# Development of a Complementation Assay to Monitor Pan-Coronavirus 3C-like Protease Activity

## TL;DR

Researchers developed a test to monitor coronavirus enzyme activity, which can help find drugs that work against multiple coronaviruses.

## Contribution

The study adapts and improves a cell-based assay to monitor 3CLpro activity across multiple human coronaviruses.

## Key findings

- The assay was successfully adapted for six human coronaviruses, including SARS-CoV and MERS-CoV.
- The Nsp4-Nsp5 cleavage site and super-active substrate provided the best performance in the assay.
- The inhibitor GC376 increased reporter activity, confirming the assay's utility for inhibitor screening.

## 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 (3CLpro) 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 3CLpro activity in cells. This assay uses a single vector that coexpresses the 3CLpro enzyme and the reporter, which consists of two luciferase fragments linked by a 3CLpro cleavage site. Cleavage of this site by 3CLpro decreases luciferase activity, whereas inhibition of 3CLpro increases the luciferase activity. Here, we adapted this assay to examine 3CLpro 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 3CLpro 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 3CLpro from multiple or novel coronaviruses.