Broad-spectrum antivirals against 3C or 3C-like proteases of picornaviruses, noroviruses, and coronaviruses

Abstract

Phylogenetic analysis has demonstrated that some positive-sense RNA viruses can be classified into the picornavirus-like supercluster, which includes picornaviruses, caliciviruses, and coronaviruses. These viruses possess 3C or 3C-like proteases (3Cpro or 3CLpro, respectively), which contain a typical chymotrypsin-like fold and a catalytic triad (or dyad) with a Cys residue as a nucleophile. The conserved key sites of 3Cpro or 3CLpro may serve as attractive targets for the design of broad-spectrum antivirals for multiple viruses in the supercluster. We previously reported the structure-based design and synthesis of potent protease inhibitors of Norwalk virus (NV), a member of the Caliciviridae family. We report herein the broad-spectrum antiviral activities of three compounds possessing a common dipeptidyl residue with different warheads, i.e., an aldehyde (GC373), a bisulfite adduct (GC376), and an α-ketoamide (GC375), against viruses that belong to the supercluster. All compounds were highly effective against the majority of tested viruses, with half-maximal inhibitory concentrations in the high nanomolar or low micromolar range in enzyme- and/or cell-based assays and with high therapeutic indices. We also report the high-resolution X-ray cocrystal structures of NV 3CLpro-, poliovirus 3Cpro-, and transmissible gastroenteritis virus 3CLpro- GC376 inhibitor complexes, which show the compound covalently bound to a nucleophilic Cys residue in the catalytic site of the corresponding protease. We conclude that these compounds have the potential to be developed as antiviral therapeutics aimed at a single virus or multiple viruses in the picornavirus-like supercluster by targeting 3Cpro or 3CLpro.

Fig 1

Structures (A) and summary of synthesis (B) of dipeptide inhibitors. GC373, GC375, and GC376 are shown, with aldehyde, α-ketoamide, and aldehyde bisulfite adduct salt, respectively, as warheads.

Fig 2

Crystal structures of apo-NV 3CLpro and NV 3CLpro in complex with GC376. (A) Noncrystallographic symmetry (NCS) dimer of NV 3CLpro showing chain A (blue) and chain B (magenta). (B) NCS dimer of apo-NV 3CLpro showing chain A (magenta) and chain B (blue) superimposed with the GC376-bound form showing chain A (cyan) and chain B (green).

Fig 3

Cocrystal structures of NV 3CLpro, PV 3Cpro, and TGEV 3CLpro in complex with GC376. (A to C) NV 3CLpro-GC376 complex. (A) F_o − F_c omit map contoured at 3σ for GC376. The aromatic ring of the inhibitor (green) was not included in the model due to disorder. (B) GC376 (red) in the S1 and S2 positions of the active site of NV 3CLpro (gray). (C) Hydrogen bonding (dashed lines) interactions between NV 3CLpro (cyan) and GC376 (gray). (D to F) PV 3Cpro-GC376 complex. (D) F_o − F_c omit map contoured at 3σ for GC376. (E) GC376 (red) in the S1 and S2 positions of the active site of PV 3Cpro (gray). (F) Hydrogen bonding (dashed lines) interactions between PV 3Cpro (magenta) and GC376 (gray). (G to I) TGEV 3CLpro-GC376 complex. (G) F_o − F_c omit map contoured at 3σ for GC376. (H) GC376 (red) in the S1 and S2 positions of the active site of TGEV 3CLpro (gray). (I) Hydrogen bonding (dashed lines) interactions between PV 3Cpro (magenta) and GC376 (blue). The red spheres in panels F and I are water molecules.

Fig 4

Conformational changes during binding of NV 3CLpro or PV 3Cpro and GC376. (A) Conformational changes in the loop containing Gln 110 in NV 3CLpro that occur upon inhibitor binding (see Movie S1 in the supplemental material for details). The apo and inhibitor-bound forms are colored magenta and cyan, respectively. The hydrogen bonds that form between Gln 110 and the inhibitor are indicated by dashed lines. (B) Conformational changes in the loops containing Leu 127, Gly 128, and Thr 142 of PV 3Cpro that occur upon ligand binding (see Movie S2 for details). The apo and ligand-bound forms are colored green and magenta, respectively. Leu 127 and Gly 128 undergo a conformational change to accommodate a water-mediated hydrogen bond. The red spheres are water molecules.

Fig 5

Inhibition of viral 3Cpro and 3CLpro by GC376. (A) Design of transition state inhibitors for viral 3CLpro or 3Cpro. (B) Suggested mechanism of the aldehyde bisulfite adduct (GC376) as an inhibitor of viral 3CLpro or 3Cpro. It may act as a prodrug of the aldehyde inhibitor (GC373).