Structure−Activity Study on a Novel Series of Macrocyclic Inhibitors of the Hepatitis C Virus NS3 Protease Leading to the Discovery of BILN 2061

Llinàs‐Brunet, Montse; Bailey, Murray; Bolger, Gordon T.; Brochu, Christian; Faucher, Anne‐Marie; Ferland, Jean Marie; Garneau, Michel; Ghiro, Élise; Gorys, Vida; Grand‐Maître, Chantal; Halmos, Ted; Lapeyre-Paquette, Nicole; Liard, Francine; Poirier, Martin; Rhéaume, Manon; Tsantrizos, Youla S.; Lamarre, Daniel

doi:10.1021/jm0342414

Cited by 191 publications

(96 citation statements)

References 21 publications

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“…1, Shire, 2), the nucleoside 2-(4-amino-pyrrolo [2,3- (Fig. 1, BILN-2061, 5), were synthesized according to procedures described previously (3,6,16,30). Recombinant IFN-␣ was purchased from PBL Biomedical Laboratories, Piscataway, NJ.…”

Section: Methodsmentioning

confidence: 99%

“…These polymerase inhibitors included two nonnucleoside (thiophene amide and diamide derivatives discovered by Shire and Boehringer Ingelheim, respectively) and one nucleoside (published by Merck) inhibitors (6,16,30). The IC 50 values for Shire-thiophene amide, Boehringer Ingelheim-diamide, and Merck-nucleoside were 0.35, 1.97, and 0.27 M, respectively, which are 68-to 492-fold less potent than A-782759 (0.004 M) in this transient-transfection assay (Table 5).…”

Section: A-782759-resistant Mutants Remained Susceptible To Other Clamentioning

confidence: 99%

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Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

Pilot‐Matias

et al. 2005

Antimicrob Agents Chemother

121

112

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Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific antihepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.Hepatitis C virus (HCV) is a leading cause of chronic liver disease, affecting over 4 million Americans and about 170 million people worldwide. The current standard of care for chronic HCV infection involves extended dosing with alpha interferon (IFN-␣) and ribavirin (10,14,18). However, these regimens have limited clinical benefit due to poor tolerability and limited efficacy (only approximately half of genotype 1 HCV-infected individuals have a sustained virological response, whereas the response rate improves significantly [ϳ80%] when genotypes 2 and 3 are treated) (9,11,35). Therefore, development of inhibitors against virally encoded targets is urgently needed.The HCV genome is a 9.6-kb single-stranded RNA of positive polarity encoding a large polyprotein, which is the precursor of at least 10 mature viral proteins: C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (2). The HCV polymerase encoded by nonstructural protein 5B (NS5B) is responsible for HCV RNA-dependent RNA polymerase (RdRp) and terminal transferase activities (4,31,32). The N-terminal domain (approximately 180 amino acids) of NS3 and the small hydrophobic NS4A protein compose a heterodimeric enzyme catalyzing the posttranslational processing of the HCV NS proteins (1, 21). Both NS5B RdRp and NS3 serine protease are believed to be components of the HCV replication complex, responsible for viral RNA replication, and have been shown to be indispensable for HCV replication in chimpanzees (26).To date, a number of distinct classes of NS5B RdRp inhibitors...

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Section: Methodsmentioning

confidence: 99%

Section: A-782759-resistant Mutants Remained Susceptible To Other Clamentioning

confidence: 99%

Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

Pilot‐Matias

et al. 2005

Antimicrob Agents Chemother

121

112

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“…Two of these, telaprevir (VX-950) and boceprevir (SCH503034), possess a ketoamide moiety that reacts with the catalytic serine nucleophile to form a reversible covalent enzyme-inhibitor adduct (20,28,38,52). In contrast, BILN2061, ITMN-191 (R7227), MK7009, and TMC435 are reversible noncovalent inhibitors of NS3/4A, and they all share the feature of a peptidomimetic macrocycle comprised of both backbone and side chain atoms (18,23,24,31,41,46). The structures of various NS3/4A inhibitor complexes show that these inhibitors bind in a similar region of the enzyme active site.…”

mentioning

confidence: 99%

In Vitro Resistance Profile of the Hepatitis C Virus NS3/4A Protease Inhibitor TMC435

Lenz¹,

Verbinnen²,

Lin³

et al. 2010

Antimicrob Agents Chemother

208

218

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TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC 50 s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to ϳ2,000-fold for those with the D168V or D168I mutation, compared to the EC 50 for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC 50 s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (<3-fold change in EC 50 s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.Hepatitis C is a blood-borne infection that can ultimately result in severe liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma (7). The chronic nature of the disease and the significant possibility of long-term liver damage have led to the current global health burden, with an estimated 180 million people being infected, of whom 130 million are chronic hepatitis C virus (HCV) carriers (54).The current standard-of-care therapy for HCV-infected patients consists of a combination of weekly injected pegylated alpha interferon (Peg-IFN-␣) and twice-daily oral ribavirin. Treatment of HCV genotype 1-infected patients with this regimen for 48 weeks has a limited success rate (a 40 to 50% sustained virological response [SVR]) and is associated with a wide range of side effects, including flu-like symptoms, anemia, and depression, leading to treatment discontinuation in a significant proportion of patients (31, 48). Therefore, specifically targeted antiviral therapies for hepatitis C (STAT-C) have been a major focus of drug discovery efforts. Treatments with several NS3/4A protease inhibitors and NS5A and NS5B polymerase inhibitors, alone or in combination with Peg-IFN-␣-ribavirin, have recently shown encouraging results in clinical trials (17,36).HCV NS3 is an essential, bifunctional, multidomain protein that possesses protease and RNA helicase activiti...

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“…These have all been used in product-based inhibitors with a C-terminal carboxylic acid [3,20]. The ACPC group has most successfully been exploited in BILN 2061 [21]. In addition, four different C-terminal groups were evaluated in our study and these can be further divided into three groups on the basis of electrophilicity and acidity: 1) the electrophilic and non-acidic pentafluoroethyl ketone (PFK) group, 2) the electrophilic and acidic ketotetrazole (KT) and ketoacid (KA) groups and 3) the non-electrophilic and acidic carboxylic acid (COOH) group.…”

mentioning

confidence: 99%

Mechanistic studies of electrophilic protease inhibitors of full length hepatic C virus (HCV) NS3

Poliakov

Sandström

Åkerblom

et al. 2007

Journal of Enzyme Inhibition and Medicinal Chemistry

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The inhibition mechanism of electrophilic peptide-based protease inhibitors of full-length hepatitis C virus (HCV) NS3 has been investigated by determining the K i -values for a series of compounds differing in the electrophilicity and acidity of the Cterminal residue at pH-values above and below the pK a of the catalytic histidine (6.85) and at two different ionic strengths. Electrophilic compounds with a pentafluoroethyl ketone group showed stronger inhibition at pH 8 than pH 6, as expected for a mechanism requiring an unprotonated catalytic histidine. However, the difference was only significant at high ionic strength. In contrast, electrophilic compounds with an acidic C-terminal group or a cyclic P1 residue showed a lower inhibitory effect at pH 8 than at pH 6, inconsistent with a mechanism-based inhibition. Moreover, all electrophilic compounds had an unexpectedly strong inhibition at pH 6, when mechanism-based inhibition is unlikely. The results suggest that for some of the electrophilic compounds the reactive group may not be properly positioned in the active site and that binding of these inhibitors is a result of non-covalent interactions. The nature of these interactions is discussed.

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Structure−Activity Study on a Novel Series of Macrocyclic Inhibitors of the Hepatitis C Virus NS3 Protease Leading to the Discovery of BILN 2061

Cited by 191 publications

References 21 publications

Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

In Vitro Resistance Profile of the Hepatitis C Virus NS3/4A Protease Inhibitor TMC435

Mechanistic studies of electrophilic protease inhibitors of full length hepatic C virus (HCV) NS3

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