Conservation of Amino Acids in Human Rhinovirus 3C Protease Correlates with Broad-Spectrum Antiviral Activity of Rupintrivir, a Novel Human Rhinovirus 3C Protease Inhibitor

Abstract

The picornavirus 3C protease is required for the majority of proteolytic cleavages that occur during the viral life cycle. Comparisons of published amino acid sequences from 6 human rhinoviruses (HRV) and 20 human enteroviruses (HEV) show considerable variability in the 3C protease-coding region but strict conservation of the catalytic triad residues. Rupintrivir (formerly AG7088) is an irreversible inhibitor of HRV 3C protease with potent in vitro activity against all HRV serotypes (48 of 48), HEV strains (4 of 4), and untyped HRV field isolates (46 of 46) tested. To better understand the relationship between in vitro antiviral activity and 3C protease-rupintrivir binding interactions, we performed nucleotide sequence analyses on an additional 21 HRV serotypes and 11 HRV clinical isolates. Antiviral activity was also determined for 23 HRV clinical isolates and four additional HEV strains. Sequence comparison of 3C proteases (n ‫؍‬ 58) show that 13 and 11 of the 14 amino acids that are involved in side chain interactions with rupintrivir are strictly conserved among HRV and HEV, respectively. These sequence analyses are consistent with the comparable in vitro antiviral potencies of rupintrivir against all HRV serotypes, HRV isolates, and HEV strains tested (50% effective concentration range, 3 to 183 nM; n ‫؍‬ 125). In summary, the conservation of critical amino acid residues in 3C protease and the observation of potent, broad-spectrum antipicornavirus activity of rupintrivir highlight the advantages of 3C protease as an antiviral target.The picornavirus family consists of over 200 medically important viruses, including human rhinoviruses (HRV) and human enteroviruses (HEV). HRV, comprising over 100 different serotypes, are a major cause of mild upper respiratory infections (reviewed in references 4, 8, and 40). Although HRV infections are usually mild and self-limiting, they can also be associated with exacerbation of disease in individuals with underlying respiratory disorders (4, 48). The HEV include over 70 viruses that are associated with diverse clinical syndromes ranging from mild, self-limiting infections to fulminant and potentially fatal disease (3,52,53). Earlier clinical studies with agents that inhibit virus attachment and/or uncoating (e.g., tremacamra, a soluble intracellular adhesion molecule-1, pirodavir, and pleconaril) and nonspecific antiviral agents such as ␣-2␤ interferon have demonstrated that prevention and early treatment of HRV colds could provide clinical benefit (3, 18-23, 53). Recently, a retrospective analysis of two multicenter clinical trials demonstrated that pleconaril, a capsid-function inhibitor (17), significantly reduced the duration and severity of picornavirus-induced colds (24). To date, however, no antiviral agents have been approved for the prevention or treatment of HRV infection.We have focused our antiviral strategy on the inhibition of 3C protease, a viral enzyme that is absolutely required for the proteolytic cleavage of viral precursor polyproteins to functional p...

In Vitro Antiviral Activity and Single-Dose Pharmacokinetics in Humans of a Novel, Orally Bioavailable Inhibitor of Human Rhinovirus 3C Protease

“…In these latter studies, pleconaril and pirodavir demonstrated a significantly wider range in antiviral potency (1,590-to 2,707-fold range in EC 50 values) and activity against most but not all HRV serotypes tested. The comparable range in antiviral activity of Compound 1 when tested against numerous different picornaviruses is consistent with DNA sequence analyses performed on 3C protease coding gene regions, which demonstrate a significant level of homology in substrate/inhibitor binding regions (3). In mechanism of action studies, Compound 1 was also shown to inhibit the HRV 14 3C-mediated proteolytic cleavage of viral precursor polyproteins into their processed viral products, confirming that the cell-based in vitro antiviral activity of Compound 1 is due to a direct inhibition of HRV 3C protease.…”

“…Moreover, Compound 1 demonstrated comparable antiviral activity against all picornaviruses tested (36-fold range in EC 50 values). While this is similar to that reported for rupintrivir (3,24), it is in contrast to that reported for inhibitors that target capsid-binding, e.g., pleconaril and pirodavir (24). In these latter studies, pleconaril and pirodavir demonstrated a significantly wider range in antiviral potency (1,590-to 2,707-fold range in EC 50 values) and activity against most but not all HRV serotypes tested.…”

“…DNA sequence comparisons among numerous HRV serotypes and several related picornaviruses have demonstrated a significant degree of homology among amino acid residues involved in key 3C protease inhibitor-binding interactions, providing an additional rationale for targeting research activities (3,20,22). Early drug discovery efforts led to the identification of rupintrivir, an intranasally administered, irreversible inhibitor of HRV 3C protease that has demonstrated broad-spectrum, potent in vitro antiviral activity against multiple HRV serotypes, HRV clinical isolates, and related picornaviruses (8,9,14,20,24,33,34).…”

Crystal Structures of Enterovirus 71 3C Protease Complexed with Rupintrivir Reveal the Roles of Catalytically Important Residues

“…Interestingly, recent studies indicated that the inhibitor is also effective against enteroviruses (6,13,24), presumably due to the structural similarity of their 3C proteases. A recent study showed that the interferon (IFN)-mediated antiviral mechanism can be compromised by the proteolytic cleavage of EV71…”

Targeting Norovirus: Strategies for the Discovery of New Antiviral Drugs

“…The inhibitors of the 3C protease of picornavirus, such as ruprintrivir, may potentially inhibit the norovirus protease given the overall similarity between the proteases of these viruses. (Binford et al, 2005;De Palma et al, 2008c;Leyssen et al, 2008).…”