In Vitro Resistance Study of Rupintrivir, a Novel Inhibitor of Human Rhinovirus 3C Protease

The novel enterovirus protease inhibitor (PI) SG85 effectively inhibits the in vitro replication of 14 rhinoviruses representative of species A and B (median 50% effective concentration, 0.04 M). A low-level SG85-resistant variant was selected that carried amino acid substitutions S127G and T143A in the 3C protease. Both substitutions are required for low-level resistance to SG85, as demonstrated by reverse genetics. Interestingly, there is no cross-resistance to SG85 and rupintrivir (another PI); a structural explanation is provided for this observation. R hinoviruses (RV) are nonenveloped, positive-sense, singlestranded RNA viruses that belong to the family of Picornaviridae, genus Enterovirus (1). Currently, more than 150 RV have been identified, which genotypically group into RV-A, RV-B, and RV-C (2). RV infections not only cause common colds but may also trigger exacerbations of asthma and chronic obstructive pulmonary disease (COPD) (3-5). Because of the high (and still rising) number of variants, development of a vaccine will be hard to achieve. Therefore, treatment with antivirals is a more realistic strategy to reduce the burden of these infections. An RV inhibitor, in particular, is needed for the prophylaxis and treatment of RVinduced exacerbations of asthma and COPD (6).The RV 3C protease (3C pro ) is a promising target for drug development efforts because of the high level of conservation in its substrate-binding site, its role as an indispensable enzyme for virus replication, and its unique cleavage specificity (after Gln), which has not been observed in any other known host cell protease (7-11). Peptidomimetics with Michael acceptor warheads permanently disable the protease by covalent binding to its catalytic site (12, 13). The peptidomimetic rupintrivir (Pfizer AG7088; Fig. 1) effectively inhibits RV and enterovirus replication in vitro but largely failed to fulfill its promise in clinical trials (14-17).Comparison of the known crystal structures of enterovirus 3C pro s revealed that the enterovirus 68 (EV68) 3C pro can be considered an intermediate between the proteases of RV02 and poliovirus (18). Therefore, it was selected for the design of broadspectrum enterovirus 3C pro inhibitors, yielding SG85, a peptidic ␣,␤-unsaturated ethyl ester with Michael acceptor properties, as the most promising candidate. SG85 is an efficient inhibitor of EV68 3Cpro and inhibits the replication of enteroviruses in cellbased assays (18,19).We demonstrate here that SG85 effectively inhibits the replication (in multicycle virus-cell-based cytopathic effect [CPE] reduction assays [20]) of 14 RV serotypes that are representative of RV-A and -B (Table 1). Median 50% effective concentrations (EC 50 s) of 0.04 Ϯ 0.02 M and 0.02 Ϯ 0.01 M were obtained against RV-A and RV-B, respectively. The 3C pro inhibitor rupintrivir (Axon Medchem, The Netherlands) was, on average, 4-fold more active (Table 1), but this varied with the type (e.g., equipotent activity against RV63 and 14-fold more potent against RV02). Akin to rupint...

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confidence: 50%

The Enterovirus 3C Protease Inhibitor SG85 Efficiently Blocks Rhinovirus Replication and Is Not Cross-Resistant with Rupintrivir

Lacroix

George

Leyssen

et al. 2015

“…A previous study identified several mutations in human rhinoviruses (HRVs) that confer resistance to treatment with rupintrivir, including the T129/130, T131, T143, N165, N130, and L136 mutations (39). By checking the crystal structure of EV71 3C pro (40), the N69S substitution unexpectedly appears to be close to but outside the active site of EV71 3C pro (Fig.…”

Section: Discussionmentioning

confidence: 99%

Peptidyl Aldehyde NK-1.8k Suppresses Enterovirus 71 and Enterovirus 68 Infection by Targeting Protease 3C

Wang

Yang

Zhai

et al. 2015

e Enterovirus (EV) is one of the major causative agents of hand, foot, and mouth disease in the Pacific-Asia region. In particular, EV71 causes severe central nervous system infections, and the fatality rates from EV71 infection are high. Moreover, an outbreak of respiratory illnesses caused by an emerging EV, EV68, recently occurred among over 1,000 young children in the United States and was also associated with neurological infections. Although enterovirus has emerged as a considerable global public health threat, no antiviral drug for clinical use is available. In the present work, we screened our compound library for agents targeting viral protease and identified a peptidyl aldehyde, NK-1.8k, that inhibits the proliferation of different EV71 strains and one EV68 strain and that had a 50% effective concentration of 90 nM. Low cytotoxicity (50% cytotoxic concentration, >200 M) indicated a high selective index of over 2,000. We further characterized a single amino acid substitution inside protease 3C (3C pro ), N69S, which conferred EV71 resistance to NK-1.8k, possibly by increasing the flexibility of the substrate binding pocket of 3Cpro . The combination of NK-1.8k and an EV71 RNA-dependent RNA polymerase inhibitor or entry inhibitor exhibited a strong synergistic anti-EV71 effect. Our findings suggest that NK-1.8k could potentially be developed for anti-EV therapy.

“…As rupintrivir was developed as an inhibitor of the 3C protease of human rhinoviruses (7,8), we assessed its potential of inhibiting the 3C-like protease of Norwalk virus by molecular modeling, based on the crystal structure of the free enzyme (11). The active site of the Norwalk virus protease bears similarities to that of the enterovirus 3C proteases (30,31); the catalytic triad is formed by residues Cys139, His30, and Glu54 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Rupintrivir was initially developed as an intranasal product to treat rhinovirus infections (7,8). Given its poor oral bioavailability, the molecule does not have the ideal pharmacokinetics profile for a prophylactic approach to prevent norovirus infections.…”

Section: Resultsmentioning

confidence: 99%

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

Rocha‐Pereira

Nascimento

et al. 2014

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...