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

Abstract

Hand-foot-and-mouth disease (HFMD), caused by enterovirus, is a threat to public health worldwide. To date, enterovirus 71 (EV71) has been one of the major causative agents of HFMD in the Pacific-Asia region, and outbreaks with EV71 cause millions of infections. However, no drug is currently available for clinical therapeutics. In our previous works, we developed a set of protease inhibitors (PIs) targeting the EV71 3C protease (3C^pro). Among these are NK-1.8k and NK-1.9k, which have various active groups and high potencies and selectivities. In the study described here, we determined the structures of the PI NK-1.8k in complex with wild-type (WT) and drug-resistant EV71 3C^pro Comparison of these structures with the structure of unliganded EV71 3C^pro and its complex with AG7088 indicated that the mutation of N69 to a serine residue destabilized the S2 pocket. Thus, the mutation influenced the cleavage activity of EV71 3C^pro and the inhibitory activity of NK-1.8k in an in vitro protease assay and highlighted that site 69 is an additional key site for PI design. More information for the optimization of the P1' to P4 groups of PIs was also obtained from these structures. Together with the results of our previous works, these in-depth results elucidate the inhibitory mechanism of PIs and shed light to develop PIs for the clinical treatment of infections caused by EV71 and other enteroviruses.

Keywords: EV71; crystal structure; inhibitor; mechanism; protease.

FIG 1

Comparison of the chemical structures of AG7088 (A), NK-1.8k (B), compound 9 (C), and NK-1.9k (D). The P1′ to P3 or P4 positions of each compound are indicated.

FIG 2

Structures of NK-1.8k bound with WT or N69S-mutated EV71 3C^pro. (A, B) Overall structure of NK-1.8k in complex with EV71 3C^pro. The polypeptide of EV71 3C^pro is shown as a colored cartoon (A) or with a colored surface (B), in which the N-terminal (N-ter) domain is colored blue. The β-ribbon, which is important for substrate or inhibitor binding, is colored red. (C, D) Densities of bound NK-1.8k in complex with WT (C) or N69S-mutated (D) EV71 3C^pro. The polypeptide of EV71 3C^pro is shown as a white cartoon. In WT (C) and N69S-mutated (D) EV71 3C^pro, NK-1.8k is presented as green and magenta sticks, respectively. Catalytic residues H40 and C147, as well as drug resistance-conferring residue N69S, are displayed as gold sticks. Both NK-1.8k molecules are covered by a 2F_o − F_c omit map at 1.1 σ, while H40 and C147 are covered by a 2F_o − F_c map at 1.6 σ (F_o is F observe and F_c is F calculate).

FIG 3

Structural comparison of EV71 3C^pro in the unliganded form and in complex with NK-1.8k. The structure of unliganded EV71 3C^pro (PDB accession no. 3OSY) was superimposed onto that of EV71 3C^pro in complex with NK-1.8k by using the CCP4 program. The polypeptides of EV71 3C^pro in the unliganded form or in complex with NK-1.8k are displayed as red and green cartoons, respectively. The structural elements with RMSDs of less than 0.7 Å are colored white. The bound NK-1.8k is shown as colored sticks.

FIG 4

Structural comparison of EV71 3C^pro in complex with AG7088 and NK-1.8k. The structure of EV71 3C^pro bound with AG7088 (PDB accession no. 3SJO) was superimposed onto the structure of EV71 3C^pro in complex with NK-1.8k by using the CCP4 program. The polypeptides of EV71 3C^pro in complex with AG7088 and NK-1.8k are displayed as gold and green cartoons, respectively. The structural elements with RMSDs of less than 0.5 Å are colored white. The bound inhibitors are shown as colored sticks. Site 69 is highlighted.

FIG 5

Binding of inhibitors to WT or drug-resistant EV71 3C^pro. The structure of WT (A) or N69S (B) EV71 3C^pro bound to NK-1.8k and the structure of 3C^pro in complex with compound 9 (PDB accession no. 5BPE) (C) or AG7088 (PDB accession no. 3SJO) (D) were superimposed and are shown in the same orientation. (Left) The polypeptides of the enzymes are displayed as cartoon diagrams, while the inhibitors and key residues are represented as colored sticks; (right) the proteins are covered with the electric potential surface. Key residues and subsites for inhibitor binding are labeled.

FIG 6

Detailed view of site 69. The structures of WT or N69S-mutated EV71 3C^pro in complex with NK-1.8k and WT EV71 3C^pro in complex with AG7088 were superimposed and are shown together (A) or individually (B to D). Polypeptides of EV71 3C^pro in the WT EV71 3C^pro–NK1.8k complex (B), the N69S EV71 3C^pro–NK1.8k complex (C), or the WT EV71 3C^pro–AG7088 complex (D) are displayed as green, light blue, and cyan sticks, respectively. Site 69 (in which drug resistance is conferred when N69 is mutated to S) and catalytic residue H40 are colored orange. The inhibitor in each complex is presented as sticks with the same color scheme as the residues. Hydrogen bonds are denoted as dotted lines with the measured distance.

FIG 7

In vitro protease activity. (A) IC₅₀ of NK-1.8k for WT and N69S proteases. The percentages of inhibitory activity of NK-1.8k at concentrations ranging from 10 to 0.00975 μM were compared with those in the absence of compound (control). AU, arbitrary units. (B) Cleavage activities of WT and N69S proteases. The intensities of the ODs were read at an excitation λ of 340 nm and an emission λ of 440 nm every 10 min (B).