Article

DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication by Preventing a Cyclophilin A Induced Cis-Trans Isomerisation in Domain II of NS5A

Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium.
PLoS ONE (Impact Factor: 3.23). 10/2010; 5(10):e13687. DOI: 10.1371/journal.pone.0013687
Source: PubMed

ABSTRACT

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025(res) replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025(res) replicons. Unlike WT, DEB025(res) replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025(res) replicon. NMR titration experiments with peptides derived from the WT or the DEB025(res) domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

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    • "By blocking the interaction between CypA and NS5A, the cyclophilin inhibitors (alisporivir, NIM811, SCY-635 or sanglifehrins) abrogate HCV replication. Based on [44] [45] [55] [57] [59]. alisporivir was able to correct/prevent HCV protein-mediated mitochondrial dysfunction by blocking CypD, and obtaining an associated effect on MPTP [70]. "
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    ABSTRACT: The cyclophilins are a group of proteins with peptidyl-prolyl isomerase enzymatic activity, localised in different cellular compartments and involved in a variety of functions related to cell metabolism and energy homeostasis, having enhanced expression in inflammation or malignancy. Cyclophilin A (CypA), the most abundantly expressed cyclophilin, is present mainly in the cytoplasm and is a host factor involved in the life cycle of multiple viruses. The extracellular fractions of CypA and CypB are potent pro-inflammatory mediators. CypD, located in mitochondria, is a key regulator of mitochondrial permeability transition pores, and is critical for necrotic cell death. Cyclosporines are the prototype cyclophilin inhibitors. Cyclic peptides, which bind and inhibit cyclophilins without having immunosuppressive properties, have been generated by chemical modifications of cyclosporin A. In addition, cyclophilin inhibitors that are structurally different from cyclosporines have been synthesized. The involvement of cyclophilins in the pathogenesis of different liver diseases has been established using both in vitro and in vivo investigations, thus indicating that cyclophilin inhibition may be of therapeutic benefit. This review summarises the evidence for potential therapeutic applications of non-immunosuppressive cyclophilin inhibitors, alone or in combination with other agents, in virus-induced liver diseases like hepatitis C, B or Delta, liver inflammation and fibrosis, acetaminophen-induced liver toxicity and hepatocellular carcinoma.
    Full-text · Article · Nov 2014 · Journal of Hepatology
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    • "By blocking the interaction between CypA and NS5A, the cyclophilin inhibitors (alisporivir, NIM811, SCY-635 or sanglifehrins) abrogate HCV replication. Based on [44] [45] [55] [57] [59]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The cyclophilins are a group of proteins with peptidyl-prolyl isomerase enzymatic activity, localised in different cellular compartments and involved in a variety of functions related to cell metabolism, energy homeostasis, having enhanced expression in inflammation or malignancy. Cyclophilin A (CypA), the most abundantly expressed cyclophilin, is present mainly in the cytoplasm and is a host factor involved in the life cycle of multiple viruses. The extracellular fractions of CypA and CypB are potent pro-inflammatory mediators. CypD is located in mitochondria, it is a key regulator of mitochondrial permeability transition pores, and is critical for necrotic cell death. Cyclosporines are the prototype cyclophilin inhibitors. Cyclic peptides, which bind and inhibit cyclophilins without having immunosuppressive properties, have been generated by chemical modifications of cyclosporin A. In addition, cyclophilin inhibitors that are structurally different from cyclosporines have been synthesized. The involvement of cyclophilins in the pathogenesis of different liver diseases has been established using both in vitro and in vivo investigations, thus indicating that cyclophilin inhibition may be of therapeutic benefit. This review summarises the evidence for potential therapeutic applications of non-immunosuppressive cyclophilin inhibitors, alone or in combination with other agents, in virus-induced liver diseases like hepatitis C, B or Delta, liver inflammation and fibrosis, acetaminophen-induced liver toxicity and hepatocellular carcinoma.
    Full-text · Article · Jul 2014
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    • "Several residues essential for replication map to CypA interaction sites As mentioned above, this region of NS5A has been shown to interact with the cellular PPIase CypA. This interaction and the PPIase activity of CypA have been shown to be of critical importance for virus replication, independent of the effects of CypA on the calcineurin pathway (Coelmont et al., 2010). Residues A311–N318, T334, G337 and C338 have been shown by NMR to undergo chemical shifts upon incubation with CypA (Hanoulle et al., 2009a; Yang et al., 2010), suggestive of a direct interaction. "
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    ABSTRACT: The NS5A protein of hepatitis C virus plays roles in both virus genome replication and the assembly of infectious virus particles. NS5A comprises three domains, separated by low complexity sequences. Whilst the function of domain I appears to be predominantly involved with genome replication, the roles of domains II and III are less well defined. It has previously been reported that a deletion spanning the majority of domain II but retaining the C-terminal 35 residues had no effect on virus production, however deletion of the entire domain II abrogated genome replication pointing to a key role for the C-terminus of this domain. Recent work has also highlighted this region as the potential binding site of the host factor cyclophilin A (CypA). To define this requirement for replication in more detail, and to investigate the involvement of CypA, we have conducted a mutagenic study of the C-terminal 30 residues of domain II within the context of both the infectious JFH-1 virus and the JFH-1 derived subgenomic replicon. We show that 12 of these residues are absolutely required for virus genome replication, the remainder either have no phenotype, or exhibit a partial reduction in genome replication. There was an absolute correlation between the data sets for virus and subgenomic replicon, indicating that this region is solely involved in the process of genome replication. Comparison of our data with the previously published analysis of the same region in genotype 1b revealed some important differences between the two genotypes of HCV.
    Full-text · Article · Jan 2013 · Journal of General Virology
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