Efficient Strategy to Design Protease Inhibitors: Application to Enterovirus 71 2A Protease

[Image: see text] One strategy to counter viruses that persistently cause outbreaks is to design molecules that can specifically inhibit an essential multifunctional viral protease. Herein, we present such a strategy using well-established methods to first identify a region present only in viral (but not human) proteases and find peptides that can bind specifically to this “unique” region by maximizing the protease–peptide binding free energy iteratively using single-point mutations starting with the substrate peptide. We applied this strategy to discover pseudosubstrate peptide inhibitors for the multifunctional 2A protease of enterovirus 71 (EV71), a key causative pathogen for hand-foot-and-mouth disease affecting young children, along with coxsackievirus A16. Four peptide candidates predicted to bind EV71 2A protease more tightly than the natural substrate were experimentally validated and found to inhibit protease activity. Furthermore, the crystal structure of the best pseudosubstrate peptide bound to the EV71 2A protease was determined to provide a molecular basis for the observed inhibition. Since the 2A proteases of EV71 and coxsackievirus A16 share nearly identical sequences and structures, our pseudosubstrate peptide inhibitor may prove useful in inhibiting the two key pathogens of hand-foot-and-mouth disease.