Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays.
Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays.
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DOI：
10.1016/j.apsb.2021.10.026
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发表时间：
2022-04
期刊：
影响因子：
--
通讯作者：
Wang J
中科院分区：
文献类型：
--
作者：
Ma C;Tan H;Choza J;Wang Y;Wang J
SARS-CoV-2主要蛋白酶（Mpro）是COVID-19最广泛开发的药物靶标之一。结构不同的化合物已被报道为Mpro抑制剂，提出了它们的靶特异性的问题。为了阐明所要求保护的Mpro抑制剂的靶特异性和细胞靶接合，我们使用无细胞FRET测定、热位移结合测定、细胞裂解物蛋白酶-Glo荧光素酶测定和基于细胞的FlipGFP测定来系统地表征它们的作用机制。总的来说，我们的研究结果表明，大多数从药物再利用中鉴定的Mpro抑制剂，包括依布硒啉、卡莫氟、双硫仑和紫草素，是混杂的半胱氨酸抑制剂，对Mpro没有特异性，而氯喹、土霉素、孟鲁司特、坎地沙坦和双嘧达莫在任何测试的试验中都不抑制Mpro。总的来说，我们的研究强调了在药物发现的早期阶段需要严格的命中验证。FlipGFP和蛋白酶-Glo荧光素酶测定，再加上FRET和热位移结合测定，应用于验证/无效的报告的SARS-CoV-2 Mpro抑制剂。
SARS-CoV-2 main protease (Mpro) is one of the most extensively exploited drug targets for COVID-19. Structurally disparate compounds have been reported as Mpro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mpro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based FlipGFP assay. Collectively, our results have shown that majority of the Mpro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mpro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mpro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery. FlipGFP and protease-Glo luciferase assays, coupled with the FRET and thermal shift binding assays, were applied to validate/invalidate the reported SARS-CoV-2 Mpro inhibitors.
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影响因子：
8.8
作者：
Bafna K;White K;Harish B;Rosales R;Ramelot TA;Acton TB;Moreno E;Kehrer T;Miorin L;Royer CA;García-Sastre A;Krug RM;Montelione GT
通讯作者：
Montelione GT
影响因子：
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通讯作者：
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作者：
Froggatt HM;Heaton BE;Heaton NS
通讯作者：
Heaton NS
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