Profiling of coronaviral Mpro and enteroviral 3Cpro specificity provides a framework for the development of broad‐spectrum antiviral compounds

Abstract 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 pro s 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.