Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (M^pro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 M^pro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance, and characterized six FRET substrates with a 2-Abz/Tyr(3-NO₂) FRET pair. The substrates 2-AbzSAVLQSGTyr(3-NO₂)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and a new substrate 2-AbzVVTLQSGTyr(3-NO₂)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at the position P5 improved the catalytic efficacy. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.