Abstract
Coronaviruses pose a global pandemic threat, making development of a pan-coronavirus inhibitor crucial for preparedness and containment in the event of a new coronavirus outbreak. The 3C-like protease (3CL(pro)) is a key target for antiviral development, as it is essential for viral replication and conserved across human coronaviruses. We previously developed an assay to monitor SARS-CoV-2 3CL(pro) activity in cells. This assay uses a single vector that coexpresses the 3CL(pro) enzyme and the reporter, which consists of two luciferase fragments linked by a 3CL(pro) cleavage site. Cleavage of this site by 3CL(pro) decreases luciferase activity, whereas inhibition of 3CL(pro) increases the luciferase activity. Here, we adapted this assay to examine 3CL(pro) activity from six other human coronaviruses: SARS-CoV, MERS-CoV, HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1. We further determined the effects of different cleavage sites to improve the signal-to-background ratio. The Nsp4-Nsp5 site and super-active substrate (SAS) resulted in the largest dynamic range for most coronaviruses in our assay. Using the broad-spectrum 3CL(pro) inhibitor GC376, we observed increased reporter activity, indicating the assay's efficacy for identifying inhibitors across multiple coronaviruses. The adaptation and improvement of the assay can facilitate the development of inhibitors against 3CL(pro) from multiple or novel coronaviruses.