Profiling of coronaviral M and enteroviral 3C specificity provides a framework for the development of broad-spectrum antiviral compounds.

The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach-HyCoSuL-to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus Ms exhibit overlapping substrate specificity in all binding pockets, whereas the 3C from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4-P2 positions. However, chemical tools such as substrates, inhibitors, and activity-based probes developed for SARS-CoV-2 M can be successfully applied to investigate the activity of the M from other coronaviruses as well as the 3C from enteroviruses. Our study provides a structural framework for the development of broad-spectrum antiviral compounds.