A Bioluminescent 3CLPro Activity Assay to Monitor SARS-CoV-2 Replication and Identify Inhibitors

Abstract

Our therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CLPro of coronaviruses, so that the luminescent biosensor is turned on upon 3CLPro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development.

A Broad Antiviral Strategy: Inhibitors of Human DHODH Pave the Way for Host-Targeting Antivirals against Emerging and Re-Emerging Viruses

“…BAY2402234 is a novel potent selective DHODHi with an IC 50 of 1.2 nM [ 86 ]. Mathieu et al screened 492 compounds inhibiting SARS-CoV-2 replication and found that BAY2402234 blocked almost 100% of SARS-CoV-2 particle production at 0.6 μM [ 87 ]. In addition, the combination of teriflunomide, IMU-838/vidofludimus, and BAY2402234 inhibited SARS-CoV-2 replication and reduced viral yield by at least two orders of magnitude in Vero E6 and Calu-3 cells infected with wildtype, the Alpha variant, and the Beta variant of SARS-CoV-2 [ 88 ].…”

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.…”

“…However, this C-terminal His-tag can lower the binding affinity of a given ligand [ 90 ]. To overcome the limitations of the in vitro screens, cell-based assays have been developed [ 73 , 80 , 83 , 84 , 87 , 88 ]. In these assays, the cells express the M-pro and the reporter used and can differentiate cytotoxicity from true M-pro inhibition [ 83 ].…”

Development of a colorimetric assay for the detection of SARS-CoV-2 3CLpro activity

“…Intracellular 3CLpro activity has previously been detected in SARS-CoV-2-infected human cell lines [ 32 , 33 ], but it remains to be determined whether the in vitro AuNP protease assay is sufficiently sensitive to detect cellular 3CLpro activity from SARS-CoV-2 infected human cell line lysates. Protein levels of 3CLpro appear to be 50-fold lower in the context of viral infection, compared with transient transfection of the protease as an exogenous transgene ( Supplementary Figure S9 ).…”