Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada.
SLAS Discov. 2021 Jul;26(6):757-765. doi: 10.1177/24725552211008863. Epub 2021 Apr 19.
Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'--methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.
新型冠状病毒（CoV）频繁爆发，当前严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）大流行尤为突出，这就需要开发出能够在全球范围内轻松有效使用的治疗方法。保守的 mRNA 加帽过程使 CoV 能够逃避其宿主免疫系统，成为抗病毒药物开发的靶点。非结构蛋白（nsp）16 与 nsp10 形成复合物，通过其 2'-甲基化活性催化冠状病毒 mRNA 加帽的最后一步。与其他甲基转移酶一样，SARS-CoV-2 nsp10-nsp16 复合物是可成药的。然而，高通量筛选（HTS）的优化测定法仍然难以实现。在这里，我们报告了在 384 孔板格式中用于 nsp10-nsp16 复合物甲基转移酶活性的基于放射性的测定法的开发、动力学特征以及 HTS 测定法的优化（Z' 因子=0.83）。考虑到 nsp16 在已知的 CoV 物种中高度保守，针对 SARS-CoV-2 nsp10-nsp16 复合物的潜在抑制剂也可能对其他新兴的致病性 CoV 有效。
