Broad-Spectrum Antivirals against 3C or 3C-Like Proteases of Picornaviruses, Noroviruses, and Coronaviruses

Abstract

dPhylogenetic 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 ␣-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.

The Enterovirus Protease Inhibitor Rupintrivir Exerts Cross-Genotypic Anti-Norovirus Activity and Clears Cells from the Norovirus Replicon

“…Such antiviral effect, although 10-fold less pronounced, was also earlier reported (10); differences in the antiviral activity in that and our study may possibly be related to either differences in methodology or in the purity of the compound. No decrease in ␤-actin levels was observed at any tested concentration, corroborating recently published data (10).…”

“…Rupintrivir inhibited in a dose-dependent manner MNV-induced CPE (EC 50 ϭ 13 Ϯ 2 M) (Fig. 3) (although no activity was reported in another study [10]). We confirmed this anti-norovirus activity by assessing the effect of rupintrivir on MNV RNA synthesis; rupintrivir inhibited the production of MNV RNA, also in a dose-dependent manner (EC 50 ϭ 10 Ϯ 1 M) (Fig.…”

“…Human noroviruses belong to the Caliciviridae family, whose protease displays a chymotrypsin-like fold with a cysteine as the active-site nucleophile and shares similarities with the 3C protease of picornaviruses (11). Rupintrivir has recently been reported to inhibit the replication of the Norwalk virus replicon (a genogroup I [GI] human norovirus) (10).…”

Development of a Gaussia Luciferase-Based Human Norovirus Protease Reporter System: Cell Type-Specific Profile of Norwalk Virus Protease Precursors and Evaluation of Inhibitors

“…Two of the NV ORF1-encoded enzymes, Pro and Pol, have been extensively studied and also targeted for antiviral therapeutic development due to their essential roles in viral replication (16)(17)(18)(19)(20)(21)(22). The NV Pro is a 3C-like cysteine protease classified in the chymotrypsin-like serine protease superfamily.…”

“…The crystal structure of NV Pro shows that it has a two-domain structure similar to those of other viral 3C-like cysteine proteases, with a catalytic triad composed of His-30, Glu-54, and Cys-139 (23,24). NV Pro cleaves at a Q/G or E/G scissile bond, with a preferred substrate specificity for an FxLQ/GP sequence in the P 4 -P 1 /P 1 =P 2 = position, where x is H, Q, N, or A. Small-molecule inhibitors of NV Pro, either designed based on its substrate specificity or screened from compound libraries, have been reported (16,(19)(20)(21)(22). However, except for those that have been tested against an NV replicon system (19,20,22), many of the NV Pro inhibitors were developed by in vitro protease assays and remain to be evaluated using a cell-based system.…”