Macrocyclic Inhibitors of the NS3 Protease as Potential Therapeutic Agents of Hepatitis C Virus Infection

Tsantrizos, Youla S.; Bolger, Gordon T.; Bonneau, Pierre; Cameron, Dale R.; Goudreau, Nathalie; Kukolj, George; LaPlante, Steven R.; Llinàs‐Brunet, Montse; Nar, Herbert; Lamarre, Daniel

doi:10.1002/anie.200390347

Cited by 169 publications

(131 citation statements)

References 26 publications

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“…Although the binding of the PI to NS3 in the polyprotein form could explain the blockage of downstream NS5A hyperphosphorylation, we, and others, do not have any direct evidence that NS3 PIs bind NS3 in the polyprotein form. Given that (i) the NS3 PI used in this study is an active-site inhibitor and the cocrystallization of NS3 and inhibitors of this chemotype has been achieved (35) and (ii) data suggest that p56 is the precursor to p58 (15,23), this would strongly support the hypothesis that the NS3 PI binds to NS3 and alters NS5A p58 formation within the same replication complex. This is consistent with results from the pulse-chase experiment showing that p58 production is still blocked by the addition of PI even when p56 is already made, suggesting that p58 is being formed from p56 in the context of a multiprotein complex, and this conversion can be disrupted by the binding of the NS3 inhibitor.…”

Section: Discussionmentioning

confidence: 56%

Identification of Hepatitis C Virus NS5A Inhibitors

Lemm¹,

O’Boyle²,

Liu³

et al. 2010

J Virol

179

198

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Using a cell-based replicon screen, we identified a class of compounds with a thiazolidinone core structure as inhibitors of hepatitis C virus (HCV) replication. The concentration of one such compound, BMS-824, that resulted in a 50% inhibition of HCV replicon replication was ϳ5 nM, with a therapeutic index of >10,000. The compound showed good specificity for HCV, as it was not active against several other RNA and DNA viruses. Replicon cells resistant to BMS-824 were isolated, and mutations were identified. A combination of amino acid substitutions of leucine to valine at residue 31 (L31V) and glutamine to leucine at residue 54 (Q54L) in NS5A conferred resistance to this chemotype, as did a single substitution of tyrosine to histidine at amino acid 93 (Y93H) in NS5A. To further explore the region(s) of NS5A involved in inhibitor sensitivity, genotype-specific NS5A inhibitors were used to evaluate a series of genotype 1a/1b hybrid replicons. Our results showed that, consistent with resistance mapping, the inhibitor sensitivity domain also mapped to the N terminus of NS5A, but it could be distinguished from the key resistance sites. In addition, we demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. Clinical proof of concept has recently been achieved with derivatives of these NS5A inhibitors, indicating that small molecules targeting a nontraditional viral protein like NS5A, without any known enzymatic activity, can also have profound antiviral effects on HCV-infected subjects.Hepatitis C virus (HCV) is the major causative agent for non-A, non-B hepatitis worldwide, which affects more than 3% of the world population. HCV establishes chronic infections in a large percentage of infected individuals, increasing the risk for developing liver cirrhosis and, in some cases, hepatocellular carcinoma. Although the current standard of care for HCV infection involves the use of PEGylated interferon and ribavirin, a large proportion of patients fail to respond to this therapy, and treatment is associated with frequent and sometimes serious side effects (9). Given the limited efficacy of the current therapy, the development of safer and more effective therapies is of tremendous importance.HCV is a positive-strand RNA virus belonging to the family Flaviviridae. The HCV genome consists of a ϳ9.6-kb RNA with a large open reading frame encoding a polyprotein of ϳ3,010 amino acids. The polyprotein is cleaved co-and posttranslationally by both cellular and viral proteases into at least 10 different products (10, 11). The viral proteins required for RNA replication include NS3, NS4A, NS4B, NS5A, and NS5B (4, 19). NS3 consists of an amino-terminal protease domain required for the cleavage of the remaining nonstructural proteins and a carboxyl-terminal helicase/NTPase domain (8,11,30). NS4A serves as a cofactor for NS3 protease and helicase activities (8). NS4B is a...

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Section: Discussionmentioning

confidence: 56%

Identification of Hepatitis C Virus NS5A Inhibitors

Lemm¹,

O’Boyle²,

Liu³

et al. 2010

J Virol

179

198

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“…2A) by using the crystal structure of an inhibitor-enzyme complex previously reported for an analog of BILN 2061 (Fig. 2B) (29). This analog displayed affinities similar to those of BILN 2061 for the NS3-NS4A proteins of genotypes 1, 2, and 3 (unpublished data).…”

Section: Resultsmentioning

confidence: 62%

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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“…In its new orientation, the Arg155 side chain is still hydrogen bonded with Asp168 and newly bonded to the carbonyl oxygen of Gln80 and acts as a binding platform for the inhibitor isoindoline moiety. Corresponding Arg155 rotamers have been reported for NS3/ 4A protease in complex with TMC435 (11), , and BILN-261 (24). It is interesting to note that the P2-extended isoindoline portion in ITMN-191, identical to inhibitor 1, binds with a slight distal-shift of about 0.75 Å in the absence of the helicase.…”

Section: Interactions With Protease Residues and Comparison To Hcv Nsmentioning

confidence: 53%

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

Schiering

D’Arcy

Villard

et al. 2011

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

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Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C infection leads to serious liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV NS3 protein is essential for viral polyprotein processing and RNA replication and hence viral replication. It is composed of an N-terminal serine protease domain and a C-terminal helicase/NTPase domain. For full activity, the protease requires the NS4A protein as a cofactor. HCV NS3/4A protease is a prime target for developing direct-acting antiviral agents. First-generation NS3/4A protease inhibitors have recently been introduced into clinical practice, markedly changing HCV treatment options. To date, crystal structures of HCV NS3/4A protease inhibitors have only been reported in complex with the protease domain alone. Here, we present a unique structure of an inhibitor bound to the full-length, bifunctional protease-helicase NS3/4A and show that parts of the P4 capping and P2 moieties of the inhibitor interact with both protease and helicase residues. The structure sheds light on inhibitor binding to the more physiologically relevant form of the enzyme and supports exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3/4A protease inhibitors. In addition, small angle X-ray scattering confirmed the observed proteasehelicase domain assembly in solution.structure-based drug design | X-ray structure | solution scattering | medicinal chemistry C hronic hepatitis C virus (HCV) infection affects more than 3% of the world's population and is a leading cause of chronic liver diseases (1). Therapeutic options have been suboptimal, especially for HCV genotype 1, the most prevalent genotype in developed countries. Recently, the addition of direct-acting antiviral agents (DAAs) to the previous standard of care (combination therapy with pegylated interferon and ribavirin) have demonstrated considerable improvement in sustained virological response rates in patients infected with HCV genotype 1 (2). With the first DAAs now having been introduced into clinical practice, it is to be expected that in the near future standard therapy will change to a triple therapy including an HCV NS3/4A protease inhibitor in combination with pegylated interferon and ribavirin.The positive-strand RNA genome of HCV encodes a polyprotein precursor, which is proteolytically processed by host and viral proteases into 10 individual structural and nonstructural (NS) proteins. The viral NS3 protease in complex with the cofactor NS4A cleaves the polyprotein at four junctions releasing the NS proteins 4A, 4B, 5A, and 5B, and therefore is essential for viral replication (3, 4). A central, hydrophobic 14-mer peptide of the 54-residue NS4A, comprising residues 21-32, is necessary and sufficient for maximal activation in vitro (5). NS3/4A has also been shown to cleave cellular proteins leading to inhibition of interferon production, thereby impairin...

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Macrocyclic Inhibitors of the NS3 Protease as Potential Therapeutic Agents of Hepatitis C Virus Infection

Cited by 169 publications

References 26 publications

Identification of Hepatitis C Virus NS5A Inhibitors

Identification of Hepatitis C Virus NS5A Inhibitors

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

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

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