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

ABSTRACT: 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.

SUBMITTER: Perveen S

PROVIDER: S-EPMC7803792 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Recently, a highly contagious novel coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has emerged, posing a global threat to public health. Identifying a potential target and developing vaccines or antiviral drugs is an urgent demand in the absence of approved therapeutic agents. The 5'-capping mechanism of eukaryotic mRNA and some viruses such as coronaviruses (CoVs) are essential for maintaining the RNA stability and protein translation in the virus. SARS-CoV-2 encodes S-adenosyl-L-methionine (SAM) dependent methyltransferase (MTase) enzyme characterized by nsp16 (2'-O-MTase) for generating the capped structure. The present study highlights the binding mechanism of nsp16 and nsp10 to identify the role of nsp10 in MTase activity. Furthermore, we investigated the conformational dynamics and energetics behind the binding of SAM to nsp16 and nsp16/nsp10 heterodimer by employing molecular dynamics simulations in conjunction with the Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) method. We observed from our simulations that the presence of nsp10 increases the favorable van der Waals and electrostatic interactions between SAM and nsp16. Thus, nsp10 acts as a stimulator for the strong binding of SAM to nsp16. The hydrophobic interactions were predominately identified for the nsp16-nsp10 interactions. Also, the stable hydrogen bonds between Ala83 (nsp16) and Tyr96 (nsp10), and between Gln87 (nsp16) and Leu45 (nsp10) play a vital role in the dimerization of nsp16 and nsp10. Besides, Computational Alanine Scanning (CAS) mutagenesis was performed, which revealed hotspot mutants, namely I40A, V104A, and R86A for the dimer association. Hence, the dimer interface of nsp16/nsp10 could also be a potential target in retarding the 2'-O-MTase activity in SARS-CoV-2. Overall, our study provides a comprehensive understanding of the dynamic and thermodynamic process of binding nsp16 and nsp10 that will contribute to the novel design of peptide inhibitors based on nsp16.

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

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