Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate.
J Biol Chem
; 298(4): 101739, 2022 04.
Article
in English
| MEDLINE | ID: covidwho-1693313
ABSTRACT
The emergence of severe acute respiratory syndrome coronavirus 2 (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 (Mpro, also called 3CLpro) 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 Mpro, then characterize the steady-state kinetics of several commonly used FRET substrates, fluorogenic substrates, and six 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 Mpro enzyme assays to facilitate the discovery and development of therapies targeting Mpro.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Enzyme Assays
/
Fluoresceins
/
Coronavirus 3C Proteases
/
SARS-CoV-2
Type of study:
Prognostic study
Limits:
Humans
Language:
English
Journal:
J Biol Chem
Year:
2022
Document Type:
Article
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