Current therapy for patients with chronic HCV infection includes high doses of IFN in combination with ribavirin. However, success rates remain low (around 50% of treated patients depending on genotype). Furthermore, the IFN/ribavirin treatment carries a significant risk of serious side effects. Thus, the development of alternative treatments is essential. The immunosuppressive drug cyclosporine A (CsA) was reported to be clinically effective against HCV infection. Moreover, recent studies provided evidence that non- immunosuppressive CsA analogs such as NIM811, Debio 025 and SCY-635 inhibit HCV RNA replication both in vitro and in vivo in an IFN-independent manner. A growing body of evidence suggests that cyclophilins (Cyps) are important for the HCV life cycle, suggesting that Cyp inhibitors (CsA, Debio 025, NIM811 or SCY- 635), by acting on intracellular Cyps, block HCV replication. Three independent studies recently demonstrated that HCV highly relies on cyclophilin A (CypA) to replicate in human hepatocytes. We showed that CypA, but not CypB, CypC and CypD, is critical for HCV replication. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which control the isomerase activity of CypA, is critical for HCV replication. We obtained several lines of evidence that a population of CypA molecules resides in a protease- resistant membrane compartment similar to that where HCV replicates. We also found that the association of CypA with this compartment does not depend on the presence of HCV proteins. Importantly, we showed that Cyp inhibitors deplete this membrane compartment of CypA. The depletion of CypA from the protected ER spherules where HCV replication occurs, may provide the first hint for the mechanism of antiviral action of Cyp inhibitors. However, the molecular requirements for CypA in HCV replication as well as the antiviral mechanisms of action of this novel class of potent anti-HCV agents - the Cyp inhibitors - are completely obscure. By selecting Cyp inhibitor-resistant HCV mutant's in vitro, studies mapped mutations into two nonstructural (NS) proteins - NS5A and NS5B. Altogether these data suggest the existence of a relationship between CypA, NS5A and NS5B that is critical for HCV replication. To test this hypothesis, we propose in this application to conduct a set of experiments aimed at fully characterizing CypA-NS5A-NS5B interactions at a molecular level;analyzing the development of HCV resistance to Cyp inhibitors;and investigating the effect of Cyp inhibitors on the composition and polymerase activities of HCV replication complexes. The ultimate goal of this application is to improve our understanding of the role of CypA in the HCV life cycle and of the in vitro and in vivo block mediated by this novel class of potent anti-HCV agents - the Cyp inhibitors.

Public Health Relevance

An estimated 170 million people worldwide are chronically infected with Hepatitis C virus. 10-20% of these will develop cirrhosis and 1-5% will develop hepatocellular carcinoma. The only currently approved therapy is weekly injection with pegylated interferon and daily oral ribavirin for 6-12 months. The therapy is associated with severe side effects and results in sustained viral clearance in only 50% of all patients. There is therefore a high need to develop new therapies with better efficacy and tolerability. By demonstrating that cyclophilin (Cyp) inhibitors have potent efficacy against HCV, our recent clinical study opens a new line of opportunity to eradicate this prime human threat. The current proposal outlines a comprehensive series of experiments aimed at determining the mechanism of action of Cyp inhibitors that should lead to the identification of new targets for inhibition of HCV. The study of the roles of Cyp - the intracellular targets of the Cyp inhibitors - in HCV replication is novel and should lead to the identification of new host and viral targets for the development of innovative anti-HCV therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI087746-05
Application #
8602812
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Koshy, Rajen
Project Start
2010-01-15
Project End
2014-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
5
Fiscal Year
2014
Total Cost
$423,002
Indirect Cost
$200,252
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Gawlik, Katarzyna; Baugh, James; Chatterji, Udayan et al. (2014) HCV core residues critical for infectivity are also involved in core-NS5A complex formation. PLoS One 9:e88866
Chatterji, Udayan; Garcia-Rivera, Jose A; Baugh, James et al. (2014) The combination of alisporivir plus an NS5A inhibitor provides additive to synergistic anti-hepatitis C virus activity without detectable cross-resistance. Antimicrob Agents Chemother 58:3327-34
Lim, Precious J; Gallay, Philippe A (2014) Hepatitis C NS5A protein: two drug targets within the same protein with different mechanisms of resistance. Curr Opin Virol 8:30-7
Gawlik, Katarzyna; Gallay, Philippe A (2014) HCV core protein and virus assembly: what we know without structures. Immunol Res 60:1-10
Baugh, James M; Garcia-Rivera, Jose A; Gallay, Philippe A (2013) Host-targeting agents in the treatment of hepatitis C: a beginning and an end? Antiviral Res 100:555-61
Bobardt, Michael; Hopkins, Sam; Baugh, James et al. (2013) HCV NS5A and IRF9 compete for CypA binding. J Hepatol 58:16-23
Gallay, Philippe A (2012) Cyclophilin inhibitors: a novel class of promising host-targeting anti-HCV agents. Immunol Res 52:200-10
Coelmont, Lotte; Hanoulle, Xavier; Chatterji, Udayan et al. (2010) DEB025 (Alisporivir) inhibits hepatitis C virus replication by preventing a cyclophilin A induced cis-trans isomerisation in domain II of NS5A. PLoS One 5:e13687
Fischer, Gunter; Gallay, Philippe; Hopkins, Sam (2010) Cyclophilin inhibitors for the treatment of HCV infection. Curr Opin Investig Drugs 11:911-8
Chatterji, Udayan; Lim, Precious; Bobardt, Michael D et al. (2010) HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors. J Hepatol 53:50-6

Showing the most recent 10 out of 11 publications