Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate
Scott Legare, Fabian Heide, Ben A. Bailey-Elkin, Jörg Stetefeld · Journal of Biological Chemistry · 2022
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 (M pro , also called 3CL pro ) 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 pro , 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 M pro enzyme assays to facilitate the discovery and development of therapies targeting M pro .