High-Resolution Substrate Specificity Profiling of SARS-CoV-2 M^pro; Comparison to SARS-CoV M^pro

Area of Science:

• Virology
• Biochemistry
• Drug Discovery

Background:

• The SARS-CoV-2 main protease (Mpro) is essential for viral polyprotein processing.
• Mpro is the target of Nirmatrelvir, a key component of the COVID-19 treatment Paxlovid.
• Understanding Mpro substrate specificity is vital for developing effective antiviral therapies.

Purpose of the Study:

• To perform high-resolution substrate specificity profiling of SARS-CoV-2 Mpro and SARS-CoV Mpro.
• To compare the substrate specificity profiles of these two related proteases.
• To assess the utility of the YESS 2.0 platform for protease substrate profiling.

Main Methods:

• Utilized the YESS 2.0 platform for combined protease and substrate expression in yeast.
• Employed fluorescence-activated cell sorting and next-generation sequencing for deep profiling.
• Analyzed cleavage sites on SARS-CoV-2 polyproteins pp1a and pp2b.

Main Results:

• SARS-CoV-2 Mpro and SARS-CoV Mpro exhibit essentially identical substrate specificity profiles.
• The YESS 2.0/NGS approach demonstrated precise and reproducible protease substrate specificity profiling.
• Qualitative predictions of relative catalytic efficiencies at different cleavage sites were achieved.

Conclusions:

• The YESS 2.0 platform is a powerful tool for high-resolution protease substrate specificity profiling.
• Identical substrate specificity suggests conserved inhibitor strategies for both SARS-CoV-2 and SARS-CoV Mpro.
• Findings will inform the design of next-generation Mpro inhibitors for improved antiviral treatments.