Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay
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Abstract
The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (M pro ). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 M pro . We have comprehensively investigated the influence of different buffer components and conditions on the assay performance, and characterized six FRET substrates with a 2-Abz/Tyr(3-NO 2 ) FRET pair. The substrates 2-AbzSAVLQSGTyr(3-NO 2 )R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and a new substrate 2-AbzVVTLQSGTyr(3-NO 2 )R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at the position P5 improved the catalytic efficacy. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.
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SciScore for 10.1101/2021.02.19.431973: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend …
SciScore for 10.1101/2021.02.19.431973: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
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