Identification of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library.
Identification of novel compounds inhibiting chikungunya virus-induced cell death by high throughput screening of a kinase inhibitor library.
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DOI：
10.1371/journal.pntd.0002471
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发表时间：
2013
影响因子：
3.8
通讯作者：
Freitas-Junior LH
中科院分区：
文献类型：
--
作者：
Cruz DJ;Bonotto RM;Gomes RG;da Silva CT;Taniguchi JB;No JH;Lombardot B;Schwartz O;Hansen MA;Freitas-Junior LH
基孔肯雅病毒（CHIKV）是一种蚊子传播的关节源性甲病毒，可引起人类急性发热性疾病，伴有关节疼痛，在许多情况下，持续数周至数年的持续性关节痛。CHIKV的重新出现导致了东半球的多次爆发，并有可能在可预见的未来扩大。不幸的是，目前没有有效的治疗方法。本研究报告了刃天青在基于细胞的高通量测定和基于图像的高含量测定中的使用，以鉴定和表征CHIKV感染的体外抑制剂。CHIKV是一种高度致细胞病变的病毒，可迅速杀死受感染的细胞。因此，通过测量刃天青的代谢还原以鉴定CHIKV相关细胞死亡的抑制剂来确定在化合物存在下用CHIKV感染的HuH-7细胞的细胞活力。使用刃天青还原测定针对HuH-7细胞的CHIKV感染筛选4，000种化合物的激酶抑制剂文库，并且还在未感染的细胞中测量细胞毒性。选择在10 µM下对CHIKV显示≥50%抑制特性的72种化合物作为主要命中物。具有苯并呋喃核心支架的四种化合物（CND 0335、CND 0364、CND 0366和CND 0415）、一种吡咯并吡啶（CND 0545）和一种噻唑-甲酰胺（CND 3514）以剂量依赖性方式抑制CHIKV相关的细胞死亡，EC 50值在2.2 μM和7.1 μM之间。基于图像分析，这6种命中化合物不抑制宿主细胞中的CHIKV复制。然而，与对照感染相比，CHIKV感染的细胞表现出不太明显的CHIKV细胞病变效应典型的凋亡泡。此外，用这些化合物处理使CHIKV感染的细胞的培养基中的病毒滴度降低高达100倍。总之，使用刃天青的这种基于细胞的高通量筛选测定与基于图像的高含量测定方法相结合，鉴定了针对CHIKV的化合物，其具有新的抗病毒活性-抑制病毒诱导的CPE -可能通过靶向参与细胞凋亡的激酶。基孔肯雅病毒（CHIKV）最近在亚洲、非洲和欧洲不同地区的爆发和全球分布的扩大需要开发有效的治疗干预措施。目前，只有两种在体外抑制病毒感染的抗病毒化合物（氯喹和利巴韦林）已用于基孔肯雅感染的临床病例。然而，这些化合物都没有在体内显示出强的功效。最近已经报道了使用基于细胞的表型方法鉴定CHIKV的新的抗病毒候选物的尝试。在这项研究中，我们开发了一种简单的基于细胞的高通量检测方法，使用刃天青来鉴定潜在的抗CHIKV化合物。该高通量测定基于通过活细胞将刃天青代谢还原为高荧光试卤灵，作为针对CHIKV诱导的CPE的活性的指标。我们筛选了属于BioFocus激酶抑制剂化学文库的4，000个小分子，发现了一组对CHIKV具有抗病毒活性的相关分子。最后，我们使用基于图像的高含量测定和常规病毒学方法（即，病毒产率降低测定、微量中和测定）。
Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis. Recent outbreaks and expanding global distribution of Chikungunya virus (CHIKV) in different regions of Asia, Africa and Europe necessitates the development of effective therapeutic interventions. At present, only two antiviral compounds (chloroquine and ribavirin) that inhibit viral infection in vitro have been used in clinical cases of chikungunya infections. However, neither of these compounds have shown strong efficacy in vivo. Recent attempts to identify new antiviral candidates for CHIKV using cell-based phenotypic approach have been reported. In this study, we developed a simple cell-based high-throughput assay using resazurin to identify potential anti-CHIKV compounds. This high-throughput assay is based on the metabolic reduction of resazurin to the highly fluorescent resorufin by viable cells as an indicator of activity against CHIKV-induced CPE. We screened 4,000 small molecules belonging to the BioFocus kinase inhibitor chemical library and found a cluster of related molecules with antiviral activity against CHIKV. Finally, we characterized the putative mode of action of these active compounds using an image-based high content assay and conventional virological methods (i.e., virus yield reduction assay, microneutralization assay).
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DOI：
10.1016/j.berh.2011.03.005
发表时间：
2011-06-01
影响因子：
5.2
作者：
Alla, Sanae Ali Ou;Combe, Bernard
通讯作者：
Combe, Bernard
影响因子：
--
作者：
Candida Monteiro, Maria;de la Cruz, Mercedes;Vicente, Francisca
通讯作者：
Vicente, Francisca
影响因子：
3.7
作者：
Bernard E;Solignat M;Gay B;Chazal N;Higgs S;Devaux C;Briant L
通讯作者：
Briant L
影响因子：
4.8
作者：
Ashfaq, Usman A.;Javed, Tariq;Riazuddin, Sheikh
通讯作者：
Riazuddin, Sheikh
影响因子：
14.9
作者：
Backman TW;Cao Y;Girke T
通讯作者：
Girke T