Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CL(pro) Inhibitors.
Development of a Cell-Based Luciferase Complementation Assay for Identification of SARS-CoV-2 3CL(pro) Inhibitors.
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SARS-CoV-2 3CL（pro）抑制剂的细胞荧光素酶互补检测方法的建立
DOI：
10.3390/v13020173
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发表时间：
2021-01-24
期刊：
影响因子：
--
通讯作者：
Hu WS
中科院分区：
文献类型：
--
作者：
Rawson JMO;Duchon A;Nikolaitchik OA;Pathak VK;Hu WS
SARS-CoV-2的3C样蛋白酶（3CLpro）被认为是COVID-19抗病毒药物开发的极好靶标，因为它对病毒复制至关重要，并且具有不同于人类蛋白酶的切割特异性。然而，由于缺乏可以在BSL-2环境中进行的基于细胞的报告基因测定，3CLpro的药物开发受到阻碍。目前鉴定3CLpro抑制剂的努力主要依赖于体外筛选，其不能解释化合物的细胞渗透性和细胞毒性，或涉及复制能力病毒的测定，其必须在BSL-3设施中进行。为了解决这些限制，我们已经开发了一种新的基于细胞的荧光素酶互补报告基因检测，以确定在BSL-2设置的SARS-CoV-2 3CLpro的抑制剂。该测定基于共表达3CLpro和通过3CLpro切割位点连接在一起的两个荧光素酶片段的慢病毒载体。3CLpro介导的切割导致互补作用的丧失和低荧光素酶活性，而3CLpro的抑制导致10倍更高水平的荧光素酶活性。荧光素酶报告基因测定可以容易地区分真正的3CLpro抑制与细胞毒性，这是一个强大的功能，可以减少筛选过程中的假阳性。使用该检测方法，我们筛选了32种小分子对SARS-CoV-2 3CLpro的活性，包括HIV蛋白酶抑制剂，HCV蛋白酶抑制剂和各种其他已报道抑制SARS-CoV-2 3CLpro的化合物。其中，只有5种在细胞中表现出对3CLpro的显著抑制：GC 376、boceprevir、Z-FA-FMK、钙蛋白酶抑制剂XII和GRL-0496。该试验将极大地促进鉴定SARS-CoV-2 3CLpro的更有效抑制剂的努力。
The 3C-like protease (3CLpro) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CLpro has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CLpro inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CLpro in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CLpro and two luciferase fragments linked together by a 3CLpro cleavage site. 3CLpro-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CLpro results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CLpro inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CLpro, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CLpro. Of these, only five exhibited significant inhibition of 3CLpro in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CLpro.
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影响因子：
64.5
作者：
Hou, Yixuan J.;Okuda, Kenichi;Baric, Ralph S.
通讯作者：
Baric, Ralph S.
针对SARS-COV-2主要蛋白酶作为Covid-19的药物开发的最新进展。
影响因子：
5
作者：
Cui W;Yang K;Yang H
通讯作者：
Yang H
影响因子：
3.7
作者：
Kim JH;Lee SR;Li LH;Park HJ;Park JH;Lee KY;Kim MK;Shin BA;Choi SY
通讯作者：
Choi SY
DOI：
10.1056/nejmoa2029849
发表时间：
2021-01-21
期刊：
The New England journal of medicine
影响因子：
--
作者：
Chen P;Nirula A;Heller B;Gottlieb RL;Boscia J;Morris J;Huhn G;Cardona J;Mocherla B;Stosor V;Shawa I;Adams AC;Van Naarden J;Custer KL;Shen L;Durante M;Oakley G;Schade AE;Sabo J;Patel DR;Klekotka P;Skovronsky DM;BLAZE-1 Investigators
通讯作者：
BLAZE-1 Investigators
影响因子：
2.9
作者：
Barrila, Jennifer;Bacha, Usman;Freire, Ernesto
通讯作者：
Freire, Ernesto