Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate

Area of Science:

• Biochemistry
• Virology
• Drug Discovery

Background:

• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a global health threat, necessitating novel antiviral therapies.
• The coronavirus main protease (Mpro), also known as 3CLpro, is a critical drug target for developing broad-spectrum coronavirus therapeutics.

Purpose of the Study:

• To characterize the biophysical and enzymatic properties of native Mpro.
• To evaluate the suitability of various FRET and fluorogenic substrates for high-throughput screening (HTS) of Mpro inhibitors.
• To develop an improved FRET substrate for more effective Mpro drug discovery.

Main Methods:

• Biophysical and enzymatic characterization of native Mpro.
• Steady-state kinetic analysis of multiple FRET and fluorogenic substrates against SARS-CoV-2 Mpro.
• Assessment of substrate performance in high-throughput screening assays.
• Design and validation of a novel 5-carboxyfluorescein-based FRET substrate.

Main Results:

• Characterization of native Mpro's enzymatic activity.
• Identification of optimal FRET and fluorogenic substrates for SARS-CoV-2 Mpro assays.
• Development of an improved 5-carboxyfluorescein-based FRET substrate with enhanced HTS suitability.
• The new substrate demonstrates reduced susceptibility to interference and false positives compared to existing options.

Conclusions:

• The study provides a robust framework for designing coronavirus Mpro enzyme assays.
• The developed FRET substrate is superior for HTS, accelerating the identification of potential Mpro-targeting antiviral drugs.
• This work facilitates the discovery and development of novel therapies against current and future coronavirus threats.