SARS-CoV-2 is the viral pathogen causing the COVID19 global pandemic. Consequently, much research has gone into the development of pre-clinical assays for the discovery of new or repurposing of FDA-approved therapies. Preventing viral entry into a host cell would be an effective antiviral strategy. One mechanism for SARS-CoV-2 entry occurs when the spike protein on the surface of SARS-CoV-2 binds to an ACE2 receptor followed by cleavage at two cut sites (priming) that causes a conformational change allowing for viral and host membrane fusion. TMPRSS2 has an extracellular protease domain capable of cleaving the spike protein to initiate membrane fusion. A validated inhibitor of TMPRSS2 protease activity would be a valuable tool for studying the impact TMPRSS2 has in viral entry and potentially be an effective antiviral therapeutic. To enable inhibitor discovery and profiling of FDA-approved therapeutics, we describe an assay for the biochemical screening of recombinant TMPRSS2 suitable for high throughput application. We demonstrate effectiveness to quantify inhibition down to subnanomolar concentrations by assessing the inhibition of camostat, nafamostat and gabexate, clinically approved agents in Japan. Also, we profiled a camostat metabolite, FOY-251, and bromhexine hydrochloride, an FDA-approved mucolytic cough suppressant. The rank order potency for the compounds tested are: nafamostat (IC50 = 0.27 nM), camostat (IC50 = 6.2 nM), FOY-251 (IC50 = 33.3 nM) and gabexate (IC50 = 130 nM). Bromhexine hydrochloride showed no inhibition of TMPRSS2. Further profiling of camostat, nafamostat and gabexate against a panel of recombinant proteases provides insight into selectivity and potency.

中文翻译：

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一种酶促 TMPRSS2 分析，用于评估临床候选者和发现抑制剂作为 COVID-19 的潜在治疗方法。

SARS-CoV-2 是导致 COVID19 全球大流行的病毒病原体。因此，许多研究已经进入临床前检测的开发，以发现新的或重新利用 FDA 批准的疗法。防止病毒进入宿主细胞将是一种有效的抗病毒策略。SARS-CoV-2 进入的一种机制发生在 SARS-CoV-2 表面的刺突蛋白与 ACE2 受体结合，然后在两个切割位点切割（引发），从而导致构象变化，从而使病毒和宿主膜融合. TMPRSS2 具有细胞外蛋白酶结构域，能够切割刺突蛋白以启动膜融合。经验证的 TMPRSS2 蛋白酶活性抑制剂将是研究 TMPRSS2 对病毒进入的影响的宝贵工具，并可能成为一种有效的抗病毒治疗剂。为了能够发现和分析 FDA 批准的治疗方法，我们描述了一种适用于高通量应用的重组 TMPRSS2 生化筛选的测定方法。我们通过评估日本临床批准的药物卡莫司他、萘莫司他和加贝司特的抑制作用，证明了将抑制量化至亚纳摩尔浓度的有效性。此外，我们还介绍了一种卡莫司他代谢物 FOY-251 和盐酸溴己新（一种 FDA 批准的粘液溶解性咳嗽抑制剂）。测试化合物的排序效力为：萘莫司他 (IC50 = 0.27 nM)、卡莫司他 (IC50 = 6.2 nM)、FOY-251 (IC50 = 33.3 nM) 和加贝酯 (IC50 = 130 nM)。盐酸溴己新对 TMPRSS2 没有抑制作用。进一步分析camostat，