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

Abstract

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 (Mpro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 Mpro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance and characterized Förster resonance energy transfer (FRET) substrates with a preference for 2-Abz/Tyr(3-NO2) FRET pairs. The substrates 2-AbzSAVLQSGTyr(3-NO2)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and 2-AbzVVTLQSGTyr(3-NO2)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at position P5 improved the catalytic efficiency. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.

A Review of the Current Landscape of SARS-CoV-2 Main Protease Inhibitors: Have We Hit the Bullseye Yet?

“…Several methods have been developed or adapted for quantifying the potency of the SARS-CoV-2 M-pro inhibitors [ 73 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 ]. These methods demonstrate the mechanism of action of antiviral drugs and do not require cells infected with SARS-CoV-2 or a laboratory with biosafety level 3 containment facilities.…”

Penicillin Derivatives Inhibit the SARS-CoV-2 Main Protease by Reaction with Its Nucleophilic Cysteine

“…To enable the identification of small-molecule M pro inhibitors for development as human therapeutics, high-throughput in vitro inhibition assays using recombinant viral M pro have been developed. 4 , 9 − 12 Most reported M pro inhibition assays employ fluorescence-based methods, though label-free assays, which directly monitor product formation/substrate depletion using mass spectrometry (MS) and SARS-CoV-2 polyprotein peptide fragments, have been reported. 13 − 15 The availability of efficient high-throughput M pro inhibition assays and libraries of bioactive and safety-assessed small molecules has enabled the identification of multiple lead M pro inhibitors, such as boceprevir ( 1 ), 11 , 16 an HCV serine protease inhibitor, 17 , 18 SDZ-224015 ( 2 ), 19 an investigational caspase-1 inhibitor, 20 and GC-376 ( 3 ), 11 , 16 , 21 for (partially) selective inhibition of M pro ( Figure 1 A–C).…”

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

“…Human coronaviruses include HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 continuously circulate in the population and mostly cause mild symptoms associated with the common cold. In contrast, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 are highly pathogenic coronaviruses causing the SARS epidemic, MERS epidemic and most recently the coronavirus disease 2019 (COVID- 19) pandemic (1). To date there have been over 220 million reported cases of COVID-19 and 4.6 million reported deaths.…”

“…Fluorogenic substrates which cleave an amino-coumarin based fluorophore attached to the carboxyl terminus of a peptide have also been used (14,15,18). A number of M pro enzyme assays have been developed using different substrates, M pro constructs, and buffer conditions (14,15,(19)(20)(21)(22). As a result, there have been inconsistent findings regarding the identification of potential M pro inhibitors (20,21,23).…”