tetano
Editor, Senior Moderator
J Biol Chem
. 2022 Feb 16;101739.
doi: 10.1016/j.jbc.2022.101739. Online ahead of print.
Improved SARS-CoV-2 main protease high throughput screening assay using a 5-carboxyfluorescein substrate
Scott Legare[SUP] 1 [/SUP], Fabian Heide[SUP] 2 [/SUP], Ben A Bailey-Elkin[SUP] 2 [/SUP], Jörg Stetefeld[SUP] 3 [/SUP]
Affiliations
Abstract
The emergence of SARS-CoV-2 as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (M[SUP]pro[/SUP], also called 3CL[SUP]pro[/SUP]) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here we report the biophysical and enzymatic characterization of native M[SUP]pro[/SUP], then characterize the steady-state kinetics of several commonly used fluorescence resonance energy transfer (FRET) substrates, fluorogenic substrates, and 6 of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate which is better suited for high throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus M[SUP]pro[/SUP] enzyme assays to facilitate the discovery and development of therapies targeting M[SUP]pro[/SUP].
Keywords: 3CL protease (3CLpro); High-throughput screening (HTS); Main protease (Mpro); SARS-CoV-2; coronavirus; enzyme kinetics; fluorescence resonance energy transfer (FRET); protease substrate; viral protease.
. 2022 Feb 16;101739.
doi: 10.1016/j.jbc.2022.101739. Online ahead of print.
Improved SARS-CoV-2 main protease high throughput screening assay using a 5-carboxyfluorescein substrate
Scott Legare[SUP] 1 [/SUP], Fabian Heide[SUP] 2 [/SUP], Ben A Bailey-Elkin[SUP] 2 [/SUP], Jörg Stetefeld[SUP] 3 [/SUP]
Affiliations
- PMID: 35182525
- DOI: 10.1016/j.jbc.2022.101739
Abstract
The emergence of SARS-CoV-2 as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (M[SUP]pro[/SUP], also called 3CL[SUP]pro[/SUP]) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here we report the biophysical and enzymatic characterization of native M[SUP]pro[/SUP], then characterize the steady-state kinetics of several commonly used fluorescence resonance energy transfer (FRET) substrates, fluorogenic substrates, and 6 of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate which is better suited for high throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus M[SUP]pro[/SUP] enzyme assays to facilitate the discovery and development of therapies targeting M[SUP]pro[/SUP].
Keywords: 3CL protease (3CLpro); High-throughput screening (HTS); Main protease (Mpro); SARS-CoV-2; coronavirus; enzyme kinetics; fluorescence resonance energy transfer (FRET); protease substrate; viral protease.