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

Abstract

Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II [GII]) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2=-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.H uman noroviruses are a major cause of food-borne illness, accountable for 50% of all-etiologies outbreaks of acute gastroenteritis (both in developing and developed countries) (1, 2). Outbreaks often occur in long-term-care facilities and hospitals where the elderly and immunocompromised can become severely ill. The vast spreading nature of this virus commonly results in hundreds of sick individuals in each setting and may lead to the closure of hospital wards (3, 4). Associations with chronic gastrointestinal problems and cases of chronic gastroenteritis caused by noroviruses are being increasingly recognized (5, 6). There is no vaccine or antiviral treatment available against norovirus infections. Rupintrivir (AG-7088) is a protease inhibitor originally developed for the treatment of human rhinovirus infections (7,8). Activity has also been demonstrated against other picornaviruses as well as against coronaviruses and caliciviruses (9, 10). 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).We here confirm and further document the inhibitory effect of rupintrivir against the human norovirus (by demonstrating that the compound is able to clear cells from the replicon) and demonstrate that the drug is antivirally active against the murine norovirus (MNV), a virus belonging to another genogroup of the genus. We study the inhibitory effect of rupintrivir against a GII 3C-like protease (in an enzymatic assay) and explain at the s...

Susceptibilities of enterovirus D68, enterovirus 71, and rhinovirus 87 strains to various antiviral compounds

“…Recently it was determined that rupintrivir inhibits noroviruses (Rocha-Pereira et al, 2014), which are not in the picornaviridae family. Perhaps this broad-spectrum activity will revitalize interest in rupintrivir as a treatment for picornaviruses, or else will serve to inspire the synthesis and development of other related compounds that may have a broader antiviral spectrum.…”

The future of antivirals

“…These viruses all have 3C or 3C-like proteases with a typical chymotrypsin-like fold and a catalytic triad (or dyad) containing a cysteine residue as a nucleophile, making them interesting targets for BSAA development. Rupintrivir for instance is an irreversible 3C-protease inhibitor that was originally developed for the treatment of human rhinovirus infections [22] and antiviral activity has also been shown against other picornaviruses, coronaviruses, and norovirus [23][24][25]. Other inhibitors of 3C-like proteases were reported with broad-spectrum antiviral activity against both feline coronaviruses and caliciviruses [26].…”

Viral replicons as valuable tools for drug discovery

“…Four nucleoside analogues were tested on the norovirus replicon, with 2 0 -C-methylcytidine showing good antiviral activity [94]. Rupintrivir, a protease inhibitor of rhinovirus 3C protease, was shown to clear cells of the norovirus replicon [95]. However, owing to the fact that norovirus infections are short-lived and selflimiting, the interest of pharma companies to develop smallmolecule inhibitors has been limited, and none of the mentioned candidates has been taken forward into clinical trials.…”