Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry.
Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry.
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DOI：
10.1177/24725552211000652
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发表时间：
2021-07
期刊：
影响因子：
--
通讯作者：
Fordyce EAF
中科院分区：
文献类型：
--
作者：
Pearson LA;Green CJ;Lin D;Petit AP;Gray DW;Cowling VH;Fordyce EAF
严重急性呼吸综合征冠状病毒-2 （SARS-CoV-2）对人类健康构成重大威胁。尽管它与相关的冠状病毒相似，但目前还没有针对COVID-19感染的特异性治疗方法，因此迫切需要为这次和未来的冠状病毒爆发开发治疗方法。在病毒RNA的5 '端形成的帽状物已被证明有助于冠状病毒逃避宿主的防御。非结构蛋白14 （nsp14）负责冠状病毒冠鸟苷的n7甲基化。该酶在冠状病毒中高度保守，是一种双功能蛋白，具有n7 -甲基转移酶和3 ‘ -5 ’外切酶活性，将nsp14与人类等同物区分开来。SARS-CoV nsp14的突变分析突出了其在病毒复制和病毒基因组翻译效率中的作用。在本文中，我们描述了利用RapidFire技术对nsp14进行高通量测定的特性和发展。该试验已用于筛选1771种食品和药物管理局（FDA）批准的药物库。由此，我们验证了硝唑尼特是nsp14甲基转移酶活性的选择性抑制剂。虽然活性不大，但该化合物可以作为进一步优化的起点。
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5′ end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3′-5′ exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.
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