Norovirus Protease Structure and Antivirals Development

Figure 1. Three-dimensional structure of HuNoV protease and its active site. (A) Ribbon representation of Norwalk protease structure (PDB ID: 2FYQ). Domain I (cyan) is connected to Domain II (lime) via a large loop (orange). The catalytic residues are shown as red stick models. The β-strands are shown as yellow characters with red outline. (B) The active site of HuNoV protease contains three residues colored red. (C) Structures of all the published protease are overall conserved with several regions exhibiting conformation flexibility, indicated by the red arrows. The five regions are: (1) the flexible surface loop from aa 33 to 36, (2) the hairpin loop from aa 107 to 113, (3) the short loop from aa 147 to 150, (4) the β-hairpin loop from aa 162 to 164, and (5) the loop from aa 122 to 134 (D) Conservation of amino acid positions in HuNoV protease structures. The figure was generated with the ConSurf server [69]. (E) The amino acid residues are colored in red for the active site, blue for the oxyanion hole, green for the S1 pocket, cyan for the S2 pocket, and magenta for the S4 pocket. The GenBank accession ID for each sequence is Norwalk, NP_786949.1; Southampton, AAA92983.1; Chiba,…

Figure 2. Proteolysis mechanism of HuNoV protease. (A) The three residues within the catalytic triad are at the rest state. (B) Binding of a substrate induces a conformational change, which leads to the removal of a proton on the thiol side chain of the Cys by the His ring. The active thiol then launches a nucleophilic attack on the carbonyl carbon of the substrate. (C) A covalent bond is formed between the substrate and the sulfur and a protease–substrate tetrahedral intermediate is formed. The His ring then donates a proton to the amide nitrogen of the substrate. (D) This leads to the breakage of the peptide bond, which releases one side of the substrate out of the active site, as indicated by the dashed arrow. The His ring then activates a water molecule, which launches a nucleophilic attack on the acyl-enzyme complex. (E) Attacking from the water molecule forms the second tetrahedral intermediate. The His ring again donates a proton to the intermediate complex. (F) The tetrahedral intermediate complex then decomposes. The substrate is released out of the active site. The enzyme is back to the native state. Red indicates an oxygen atom. Blue indicates a nitrogen atom. Green indicates a…

Figure 3. Interactions of the active site residues are affected by the Arg112 residue. (A) Comparison of the active sites of GI.1 Norwalk (cyan) and GII.4 Houston (green) depicts a different orientation of the His30 residue. In GI.1 Norwalk protease, the Arg112 residue is positioned away from the active site, therefore allowing His30 to interact with Glu54. However, in GII.4 Houston protease, such interaction between His30 and Glu54 is not observed, as the Arg112 residue is pointed toward the active site and interacts with His30. (B) Active site comparison within the GI genogroup protease (Norwalk, cyan; Chiba, yellow; Southampton, orange) indicates that the Arg112 residue (within the grey box) of the GI.2 Southampton protease is also pointed toward the active site. (C) Active site comparison within the GII genogroup protease (Houston, green; Minerva, grey) indicates that even though Houston and Minerva are in the same genogroup, the orientation of the active site residues are different due to the position of the Arg112 residue (within the grey box).

Figure 4. Protease cleavage sites and the substrate-binding pockets. (A) Cleavage sites of five HuNoV proteases. Each box indicating a non-structural protein contains the P4–P1 residues in its right side and the P1′–P4′ residues in its left side. P1 and P1′ residues are colored in red. (B) Surface representation of the substrate-binding pockets of Norwalk virus and (C) of Houston virus. (D–G) Structural comparison of Norwalk (PDB ID: 2FYQ) and HOV (PDB ID: 6NIR) proteases. (D,E) The residues mediating the binding specificity of the S1 pocket and the active site residues are colored in green and red, respectively. The structural differences between Norwalk and HOV are colored in cyan and yellow. (F,G) The residues mediating the binding specificity of the S2 pocket and the active site residues are colored in cyan and red, respectively. The structural differences of these proteases are colored in yellow.

Figure 5. Binding and recognition of substrates and inhibitors to the HuNoV protease. (A) Binding of the TALE substrate (orange) to the Norwalk protease (cyan) (PDB ID: 4IN2). Water molecules are represented as red spheres. (B) Binding of the INFE substrate (orange) to the Norwalk protease (cyan) (PDB ID: 4IN1). (C) Binding of the Syc59 inhibitor (yellow) to the Norwalk protease (cyan) (PDB ID: 4INH). (D) Binding of the macrocyclic inhibitor 1 (yellow) to the Norwalk protease (cyan) (PDB ID: 6BIB). Oxygen and nitrogen atoms are labeled in red and blue, respectively.

Figure 6. Conformational changes of the substrate-binding pockets induced by the binding of two substrates. (A) In the native unbound protease, the S2 pocket colored in cyan is in the closed conformation. The active site is colored in red and the oxyanion hole is colored in blue. (B) Binding of a TALE substrate (yellow) opens up the S2 pocket (PDB ID: 4IN2). (C) Binding of an INFE substrate (orange) further opens the S2 pocket (PDB ID: 4IN1). (D) The S4 pocket colored in magenta is widely open as the S2 pocket is closed. (E) The S4 pocket narrows when the TALE substrate (yellow) binds. (F) The S4 pocket becomes narrower when the INFE substrate (orange) binds.

Figure 7. Oxyanion hole of the HuNoV protease. (A) The oxyanion hole colored in blue is next to the active site colored in red. (B) The amino acid residues involved in the hydrogen bonding to stabilize the oxyanion hole are colored in deep blue (Asp138) and yellow (Arg89). (C) In the native unbound protease, the active site colored in red interacts with the oxyanion hole colored in white through a water molecule (red sphere). (D) In the protease bound with a TALE substrate (yellow), the substrate interacts with the oxyanion hole colored in white though two hydrogen bonds. Oxygen and nitrogen atoms are colored in red and blue, respectively.

Figure 8. Inhibition potency of representative Norwalk protease inhibitors. The chemical structures and inhibition potencies of representative linear aldehyde peptidomimetic inhibitors (compound 4 and 7), linear hydroxyphosphonate peptidomimetic inhibitor (compound 7d), linear cyclohexylalanine peptidomimetic inhibitor (compound 16), linear lactam peptidomimetic inhibitor (compound 19), linear cyanoacrylamide peptidomimetic inhibitor (compound 43), prodrug GC376, triazole-based and oxadiazole-based macrocyclic inhibitors, and commercially available inhibitors (chymostatin, WP1130, rupintrivir).

Figure 9. Peptidomimetic inhibitor exhibits differential inhibitory activity to proteases of different genogroups. (A) The inhibition potency of Syc-10 to GI.1. Syc-10 was identified and synthesized specifically to GI.1 protease. (B) The inhibition potency of Syc-10 to GII.4 protease is one-fold lower. (C) The inhibition potency of Syc-10 to a GII.4 Arg112 mutant.