A High-Throughput RNA Displacement Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex toward Developing Therapeutics for COVID-19.

Szczegóły
Abstrakt

Tytuł:: A High-Throughput RNA Displacement Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex toward Developing Therapeutics for COVID-19.
Autorzy:: Perveen S; Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
Khalili Yazdi A; Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
Devkota K; Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
Li F; Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
Ghiabi P; Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
Hajian T; Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
Loppnau P; Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
Bolotokova A; Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.
Vedadi M; Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada.; Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada.
Źródło:: SLAS discovery : advancing life sciences R & D [SLAS Discov] 2021 Jun; Vol. 26 (5), pp. 620-627. Date of Electronic Publication: 2021 Jan 10.
Typ publikacji:: Journal Article; Research Support, Non-U.S. Gov't
Język:: English
Imprint Name(s):: Original Publication: Thousand Oaks, CA : SAGE Publications, [2017]-
MeSH Terms:: High-Throughput Screening Assays*
Antiviral Agents/*pharmacology
RNA, Viral/*antagonists & inhibitors
SARS-CoV-2/*drug effects
Small Molecule Libraries/*pharmacology
Viral Nonstructural Proteins/*antagonists & inhibitors
Viral Regulatory and Accessory Proteins/*antagonists & inhibitors
Adenosine/analogs & derivatives ; Adenosine/pharmacology ; Binding, Competitive ; COVID-19/virology ; Enzyme Inhibitors/pharmacology ; Fluorescence Polarization ; Gene Expression Regulation ; Host-Pathogen Interactions/drug effects ; Humans ; Methyltransferases ; Protein Binding ; RNA Caps/antagonists & inhibitors ; RNA Caps/genetics ; RNA Caps/metabolism ; RNA, Viral/genetics ; RNA, Viral/metabolism ; SARS-CoV-2/genetics ; SARS-CoV-2/metabolism ; Signal Transduction ; Viral Nonstructural Proteins/genetics ; Viral Nonstructural Proteins/metabolism ; Viral Regulatory and Accessory Proteins/genetics ; Viral Regulatory and Accessory Proteins/metabolism ; Virus Replication ; COVID-19 Drug Treatment
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Grant Information:: United Kingdom WT_ Wellcome Trust
Contributed Indexing:: Keywords: COVID-19; SARS-CoV-2; coronavirus; nsp10; nsp16
Substance Nomenclature:: 0 (Antiviral Agents)
0 (Enzyme Inhibitors)
0 (NSP10 protein, SARS-CoV-2)
0 (NSP16 protein, SARS-CoV-2)
0 (RNA Caps)
0 (RNA, Viral)
0 (Small Molecule Libraries)
0 (Viral Nonstructural Proteins)
0 (Viral Regulatory and Accessory Proteins)
EC 2.1.1.- (Methyltransferases)
K72T3FS567 (Adenosine)
W2U467CIIL (sinefungin)
Entry Date(s):: Date Created: 20210111 Date Completed: 20210607 Latest Revision: 20240211
Update Code:: 20240211
PubMed Central ID:: PMC7803792
DOI:: 10.1177/2472555220985040
PMID:: 33423577
: Czasopismo naukowe

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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