Evaluating the 3C-like protease activity of SARS-Coronavirus: recommendations for standardized assays for drug discovery.
Evaluating the 3C-like protease activity of SARS-Coronavirus: recommendations for standardized assays for drug discovery.
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DOI：
10.1016/j.virusres.2007.02.015
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发表时间：
2008-04
期刊：
影响因子：
5
通讯作者：
Mesecar, Andrew D.
中科院分区：
文献类型：
--
作者：
Grum-Tokars, Valerie;Ratia, Kiira;Begaye, Adrian;Baker, Susan C.;Mesecar, Andrew D.
关键词：
虽然导致严重急性呼吸综合征（SARS-CoV）的致命冠状病毒的最初爆发被公共卫生措施控制，但SARS-CoV疫苗和抗病毒剂的开发对于改善未来爆发的控制和治疗至关重要。SARS-CoV抗病毒药物开发的一个潜在靶点是3C样蛋白酶（3CLpro）。这种酶是一个有吸引力的目标，因为它是病毒复制所必需的，并且因为现在有许多SARS-CoV 3CLpro的高分辨率X射线结构，使得基于结构的药物设计成为可能。因此，SARS-CoV 3CLpro已成为世界范围内众多药物发现工作的焦点，但因此，已经独立开发了各种不同的3CLpro表达构建体和动力学测定，使得潜在抑制剂之间的评价和比较成为问题。在这里，我们回顾了文献，重点是不同的SARS冠状病毒3CLpro表达结构和测定酶活性。此外，我们提供的实验证据表明，当非天然序列或亲和标签被添加到酶的N-或C-末端，或当在测定中使用的酶的浓度低于3CLpro二聚体的平衡解离常数时，3CLpro酶的活性显著降低。我们首次证明了设计用于常规分析和高通量筛选的高灵敏度和新型基于Alexa 488-QSY 7 FRET的肽底物的实用性，并表明从基于FRET的测定中确定的动力学常数（未校正内部过滤器效应）可能会导致伪影。最后，我们评估了常见的检测成分，包括DTT，NaCl，EDTA和DMSO对酶活性的影响，我们推荐了标准化的检测条件和结构，用于常规的SARS-CoV 3CLpro检测，以促进全球正在开发的SARS-CoV 3CLpro抑制剂之间的直接比较。
Although the initial outbreaks of the deadly coronavirus that causes severe acute respiratory syndrome (SARS-CoV) were controlled by public health measures, the development of vaccines and antiviral agents for SARS-CoV is essential for improving control and treatment of future outbreaks. One potential target for SARS-CoV antiviral drug development is the 3C-like protease (3CLpro). This enzyme is an attractive target since it is essential for viral replication, and since there are now a number of high resolution X-ray structures of SARS-CoV 3CLpro available making structure-based drug-design possible. As a result, SARS-CoV 3CLpro has become the focus of numerous drug discovery efforts worldwide, but as a consequence, a variety of different 3CLpro expression constructs and kinetic assays have been independently developed making evaluation and comparison between potential inhibitors problematic. Here, we review the literature focusing on different SARS-CoV 3CLpro expression constructs and assays used to measure enzymatic activity. Moreover, we provide experimental evidence showing that the activity of 3CLpro enzymatic is significantly reduced when non-native sequences or affinity-tags are added to the N- or C-termini of the enzyme, or when the enzyme used in assays is at concentrations below the equilibrium dissociation constant of the 3CLpro dimer. We demonstrate for the first time the utility of a highly sensitive and novel Alexa488-QSY7 FRET-based peptide substrate designed for routine analysis and high-throughput screening, and show that kinetic constants determined from FRET-based assays that are uncorrected for inner-filter effects can lead to artifacts. Finally, we evaluated the effects of common assay components including DTT, NaCl, EDTA and DMSO on enzymatic activity, and we recommend standardized assay conditions and constructs for routine SARS-CoV 3CLpro assays to facilitate direct comparisons between SARS-CoV 3CLpro inhibitors under development worldwide.
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冠状病毒主蛋白酶的结构揭示了甲over依蛋白酶折叠与额外的α-螺旋结构域的组合。
影响因子：
11.4
作者：
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通讯作者：
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DOI：
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发表时间：
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期刊：
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影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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