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首页> 美国卫生研究院文献>Antimicrobial Agents and Chemotherapy >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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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

机译：使用基于分裂蛋白的细胞-细胞融合测定法鉴定Nafamostat作为中东呼吸系统综合症冠状病毒S蛋白介导的膜融合的有效抑制剂。

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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.
机译：中东呼吸综合征（MERS）是一种新兴的传染病，其死亡率相对较高，约为40％。 MERS是由MERS冠状病毒（MERS-CoV）感染引起的，目前尚无可预防MERS-CoV感染的特定药物或疫苗。 MERS-CoV是一种包膜病毒，其包膜蛋白（S蛋白）在质膜或内体膜处介导膜融合。宿主蛋白酶（如弗林蛋白酶，跨膜蛋白酶丝氨酸2（TMPRSS2）和组织蛋白酶）的多种蛋白水解作用使S蛋白具有融合能力。定位于质膜的TMPRSS2是一种丝氨酸蛋白酶，在受体结合后阶段负责S的蛋白水解。在这里，我们使用表达基于海肾荧光素酶（RL）的分裂报告基因的细胞系，以TMPRSS2依赖的方式开发了基于细胞的S融合检测。 S在效应细胞中稳定表达，并且相应的S受体CD26在靶细胞中与TMPRSS2稳定共表达。通过确定RL活性，定量测量了这些效应子与靶细胞之间的膜融合。该测定法针对384孔格式进行了优化，在筛选约1,000种已获美国食品和药物管理局批准使用的药物时，将那发莫司他（一种丝氨酸蛋白酶抑制剂）鉴定为S介导的膜融合的有效抑制剂。 Nafamostat还可以在体外阻断MERS-CoV感染。我们的测定方法有可能促进发现新的MERS-CoV膜融合抑制剂以及依赖于TMPRSS2活性的其他病毒。

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