Profiling of coronaviral M^pro and enteroviral <scp>3C^pro</scp> 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 M^pros exhibit overlapping substrate specificity in all binding pockets, whereas the 3C^pro 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^pro can be successfully applied to investigate the activity of the M^pro from other coronaviruses as well as the 3C^pro from enteroviruses. Our study provides a structural framework for the development of broad-spectrum antiviral compounds.