Identification and Mechanism of Action of a Novel Small-Molecule Inhibitor of Arenavirus Multiplication.
Identification and Mechanism of Action of a Novel Small-Molecule Inhibitor of Arenavirus Multiplication.
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新型沙粒病毒增殖小分子抑制剂的鉴定和作用机制。
DOI：
10.1128/jvi.01587-15
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发表时间：
2015
影响因子：
5.4
通讯作者：
Lama,Juan
中科院分区：
文献类型：
--
作者：
Ngo,Nhi;Henthorn,KristinaSchimmelpfeng;Cisneros,MariaIsabel;Cubitt,Beatrice;Iwasaki,Masaharu;delaTorre,JuanC;Lama,Juan
几种阿拉伯病毒在人类中引起出血热疾病，并在这些病毒流行的地区代表着重要的公共卫生问题。此外，有证据表明，世界范围内分布的原型性淋巴细胞性脉络膜脑膜炎病毒(LCMV)是一种重要的被忽视的人类病原体。目前还没有获得许可的阿拉伯病毒疫苗，目前的抗病毒治疗仅限于使用利巴韦林，而利巴韦林只有部分有效。因此，对新的抗ARA病毒疗法的需求还没有得到满足。在这里，我们报道了一种新型重组LCM病毒的产生，并将其用于开发一种基于细胞的高通量筛选，以快速识别LCMV增殖的抑制物。我们使用这一新的方法来筛选30,400个小分子的文库，并鉴定出化合物F3406(化学name:N-[3，5-bis(fluoranyl)phenyl]-2-[5，7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1，3]thiazolo[4，5-d]pyrimidin-4-yl]ethanamide)，)，它在没有细胞毒性的情况下具有很强的抗巨细胞病毒活性。作用机制研究表明，F3406通过特异性干扰LCMV糖蛋白GP2介导的内吞体内pH依赖的融合来抑制LCMV细胞进入，该融合需要将病毒核糖核蛋白释放到细胞质中，以启动病毒基因组的转录和复制。我们发现GP2跨膜区中的M437残基对病毒对F3406的易感性至关重要。出血热出血热病毒(HFA)是一种重要的人类病原体，在这些病毒流行的地区会导致高发病率和死亡率。此外，有证据表明，世界范围内分布的原型性ArenaVirus淋巴细胞性脉络膜脑膜炎病毒(LCMV)是一种被忽视的人类临床病原体。由于缺乏美国食品和药物管理局(FDA)许可的ArenaVirus疫苗，以及目前的抗ArenaVirus治疗仅限于标签外使用仅部分有效的利巴韦林，ArenaVirus感染引起的担忧加剧。在这里，我们描述了一种新的重组LCMV及其用于建立一种适合HTS的基于细胞的检测方法，以快速识别病毒增殖的抑制物。我们在这项工作中描述的概念和实验策略为快速鉴定和表征新型抗HFA药物提供了基础。
Several arenaviruses cause hemorrhagic fever disease in humans and represent important public health problems in the regions where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important neglected human pathogen. There are no licensed arenavirus vaccines and current antiarenavirus therapy is limited to the use of ribavirin that is only partially effective. Therefore, there is an unmet need for novel antiarenaviral therapeutics. Here, we report the generation of a novel recombinant LCM virus and its use to develop a cell-based high-throughput screen to rapidly identify inhibitors of LCMV multiplication. We used this novel assay to screen a library of 30,400 small molecules and identified compound F3406 (chemical name:N-[3,5-bis(fluoranyl)phenyl]-2-[5,7-bis(oxidanylidene)-6-propyl-2-pyrrolidin-1-yl-[1,3]thiazolo[4,5-d]pyrimidin-4-yl]ethanamide), which exhibited strong anti-LCMV activity in the absence of cell toxicity. Mechanism-of-action studies revealed that F3406 inhibited LCMV cell entry by specifically interfering with the pH-dependent fusion in the endosome compartment that is mediated by LCMV glycoprotein GP2 and required to release the virus ribonucleoprotein into the cell cytoplasm to initiate transcription and replication of the virus genome. We identified residue M437 within the transmembrane domain of GP2 as critical for virus susceptibility to F3406.IMPORTANCEHemorrhagic fever arenaviruses (HFA) are important human pathogens that cause high morbidity and mortality in areas where these viruses are endemic. In addition, evidence indicates that the worldwide-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. Concerns posed by arenavirus infections are aggravated by the lack of U.S. Food and Drug Administration-licensed arenavirus vaccines and current antiarenaviral therapy being limited to the off-label use of ribavirin that is only partially effective. Here we describe a novel recombinant LCMV and its use to develop a cell-based assay suitable for HTS to rapidly identify inhibitors arenavirus multiplication. The concepts and experimental strategies we describe in this work provide the bases for the rapid identification and characterization of novel anti-HFA therapeutics.