Crystal Structures of Enterovirus 71 3C Protease Complexed with Rupintrivir Reveal the Roles of Catalytically Important Residues

Abstract

EV71 is the primary pathogenic cause of hand-foot-mouth disease (HFMD), but an effective antiviral drug currently is unavailable. Rupintrivir, an inhibitor against human rhinovirus (HRV), has potent antiviral activities against EV71. We determined the high-resolution crystal structures of the EV71 3C pro /rupintrivir complex, showing that although rupintrivir interacts with EV71 3C pro similarly to HRV 3C pro , the C terminus of the inhibitor cannot accommodate the leaving-group pockets of EV71 3C pro . Our structures reveal that EV71 3C pro possesses a surface-recessive S2 pocket that is not present in HRV 3C pro that contributes to the additional substrate binding affinity. Combined with mutagenic studies, we demonstrated that catalytic Glu71 is irreplaceable for maintaining the overall architecture of the active site and, most importantly, the productive conformation of catalytic His40. We discovered the role of a previously uncharacterized residue, Arg39 of EV71 3Cpro , that can neutralize the negative charge of Glu71, which may subsequently assist deprotonation of His40 during proteolysis.

Structure of the Enterovirus 71 3C Protease in Complex with NK-1.8k and Indications for the Development of Antienterovirus Protease Inhibitor

“…5C). In contrast, this leaving-group side pocket in HRV 3C pro is partially occupied by the ethyl ester of AG7088 (22). Because the P1= position is replaced by an aldehyde group in NK-1.8k or a cyanohydrin group in compound 9, the structural finding that the leaving-group side pocket is in a free state is not surprising (Fig.…”

“…In the structures of EV71 3C pro in complex with NK-1.8k, this ␤-ribbon changes to a closed conformation and seals the bottom of the substrate-binding groove (Fig. 2B), which is similar to the conformation when binding substrate or inhibitors (22,23).…”

“…5). H40 is a key residue that not only participates in the catalytic reaction but also structurally forms the ceiling of the S2 site by its imidazole group (22). In the structures of the WT or N69S EV71 3C pro complex with NK-1.8k and compound 9, the side chain of H40 maintains the same orientation that it maintains in natural substrate-bound or unliganded EV71 3C pro .…”

Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D

“…To explore whether the protease activity of EV71 3C is required for the cleavage of GSDMD, we evaluated the impact of rupintrivir, which is an inhibitor of 3C protease with a broad spectrum of activity against picornaviruses (37)(38)(39). As shown in Fig.…”

“…In order to further verify this issue, we carried out mutational analysis. His40, Glu71, and Cys147 are key amino acids which form the catalytic triad of EV71 3C (39). H40D and C147S substitutions in the active site of EV71 3C disrupt the protease activity.…”