Perveen Sumera, Khalili Yazdi Aliakbar, Devkota Kanchan, Li Fengling, Ghiabi Pegah, Hajian Taraneh, Loppnau Peter, Bolotokova Albina, Vedadi Masoud
Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada.
SLAS Discov. 2021 Jun;26(5):620-627. doi: 10.1177/2472555220985040. Epub 2021 Jan 10.
SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process, including the nonstructural protein 16 (nsp16), which is an -adenosyl-l-methionine (SAM)-dependent 2'--methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in a 384-well format with a Z' factor of 0.6, suitable for high-throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, the product of the reaction, -adenosyl-l-homocysteine (SAH), and a common methyltransferase inhibitor, sinefungin, using isothermal titration calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high-throughput method for screening the nsp10-nsp16 complex for RNA competitive inhibitors toward developing COVID-19 therapeutics.
导致新冠肺炎的新冠病毒2型（SARS-CoV-2）通过对其RNA进行加帽来逃避免疫系统。这一过程保护病毒RNA，对其复制至关重要。多种病毒蛋白参与了这一RNA加帽过程，包括非结构蛋白16（nsp16），它是一种依赖S-腺苷-L-甲硫氨酸（SAM）的2'-O-甲基转移酶。当与另一种非结构蛋白nsp10形成复合物时，nsp16具有显著活性，nsp10在其稳定性和活性中起关键作用。在此，我们报告了一种基于荧光偏振（FP）的RNA置换分析方法的开发，用于检测384孔板中的nsp10-nsp16复合物，Z'因子为0.6，适用于高通量筛选。在此过程中，我们将nsp10-nsp16复合物纯化至纯度高于95%，并使用等温滴定量热法（ITC）确认其与甲基供体SAM、反应产物S-腺苷-L-高半胱氨酸（SAH）以及一种常见的甲基转移酶抑制剂西奈芬净的结合。通过筛选一个包含1124种类药物化合物的文库，进一步验证了该分析方法。该分析方法为筛选nsp10-nsp16复合物的RNA竞争性抑制剂以开发新冠肺炎治疗药物提供了一种经济高效的高通量方法。
