Resistance Mutations Define Specific Antiviral Effects for Inhibitors of the Hepatitis C Virus p7 Ion Channel

Section of Oncology and Clinical Research, Leeds Institute of Molecular Medicine, St. James's University Hospital, Leeds, United Kingdom.
Hepatology (Impact Factor: 11.06). 07/2011; 54(1):79-90. DOI: 10.1002/hep.24371
Source: PubMed


The hepatitis C virus (HCV) p7 ion channel plays a critical role during infectious virus production and represents an important new therapeutic target. Its activity is blocked by structurally distinct classes of small molecules, with sensitivity varying between isolate p7 sequences. Although this is indicative of specific protein-drug interactions, a lack of high-resolution structural information has precluded the identification of inhibitor binding sites, and their modes of action remain undefined. Furthermore, a lack of clinical efficacy for existing p7 inhibitors has cast doubt over their specific antiviral effects. We identified specific resistance mutations that define the mode of action for two classes of p7 inhibitor: adamantanes and alkylated imino sugars (IS). Adamantane resistance was mediated by an L20F mutation, which has been documented in clinical trials. Molecular modeling revealed that L20 resided within a membrane-exposed binding pocket, where drug binding prevented low pH-mediated channel opening. The peripheral binding pocket was further validated by a panel of adamantane derivatives as well as a bespoke molecule designed to bind the region with high affinity. By contrast, an F25A polymorphism found in genotype 3a HCV conferred IS resistance and confirmed that these compounds intercalate between p7 protomers, preventing channel oligomerization. Neither resistance mutation significantly reduced viral fitness in culture, consistent with a low genetic barrier to resistance occurring in vivo. Furthermore, no cross-resistance was observed for the mutant phenotypes, and the two inhibitor classes showed additive effects against wild-type HCV. CONCLUSION: These observations support the notion that p7 inhibitor combinations could be a useful addition to future HCV-specific therapies.

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Available from: Stephen d. c. Griffin, Apr 17, 2015
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    • "Bundle models of p7 of GT 1a were generated using the abovementioned protocols [32] [33]. The hexameric bundle of GT 1b was generated using a monomeric NMR structure [40] and positioning six copies around a pseudo sixfold symmetry axis. The bundle of GT 5a derives from NMR spectroscopic data with the protein in an oligomerized (hexamer) form [29]. "
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    ABSTRACT: The 63 amino acid polytopic membrane protein, p7, encoded by hepatitis C virus (HCV) is involved in the modulation of electrochemical gradients across membranes within infected cells. Structural information relating to p7 from multiple genotypes has been generated in silico (e.g. genotype (GT) 1a), as well as recently reported experimental monomeric and hexameric structures (GTs 1b and 5a, respectively). However, sequence diversity and structural differences mean that comparison of their channel gating behaviour has not thus far been simulated. Here, a molecular dynamics model of the monomeric GT 1a protein is optimized and assembled into a hexameric bundle for comparison with both the 5a hexamer structure and another hexameric bundle generated using the 1b monomer structure. All bundles tend to turn into a compact structure during MD simulations (Gromos96 (ffG45a3)) in hydrated lipid bilayers, as well as when simulated at 'low pH', which may trigger channel opening according to some functional studies. Both GT 1a and 1b channel models are gated via movement of the parallel aligned helices, yet the scenario for the GT 5a protein is more complex, with a short N-terminal helix being involved. However, all bundles display pulsatile dynamics identified by monitoring water dynamics within the pore. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Mar 2015 · Biochimica et Biophysica Acta (BBA) - Biomembranes
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    • "These drugs are developed to target soluble proteins. Promising small molecule drugs are on its way targeting a membrane protein encoded by HCV, namely p7, a channel forming protein (Pavlovi c et al., 2003; Luscombe et al., 2010; Foster et al., 2011). Viral channel forming proteins (VCPs) are found in a series of viruses (Fischer and Sansom, 2002; Gonzales and Carrasco, 2003; Wang et al., 2010; Nieva et al., 2012; Fischer et al., 2012; OuYang and Chou, 2014). "
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    ABSTRACT: Protein p7 of HCV is a 63 amino acid channel forming membrane protein essential for the progression of viral infection. With this momentousness, p7 emerges as an important target for antiviral therapy. A series of small molecule drugs, such as amantadine, rimantadine, amiloride, hexamethylene amiloride, NN-DNJ and BIT225 have been found to affect the channel activity. These compounds are docked against monomeric and hexameric structures of p7 taken at various time steps from a molecular dynamics simulation of the protein embedded in a hydrated lipid bilayer. The energetics of binding identifies the guanidine based ligands as the most potent ligands. The adamantanes and NN-DNJ show weaker binding energies. The lowest energy poses are those at the site of the loop region for the monomer and hexamer. For the latter, the poses show a tendency of the ligand to face the lumen of the pore. The mode of binding is that of a balance between hydrophobic interactions and hydrogen bond formation with backbone atoms of the protein.
    Full-text · Article · Nov 2014 · Computational Biology and Chemistry
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    • "These proteins are known to modulate membrane permeability to support virus entry, assembly or release (viroporins reviewed in (Nieva et al., 2012)). Depending on the genotype, p7 activity can be hindered by multiple ion-channel blockers, such as amantadine, rimantadine and iminosugar derivatives (Griffin et al., 2003, 2008; Pavlovic et al., 2003; Steinmann et al., 2007; Foster et al., 2011). In addition, p7 is dispensable for viral RNA replication, but is important for in vivo infectivity in the chimpanzee model, and for production of infectious HCVcc (Jones et al., 2007; Steinmann et al., 2007; Sakai et al., 2003; Brohm et al., 2009; Atoom et al., 2013). "
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    ABSTRACT: Hepatitis C virus (HCV) is a major global health burden with 2-3% of the world׳s population being chronically infected. Persistent infection can lead to cirrhosis and hepatocellular carcinoma. Recently available treatment options show enhanced efficacy of virus clearance, but are associated with resistance and significant side effects. This warrants further research into the basic understanding of viral proteins and their pathophysiology. The p7 protein of HCV is an integral membrane protein that forms an ion-channel. The role of p7 in the HCV life cycle is presently uncertain, but most of the research performed to date highlights its role in the virus assembly process. The aim of this review is to provide an overview of the literature investigating p7, its structural and functional details, and to summarize the developments to date regarding potential anti-p7 compounds. A better understanding of this protein may lead to development of a new and effective therapy.
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