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

Perveen, Sumera; Yazdi, Aliakbar Khalili; Devkota, Kanchan; Li, Fengling; Ghiabi, Pegah; Loppnau, P.; Bolotokova, Albina; Vedadi, Masoud

doi:10.1177/2472555220985040

Cited by 26 publications

(30 citation statements)

References 34 publications

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“…22 Most recently, an HTS RNA displacement assay has been reported for the SARS-CoV-2 nsp10-nsp16 complex that will detect RNA-competitive inhibitors. 31 Overall, the nsp10-nsp16 complex activity assays reported to-date are low throughput. [22][23][24]38 Here we reported development of a radioactivity-based assay for screening the SARS-CoV-2 nsp10-nsp16 complex in a 384-well format.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…The available evidence indicates that many other CoVs currently in various animals are preadapted to likely infect humans at some point in time in the future and cause new pandemics. 42 – 44 Considering the natural diversity of CoVs across the globe 1 and the close interactions of humans with wild and domesticated animals, these future pandemics may not be prevented by the current vaccines. 36 This further highlights the importance of developing potent inhibitors against coronaviral proteins that are conserved across this family of viruses to develop pan-CoV therapeutics.…”

Section: Discussionmentioning

confidence: 99%

“…Expression and purification of the SARS-CoV-2 nsp10-nsp16 complex was recently described. 31 Briefly, nsp16 (S 1 -N 298 ) and nsp10 (A 1 -Q 139 ) were separately expressed in Escherichia coli BL21(DE3) RIL and purified to near homogeneity. The nsp10-nsp16 complex was prepared using the purified proteins in an 8 (nsp10) to 1 (nsp16) molar ratio; dialyzed in storage buffer containing 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 0.5 mM TCEP, and 5% glycerol; and flash frozen.…”

Section: Methodsmentioning

confidence: 99%

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A High-Throughput Radioactivity-Based Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex

Yazdi

Devkota

et al. 2021

SLAS Discovery

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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′- O-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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A High-Throughput Radioactivity-Based Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex

Yazdi

Devkota

et al. 2021

SLAS Discovery

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“…The S-ecto ( 53 ), expressed in mammalian EXPI293 cells, and N-dimer domain, N-RBD, NSP3-unique, NSP3-ADRP, NSP3-NAB, NSP3-PLPro, NSP4-CTD, NSP5, NSP7, NSP8, NSP9, NSP10, and NSP16 ( 54 ) expressed in bacterial Escherichia coli cells were supplied by the Toronto Open Access Covid-19 Protein Manufacturing Center (comprising BioZone and the Structural Genomics Consortium, Toronto, Canada) under an Open Science Trust Agreement: http://www.thesgc.org/click-trust The Center received funding from the Toronto COVID-19 Action Fund. See Supplemental Table 1 for a complete list of proteins.…”

Section: Methodsmentioning

confidence: 99%

Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern

Voss¹,

Esmail²,

Liu³

et al. 2021

JCI Insight

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BACKGROUND. The role of humoral immunity in the coronavirus disease 2019 (COVID-19) is not fully understood owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome. METHODS. Using SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution. RESULTS. We identified linear epitopes from the Spike (S) and Nucleocapsid (N) protein and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S(811-825), S(881-895) and N(156-170) epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes. CONCLUSIONS. Epitope-resolved antibody testing not only affords a high-resolution alternativeto conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, it may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern (VOC) in both the peptide array and latex agglutination formats.

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Enzymatic Assays to Explore Viral mRNA Capping Machinery

Kasprzyk

Jemielity

2021

ChemBioChem

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In eukaryotes, mRNA is modified by the addition of the 7‐methylguanosine (m 7 G) 5′ cap to protect mRNA from premature degradation, thereby enhancing translation and enabling differentiation between self (endogenous) and non‐self RNAs (e. g., viral ones). Viruses often develop their own mRNA capping pathways to augment the expression of their proteins and escape host innate immune response. Insights into this capping system may provide new ideas for therapeutic interventions and facilitate drug discovery, e. g., against viruses that cause pandemic outbreaks, such as beta‐coronaviruses SARS‐CoV (2002), MARS‐CoV (2012), and the most recent SARS‐CoV‐2. Thus, proper methods for the screening of large compound libraries are required to identify lead structures that could serve as a basis for rational antiviral drug design. This review summarizes the methods that allow the monitoring of the activity and inhibition of enzymes involved in mRNA capping.

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

Cited by 26 publications

References 34 publications

A High-Throughput Radioactivity-Based Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex

A High-Throughput Radioactivity-Based Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex

Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern

Enzymatic Assays to Explore Viral mRNA Capping Machinery

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