期刊
SLAS DISCOVERY
卷 26, 期 5, 页码 620-627出版社
ELSEVIER SCIENCE INC
DOI: 10.1177/2472555220985040
关键词
COVID-19; nsp16; coronavirus; SARS-CoV-2; nsp10
资金
- University of Toronto COVID-19 Action Initiative-2020
- Takeda California, Inc.
- COVID-19 Mitacs Accelerate postdoctoral awards
- AbbVie
- Bayer Pharma AG
- Boehringer Ingelheim
- Canada Foundation for Innovation
- Eshelman Institute for Innovation
- Genentech
- Genome Canada through Ontario Genomics Institute [OGI-196]
- EU/EFPIA/OICR/McGill/KTH
- Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant) [875510]
- Janssen
- Merck KGaA (aka EMD in Canada)
- Merck KGaA (aka EMD in United States)
- Merck Co.
- Pfizer
- Sao Paulo Research Foundation-FAPESP
- Takeda
- Wellcome [106169/ZZ14/Z]
The study presents a fluorescence polarization-based RNA displacement assay for the nsp10-nsp16 complex, suitable for high-throughput screening. Through purification of the complex, confirmation of binding using isothermal titration calorimetry, and validation with a library of drug-like compounds, the assay was shown to be a cost-effective method for screening potential RNA competitive inhibitors for the development of COVID-19 therapeutics.
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 S-adenosyl-l-methionine (SAM)-dependent 2 '-O-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, S-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.
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