Peptide-based inhibitors of the hepatitis C virus serine protease

Llinàs‐Brunet, Montse; Bailey, Murray; Fazal, Gulrez; Goulet, Sylvie; Halmos, Ted; LaPlante, Steven R.; Maurice, Roger; Poirier, Martin; Poupart, Marc‐André; Thibeault, Diane; Wernic, Dominik; Lamarre, Daniel

doi:10.1016/s0960-894x(98)00299-6

Cited by 169 publications

(121 citation statements)

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“…A breakthrough in this arena was the unusual observation that N-terminal cleavage products (i.e., the nonprime side) bind NS3-4A and competitively block substrate recognition. 22,23 Combinatorial chemistry was then used to further optimize and derive increasingly potent inhibitors, such as compound 1, which was used in this study.…”

Section: Commentsmentioning

confidence: 99%

Evasive Maneuvers by Hepatitits C Virus

Lindenbach

Rice

2003

Hepatology

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The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use of HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

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Section: Commentsmentioning

confidence: 99%

Evasive Maneuvers by Hepatitits C Virus

Lindenbach

Rice

2003

Hepatology

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“…Early drug design efforts were hampered by the relatively shallow, featureless architecture of the protease active site. The eventual observation of N-terminal product inhibition served as a stepping stone for the discovery of more potent peptidomimetic inhibitors (9,10). Over the past decade, pharmaceutical companies have further developed these lead compounds.…”

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confidence: 99%

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Romano

Ali

Royer

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

182

245

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Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates.drug design | hepatitis C | substrate envelope D rug resistance is a major obstacle in the treatment of quickly evolving diseases. Hepatitis C virus (HCV) is a genetically diverse Hepacivirus of the Flaviviridae family infecting an estimated 180 million people worldwide (1). The viral RNA genome is translated as a single polyprotein and subsequently processed by host-cell and viral proteases into structural (C, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (2). The viral RNA-dependent RNA polymerase, NS5B, is inherently inaccurate and misincorporation of bases accounts for a very high mutation rate (3). While some mutations are neutral, others will alter the viability of the virus and propagate with varying efficiencies in each patient. Thus HCV infected individuals will develop a heterogeneous population of virus variants known as quasispecies (4). As patients begin treatment, the selective pressures of antiviral drugs will favor drug resistant variants (5). Therefore, an inhibitor must not only recognize one protein variant, but an ensemble of related enzymes. A detailed understanding of the atomic mechanisms of resistance is essential to effectively combat drug resistance against HCV antivirals.The essential HCV NS3/4A protease is an attractive therapeutic target responsible for cleaving at least four sites along the viral polyprotein. These sites share little sequence homology except for an acid at position P6, Cys or Thr at P1, and Ser or Ala at P1′ (Table S1). The first cleavage event at the 3-4A junction occurs in cis as a unimolecular process, while the remaining substrates are processed bimolecularly in trans. The NS3/4A protease also cleaves the human cellular targets TRIF and MAVS, which confounds the innate immune response to viral infection (6-8).Early drug design efforts were hampered by the relatively shallow, featureless architecture of the protease active site. The eventual observation of N-terminal product inhibition served as a stepping stone f...

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“…We targeted the serine protease activity responsible for viral maturation (26), which has been shown to be essential for HCV replication in vivo (11). Inhibitors of the HCV serine protease were designed through a substrate-based approach (15)(16)(17) which led to the discovery of the macrocyclic tripeptide inhibitor BILN 2061 (12). BILN 2061 is a potent and competitive inhibitor of the NS3 proteases of genotypes 1a and 1b, with inhibition constant (K i In this study, different natural variants of HCV NS3 protease are evaluated with regard to their kinetic properties and their sensitivity to BILN 2061.…”

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Sensitivity of NS3 Serine Proteases from Hepatitis C Virus Genotypes 2 and 3 to the Inhibitor BILN 2061

Thibeault

Bousquet

Gingras

et al. 2004

J Virol

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Hepatitis C virus (HCV) displays a high degree of genetic variability. Six genotypes and more than 50 subtypes have been identified to date. In this report, kinetic profiles were determined for NS3 proteases of genotypes 1a, 1b, 2ac, 2b, and 3a, revealing no major differences in activity. In vitro sensitivity studies with BILN 2061 showed a decrease in affinity for proteases of genotypes 2 and 3 (K i , 80 to 90 nM) compared to genotype 1 enzymes (K i , 1.5 nM). To understand the reduced sensitivity of genotypes 2 and 3 to BILN 2061, active-site residues in the proximity of the inhibitor binding site were replaced in the genotype-1b enzyme with the corresponding genotype-2b or -3a residues. The replacement of five residues at positions 78, 79, 80, 122, and 132 accounted for most of the reduced sensitivity of genotype 2b, while replacement of residue 168 alone could account for the reduced sensitivity of genotype 3a. BILN 2061 remains a potent inhibitor of these nongenotype-1 NS3-NS4A proteins, with K i values below 100 nM. This in vitro potency, in conjunction with the good pharmacokinetic data reported for humans, suggests that there is potential for BILN 2061 as an antiviral agent for individuals infected with non-genotype-1 HCV.According to the latest World Health Organization estimates, more than 170 million individuals may be infected with hepatitis C virus (HCV). Chronic infection, observed in about 85% of cases, could lead to progressive hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (7). HCV belongs to the Flaviviridae family. Its positive-strand RNA genome contains 9,600 nucleotides and encodes a ϳ3,100-amino-acid protein that is posttranslationally processed by host-and virally encoded proteases into structural (C, E1, E2, p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (23). The nonstructural (NS) proteins include enzymes necessary for protein maturation (NS2/3 and NS3 proteases) and viral replication (NS3 helicase/nucleoside triphosphatase and NS5B RNA polymerase).The high rate of viral production linked to the low fidelity of the RNA polymerases (5, 6) leads to genetic heterogeneity of HCV in infected patients (20). Natural variants of HCV are currently classified into 6 genotypes and more than 50 subtypes (25). The genotypes differ by as much as 34% in their nucleotide sequences, resulting in approximately 30% amino acid sequence divergence between the encoded polyproteins, while subtypes can differ by as much as 23% of their nucleotide sequence. The degree of sequence variability also varies for the different subgenomic regions. For example, the core and the 3Ј and 5Ј nontranslated regions are more conserved, whereas the envelope region displays more variability (24, 31). Sequences coding for the NS3 protease domain and the NS5B polymerase show degrees of variability comparable to that for the complete genome.The HCV infections most frequently encountered are caused by genotypes 1, 2, and 3 (18). In Europe, Japan, and the United States, more than 70% of the HCV-posi...

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Peptide-based inhibitors of the hepatitis C virus serine protease

Cited by 169 publications

References 18 publications

Evasive Maneuvers by Hepatitits C Virus

Evasive Maneuvers by Hepatitits C Virus

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Sensitivity of NS3 Serine Proteases from Hepatitis C Virus Genotypes 2 and 3 to the Inhibitor BILN 2061

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