A rapid and robust luciferase-based reporter system to assess SARS-CoV-2 protease activity.

Despite effective antiviral drugs that have emerged to combat SARS-CoV-2 infections, novel therapeutic strategies are required to better address the ongoing and future evolutions of the virus. Targeting viral proteases, such as the main protease (Mpro), remains a promising approach. Here, we present a rapid and sensitive luminescence-based reporter system, the i-NSP4/5-Gluc2, to assess Mpro activity. This system employs Gaussia luciferase (Gluc) fused to a pro-interleukin 1β (pro-IL-1β) fragment containing a specific Mpro cleavage site. Upon Mpro cleavage, Gluc is released and secreted, generating a luminescent signal outside the cells. By optimizing the system's design and experimental conditions, we achieved high sensitivity and specificity. The i-NSP4/5-Gluc2 system was validated using the Mpro inhibitor Nirmatrelvir and successfully identified potential Mpro inhibitors from a small library of 46 compounds, as proof of concept. Notably, 13 out of 14 new compounds identified by the i-NSP4/5-Gluc2 assay exhibited potent antiviral activity against live SARS-CoV-2, highlighting the system's accuracy and predictive power. This BSL2-compatible, high-throughput approach facilitates rapid and efficient screening of antiviral compounds, accelerating the development of effective therapeutics against SARS-CoV-2 and future viral pandemics.