A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors.
A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors.
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DOI：
10.1016/j.antiviral.2022.105272
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发表时间：
2022-05
影响因子：
7.6
通讯作者：
Naffakh N
中科院分区：
文献类型：
--
作者：
Chen KY;Krischuns T;Varga LO;Harigua-Souiai E;Paisant S;Zettor A;Chiaravalli J;Delpal A;Courtney D;O'Brien A;Baker SC;Decroly E;Isel C;Agou F;Jacob Y;Blondel A;Naffakh N
迫切需要针对 SARS-CoV-2 的有效药物来治疗严重感染病例并用于预防性使用。主要病毒蛋白酶（nsp5 或 3CLpro）代表了药物开发的一个有吸引力且可能广谱的靶点，因为它对病毒生命周期至关重要，并且在 β 冠状病毒中高度保守。灵敏且高效的高通量筛选方法是药物发现的关键。在这里，我们报告了一种信号增益、高度敏感的基于细胞的荧光素酶测定的开发，用于监测 SARS-CoV-2 nsp5 活性，并表明它适合以 384 孔形式筛选化合物。小型化和自动化的好处是可以对野生型和催化失活的 nsp5 并行进行筛选，从而提高测定的选择性。我们对内部化学数据库中的一组 14,468 种化合物进行了基于分子对接的筛选，选择了 359 种候选 nsp5 抑制剂并进行了实验测试。我们在基于细胞的检测和体外纯化的 nsp5 蛋白中鉴定出两种具有抗 nsp5 活性的分子，并抑制 A549-ACE2 细胞中的 SARS-CoV-2 复制，EC50 值在 4–8 μM 范围内。这里描述的高通量兼容测定将允许筛选 SARS-CoV-2 nsp5 抑制剂的大规模化合物库。此外，我们提供的证据表明，该测定可以适用于依赖病毒蛋白酶的其他冠状病毒和病毒。
Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for the screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified two molecules which show anti-nsp5 activity, both in our cell-based assay and in vitro on purified nsp5 protein, and inhibit SARS-CoV-2 replication in A549-ACE2 cells with EC50 values in the 4–8 μM range. The here described high-throughput-compatible assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.
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影响因子：
17.1
作者：
通讯作者：
--
影响因子：
5.3
作者：
Hu, Yanmei;Ma, Chunlong;Wang, Jun
通讯作者：
Wang, Jun
影响因子：
5.4
作者：
Froggatt HM;Heaton BE;Heaton NS
通讯作者：
Heaton NS
影响因子：
3.7
作者：
Chuck CP;Chow HF;Wan DC;Wong KB
通讯作者：
Wong KB
DOI：
10.1126/science.abg5827
发表时间：
2021-08-20
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Drayman N;DeMarco JK;Jones KA;Azizi SA;Froggatt HM;Tan K;Maltseva NI;Chen S;Nicolaescu V;Dvorkin S;Furlong K;Kathayat RS;Firpo MR;Mastrodomenico V;Bruce EA;Schmidt MM;Jedrzejczak R;Muñoz-Alía MÁ;Schuster B;Nair V;Han KY;O'Brien A;Tomatsidou A;Meyer B;Vignuzzi M;Missiakas D;Botten JW;Brooke CB;Lee H;Baker SC;Mounce BC;Heaton NS;Severson WE;Palmer KE;Dickinson BC;Joachimiak A;Randall G;Tay S
通讯作者：
Tay S