compound libraries has been developed, optimized and validated. The active compounds identified in the screen represent different classes of molecules, including aryl sulfonylpyrrolidines which have not been previously identified as having anti-hRSV activity.