Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase.
Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase.
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基于酵母的检测方法，用于高通量筛选冠状病毒 RNA 帽鸟嘌呤-N7-甲基转移酶抑制剂
DOI：
10.1016/j.antiviral.2014.02.002
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发表时间：
2014-04
影响因子：
7.6
通讯作者：
Guo D
中科院分区：
文献类型：
--
作者：
Sun Y;Wang Z;Tao J;Wang Y;Wu A;Yang Z;Wang K;Shi L;Chen Y;Guo D
建立了一种基于酵母的冠状病毒N7-MTases抑制剂筛选系统。Sinefungin不是一种理想的抗病毒抑制剂。观察到三种天然提取物特异性抑制冠状病毒的N7-MTases。5′-帽结构是真核生物mRNA的一个独特特征，通过提供区分自身或非自身mRNA的分子标记，对RNA稳定性和蛋白质翻译至关重要。真核病毒通常修饰其RNA的5′端以模拟细胞mRNA结构，从而促进病毒在宿主细胞中的复制。然而，病毒加帽装置的分子组织和生化机制通常不同于其细胞对应物，这使得病毒加帽酶成为药物发现的有吸引力的靶点。我们的前期工作表明，SARS冠状病毒（SARS-CoV）的非结构蛋白14代表了一种结构新颖和独特的鸟嘌呤-N7-甲基转移酶（N7-MTase），能够在功能上补充酵母细胞N7-MTase。在本研究中，我们开发了一种基于酵母的系统，用于鉴定和筛选针对冠状病毒N7-MTase的抑制剂，使用96孔和384孔微量滴定板。MTase抑制剂以前确定的体外生化试验进行了测试，和一些，如sinefungin，有效地抑制N7-MTase在酵母系统。然而，其他化合物，如ATA和DHHcy，没有发挥抑制作用的细胞内的情况。这些结果验证了用于抑制剂筛选的酵母测定系统，但也证明了基于细胞的测定和体外生化测定之间的差异。应用酵母系统筛选3000种天然产物提取物，观察到三种提取物比人N7-MTase更有效地抑制冠状病毒的活性。
A yeast-based system for screening of coronavirus N7-MTases inhibitors was developed. Sinefungin is not an ideal antiviral inhibitor. Three natural extracts were observed to specifically inhibit the N7-MTases of coronavirus. The 5′-cap structure is a distinct feature of eukaryotic mRNAs and is important for RNA stability and protein translation by providing a molecular signature for the distinction of self or non-self mRNA. Eukaryotic viruses generally modify the 5′-end of their RNAs to mimic the cellular mRNA structure, thereby facilitating viral replication in host cells. However, the molecular organization and biochemical mechanisms of the viral capping apparatus typically differ from its cellular counterpart, which makes viral capping enzymes attractive targets for drug discovery. Our previous work showed that SARS coronavirus (SARS-CoV) non-structural protein 14 represents a structurally novel and unique guanine-N7-methyltransferase (N7-MTase) that is able to functionally complement yeast cellular N7-MTase. In the present study, we developed a yeast-based system for identifying and screening inhibitors against coronavirus N7-MTase using both 96-well and 384-well microtiter plates. The MTase inhibitors previously identified by in vitro biochemical assays were tested, and some, such as sinefungin, effectively suppressed N7-MTase in the yeast system. However, other compounds, such as ATA and AdoHcy, did not exert an inhibitory effect within a cellular context. These results validated the yeast assay system for inhibitor screening yet also demonstrated the difference between cell-based and in vitro biochemical assays. The yeast system was applied to the screening of 3000 natural product extracts, and three were observed to more potently inhibit the activity of coronavirus than human N7-MTase.
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影响因子：
6.7
作者：
Decroly E;Debarnot C;Ferron F;Bouvet M;Coutard B;Imbert I;Gluais L;Papageorgiou N;Sharff A;Bricogne G;Ortiz-Lombardia M;Lescar J;Canard B
通讯作者：
Canard B
影响因子：
64.8
作者：
Abbas YM;Pichlmair A;Górna MW;Superti-Furga G;Nagar B
通讯作者：
Nagar B
病毒mRNA帽的2'-O甲基化通过IFIT家族成员逃避了宿主的限制。
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
7.6
作者：
Milani M;Mastrangelo E;Bollati M;Selisko B;Decroly E;Bouvet M;Canard B;Bolognesi M
通讯作者：
Bolognesi M
影响因子：
--
作者：
Chrebet, G;Wisniewski, D;Parent, S
通讯作者：
Parent, S