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.

FIG. 1.

Interactions between HRV 2 3C protease and rupintrivir. All amino acid residues having side chains interacting directly with the bound inhibitor are included (Phe 25, His 40, Glu 71, Ile 125, Leu 127, Ser 128, Asn 130, Thr 142, Cys 147, His 161, Gly 163, Gly 164, Asn 165, and Phe 170). Amino acid residues (Ser 144, Gly 145, and Val 162) having only polypeptide main chain interactions are shown. Other amino acid residues (Asn 22, Gly 23, Lys 24, Leu 126, Lys 143, and Tyr 146) having only polypeptide main chain interactions are not shown. Hydrogen bonds are indicated by dashed lines. The covalent bond between the sulfur γ atom (SG) of Cys 147 and carbon 19 atom (C19) of the inhibitor is shown by a solid line. The drawing was prepared by using the program LIGPLOT (60).

FIG. 2.

Amino acid sequences of 3C proteases from HRV and HEV (n = 58) including PV (poliovirus), CVB, coxsackievirus A (CAV), EV (echovirus serotypes 6, 9, 11, 12, or 30 or enterovirus serotypes 70 or 71), HRV clinical isolates (designated 100, 103, 108, 113, 119, 138, J, and Q), and HRV field isolates (M24, M36, and MR). Dashes represent homologous sequences. Fourteen amino acids of 3C protease that have side chain interactions with rupintrivir, including the catalytic triad (His 40, Cys 147, and Glu 71), are boxed in gray.

FIG. 2.

Amino acid sequences of 3C proteases from HRV and HEV (n = 58) including PV (poliovirus), CVB, coxsackievirus A (CAV), EV (echovirus serotypes 6, 9, 11, 12, or 30 or enterovirus serotypes 70 or 71), HRV clinical isolates (designated 100, 103, 108, 113, 119, 138, J, and Q), and HRV field isolates (M24, M36, and MR). Dashes represent homologous sequences. Fourteen amino acids of 3C protease that have side chain interactions with rupintrivir, including the catalytic triad (His 40, Cys 147, and Glu 71), are boxed in gray.

FIG. 3.

Phylogenetic tree of HRV and HEV based on homology of 3C protease amino acid residues was generated by MegAlign by using Clustal W analysis as described in Materials and Methods. The length of each pair of branches represents the distance between the sequences, and the horizontal axis indicates the approximate percent dissimilarities of the 3C protease amino acid sequences.