Antiviral Drug Discovery: Norovirus Proteases and Development of Inhibitors

Figure 1. Proteolysis of the scissile bond between P1 and P1’. Standard nomenclature P1… Pn and P1’… Pn’ for amino acid residues of the substrates. S1... Sn and S1’… Sn’ are the corresponding binding sites on the enzyme (per Berger and Schechter nomenclature (3)).

Figure 2. Examples of commercially available protease inhibitors and their targets. Dabigatran and Rivaroxaban are anti-coagulants; Lisinopril is for hypertension; Sitagliptin is for diabetes; and Bortezomib is for multiple myeloma.

Figure 3. Crystal structure of HIV-1 protease (A), and its cleavage sites (B). The catalytic site resides between two monomers (blue and green), and Asp25 and Asp25’ (red) from each dimer orchestrate the cleavages (PDB: 2NMZ). The flap region and dimerization domain are also shown. Cleavage sites (amino acids) of mature proteins of HIV-1 are listed. The red color indicates P1 specificity.

Figure 4. FDA-approved HIV protease inhibitors with approved year and EC50 values against HIV-1 in cell culture.

Figure 5. Crystal structure of hepatitis C virus (HCV) NS3/4A (genotype 1), and its cleavage sites. (A) The two β-barrel sub-domains of the NS3/4A protease domain are indicated in a ribbon diagram (adapted from PDB:1A1R). His57, Asp99, and Ser139, which form the catalytic triad, are shown in ball-and-stick representation. The protease structural zinc ion (blue sphere) is indicated. The NS4A peptide is rendered as a light orange ribbon. (B) Cleavage sites (amino acids) between mature proteins of HCV genotype 1 are listed. Red color indicates P1 specificity.

Figure 6. FDA-approved HCV protease inhibitors with approved year and EC50 values against genotype 1 in cell culture (replicon harboring cells).

Figure 7. Crystal structure of NV 3CLPro (PDB: 2LNC) (A) and its cleavage sites (B). The active site resides between the two domains and the catalytic triad with Cys139, His30 and Glu54 are shown. Cleavage sites (amino acids) between mature proteins of Norwalk virus are listed. The red color indicates P1 specificity.

Figure 8. Examples of optimization strategy employed for the dipeptidyl compounds against norovirus from one of the initial hits (GC373). Potency increased by making changes in R1 and R2. Red color indicates the most optimized GC583.

Figure 9. X-ray crystal structure of NV 3CLPro and GC543 (A,C, PDB: 4XBC) and GC583 (B,D, PDB: 4XBB). The structures revealed that increased potency is correlated to interactions between the S4 subsite and the cap residue. The m-Cl benzyl group in the cap allows for interactions with hydrophobic residues in S4.

Figure 10. Examples of macrocyclic NV 3CLPro inhibitors. (A–C). Three different classes (A–C) of macrocyclic inhibitors were synthesized. The P2 position, and the length and nature of the linker were examined for optimal efficacy against the enzyme and viral replication. (D–E) X-ray co-crystallization of NV 3CLPro and an oxadiazole-based macrocycle (B, PDB: 5DG6).