[Preparation and characterization of a fluorogenic ddRFP-M biosensor as a specific SARS-CoV-2 main protease...

Area of Science:

• Biochemistry and Molecular Biology
• Protein Engineering
• Drug Discovery

Context:

• Conventional peptide substrates for SARS-CoV-2 main protease (Mpro) present challenges including high cost, unstable kinetics, and complex synthesis.
• There is a critical need for cost-effective and stable Mpro substrates for effective pharmacological research and drug development.

Purpose:

• To design and characterize a novel, functional, and stable Mpro substrate for in vitro evaluation of Mpro inhibitors.
• To utilize a dimerization-dependent red fluorescent protein (ddRFP) platform for creating a sensitive and reliable biosensor.

Summary:

• A codon-optimized DNA fragment encoding two RFP domains linked by an Mpro cleavage sequence was constructed and expressed in E. coli.
• The resulting ddRFP-M protein was purified and demonstrated specificity, sensitivity, and reliability in assessing Mpro inhibition by ensitrelvir and baicalein.
• This fluorogenic substrate provides a robust and accessible method for Mpro inhibitor screening.

Impact:

• Provides an affordable and stable alternative to conventional Mpro substrates, facilitating broader research.
• Enables efficient in vitro assessment of SARS-CoV-2 Mpro inhibitors, potentially accelerating antiviral drug development.
• The ddRFP-M biosensor offers a reliable tool for biochemical assays and drug screening platforms.