A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry.
A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry.
复制标题
DOI：
10.1016/j.slasd.2021.12.005
复制
发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Chen CZ
中科院分区：
文献类型：
--
作者：
Xu M;Pradhan M;Gorshkov K;Petersen JD;Shen M;Guo H;Zhu W;Klumpp-Thomas C;Michael S;Itkin M;Itkin Z;Straus MR;Zimmerberg J;Zheng W;Whittaker GR;Chen CZ
随着COVID-19大流行在全球持续，对抗SARS-CoV-2感染的有效小分子疗法仍然缺乏。高通量筛选方法是发现和优化小分子SARS-CoV-2抑制剂的关键。在这项工作中，我们应用病毒假型建立了一个基于细胞的SARS-CoV-2进入检测。在这里，假型化颗粒（PP）在包被鼠白血病病毒（MLV）gag-pol多聚蛋白和荧光素酶报告RNA的膜中含有SARS-CoV-2刺突。在向HEK 293-ACE 2细胞中加入PP后，SARS-CoV-2刺突蛋白与细胞表面的ACE 2受体结合，导致宿主蛋白酶引发这些颗粒的内吞作用，以及颗粒包膜和细胞膜之间的膜融合。然后，内化的荧光素酶报告基因在细胞中表达，产生发光读数，作为刺突介导的进入细胞的替代物。该SARS-CoV-2 PP进入试验可在生物安全2级防护实验室中进行，以进行高通量筛选。从5，158种批准的药物和候选药物中，我们的筛选工作确定了7种抑制SARS-CoV-2-S PP进入的活性化合物。在这七种化合物中，有六种化合物在细胞病变效应试验中对活的复制型SARS-CoV-2病毒具有活性。我们的研究结果表明，该方法的实用性，在发现和开发的SARS-CoV-2进入抑制剂，以及抗SARS-CoV-2化合物的机制研究。此外，制备了用来自SARS-CoV-2 B.1.1.7和B.1.351变体的刺突蛋白假型化的颗粒，并用于评估病毒进入抑制剂的治疗效果。
Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.
登录
查看更多内容
影响因子：
1.2
作者：
Millet, Jean K.;Tang, Tiffany;Whittaker, Gary R.
通讯作者：
Whittaker, Gary R.
DOI：
10.2174/1875397301004010057
发表时间：
2010-10-21
期刊：
Current chemical genomics
影响因子：
--
作者：
Wang Y;Jadhav A;Southal N;Huang R;Nguyen DT
通讯作者：
Nguyen DT
影响因子：
64.8
作者：
Riva L;Yuan S;Yin X;Martin-Sancho L;Matsunaga N;Pache L;Burgstaller-Muehlbacher S;De Jesus PD;Teriete P;Hull MV;Chang MW;Chan JF;Cao J;Poon VK;Herbert KM;Cheng K;Nguyen TH;Rubanov A;Pu Y;Nguyen C;Choi A;Rathnasinghe R;Schotsaert M;Miorin L;Dejosez M;Zwaka TP;Sit KY;Martinez-Sobrido L;Liu WC;White KM;Chapman ME;Lendy EK;Glynne RJ;Albrecht R;Ruppin E;Mesecar AD;Johnson JR;Benner C;Sun R;Schultz PG;Su AI;García-Sastre A;Chatterjee AK;Yuen KY;Chanda SK
通讯作者：
Chanda SK
影响因子：
5.4
作者：
Johnson MC;Lyddon TD;Suarez R;Salcedo B;LePique M;Graham M;Ricana C;Robinson C;Ritter DG
通讯作者：
Ritter DG
影响因子：
64.8
作者：
Hoffmann, Markus;Moesbauer, Kirstin;Poehlmann, Stefan
通讯作者：
Poehlmann, Stefan