Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay
Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay
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DOI：
10.1128/aac.01043-16
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发表时间：
2016-11-01
影响因子：
4.9
通讯作者：
Matsuda, Zene
中科院分区：
文献类型：
--
作者：
Yamamoto, Mizuki;Matsuyama, Shutoku;Matsuda, Zene
中东呼吸综合征 (MERS) 是一种新出现的传染病，死亡率相对较高，约为 40%。 MERS是由MERS冠状病毒（MERS-CoV）感染引起的，目前尚无特效药物或疫苗可预防MERS-CoV感染。 MERS-CoV是一种有包膜病毒，其包膜蛋白（S蛋白）介导质膜或内体膜的膜融合。宿主蛋白酶（例如弗林蛋白酶、跨膜蛋白酶丝氨酸 2 (TMPRSS2) 和组织蛋白酶）的多重蛋白水解作用，导致 S 蛋白具有融合能力。 TMPRSS2 定位于质膜，是一种丝氨酸蛋白酶，负责受体结合后阶段的 S 蛋白水解。在这里，我们使用表达基于海肾荧光素酶 (RL) 的分裂报告蛋白的细胞系，以 TMPRSS2 依赖性方式开发了一种基于细胞的 S 融合测定。 S在效应细胞中稳定表达，S的相应受体CD26与靶细胞中的TMPRSS2稳定共表达。通过测定 RL 活性来定量测量这些效应细胞和靶细胞之间的膜融合。该测定针对 384 孔格式进行了优化，在美国食品和药物管理局批准使用的约 1,000 种药物的筛选中，nafamostat（一种丝氨酸蛋白酶抑制剂）被确定为 S 介导的膜融合的有效抑制剂。 Nafamostat 还在体外阻断了 MERS-CoV 感染。我们的测定有可能促进 MERS-CoV 以及依赖 TMPRSS2 活性的其他病毒膜融合的新抑制剂的发现。
Middle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressing Renilla luciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infection in vitro. Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.