Cyclophilin Inhibitors Research Articles

Cyclophilin Inhibitors as a Novel HCV Therapy

A critical role of Cyclophilins, mostly Cyclophilin A (CyPA), in the replication of HCV is supported by a growing body of in vitro and in vivo evidence. CyPA probably interacts directly with nonstructural protein 5A to exert its effect, through its peptidyl-prolyl isomerase activity, on maintaining the proper structure and function of the HCV replicase. The major proline substrates are located in domain II of NS5A, centered around a “DY” dipeptide motif that regulates CyPA dependence and CsA resistance. Importantly, Cyclosporine A derivatives that lack immunosuppressive function efficiently block the CyPA-NS5A interaction and inhibit HCV in cell culture, an animal model, and human trials. Given the high genetic barrier to development of resistance and the distinctness of their mechanism from that of either the current standard of care or any specifically targeted antiviral therapy for HCV (STAT-C), CyP inhibitors hold promise as a novel class of anti-HCV therapy.

The Domain 2 of the HCV NS5A Protein Is Intrinsically Unstructured

We present here our current understanding of the NS5A-D2 domain of the hepatitis C virus. Whereas this protein domain is globally unstructured as assessed by macroscopic techniques such as size exclusion chromatography, circular dichroism and homonuclear NMR spectroscopy, high resolution triple resonance spectroscopy allows the identification of a small region of residual structure. This region corresponds moreover to the most conserved sequence over the different genotypes of the virus, underscoring its functional importance. We show that it forms an anchoring point for the host cell cyclophilin prolyl cis/trans isomerase, providing a molecular basis for the use of cyclophilin inhibitors in an antiviral strategy.

Debio-025 is more effective than prednisone in reducing muscular pathology in mdx mice

Muscular dystrophy results in the progressive wasting and necrosis of skeletal muscle. Glucocorticoids such as prednisone have emerged as a front-line treatment for many forms of this disease. Recently, Debio-025, a cyclophilin inhibitor that desensitizes the mitochondrial permeability pore and subsequent cellular necrosis, was shown to improve pathology in three different mouse models of muscular dystrophy. However it is not known if Debio-025 can work in conjunction with prednisone, or how it compares against prednisone in mitigating disease in dystrophic mouse models. Here we show that Debio-025 reduced the variations in myofiber cross-sectional areas, decreased fibrosis, and decreased infiltration of activated macrophages more efficiently than prednisone. However the use of prednisone and Debio-025 together had no additional effect on these histopathological indexes. Orally administered Debio-025 also reduced creatine kinase blood levels and improved grip strength in mdx mice after 6 weeks of treatment, and the combination of Debio-025 with prednisone increased muscle function slightly better than prednisone alone. Thus, our results suggest that Debio-025 is as, effective as or slightly better than, prednisone in mitigating muscular dystrophy in the mdx mouse model of disease.

New anti HCV drugs in the pipeline

Several novel therapeutic approaches are currently in the pipeline for HCV treatment. They include new target host immune system as novel interferon, toll-like receptor agonists, therapeutic vaccines or interleukins, new target cell replication drug as ribavirin analogues, cyclophilin inhibitors, alpha glucosidase inhibitors, dephosphorylationinhibitors of eukaryotic initiation factors 2alpha, antisense molecule, entry inhibitors and specifically targeted antiviral therapy (STAT-C) such as viral enzyme inhibitor ( protease and polymerase). Several STAT-C molecules are currently in development and will soon be at hand and will offer new treatment opportunities to patients infected with hepatitis C. Promising results leading to more than 70% of sustained virological response in genotype 1 naive patients have been reported with two proteases inhibitors (telaprevir and boceprevir) in combination with pegylated interferon and ribavirin that are currently in phase III. These studies also demonstrated the potential to shorten treatment duration in those with a rapid viral response and the realistic hope for retreatment (40 and 75%) in previous non responders or relapsers to interferon based therapies. In addition there is indication that STAT-C drugs may help to overcome negative host factors that have historically been associated with poor response rates (such as ethnicity, insulin resistance, steatosis and cirrhosis). However these trials also emphasize the limitations of protease inhibitors and viral resistance data have provided important new lessons for small molecule drug development. In the near feature, it is likely that IFN-based therapy plus ribavirin will remain the backbone of the treatment of chronic hepatitis C. PEG-IFN and ribavirin are needed in order to prevent HCV resistance to STAT-C drugs and subsequently increase SVR. Genotypic and phenotypic resistance tests will also enter the therapeutic arena. Once several STAT-C agents without cross resistance become available, treatment strategies will include a combination of several drugs with different mechanisms of action (protease inhibitors and polymerase inhibitors) that could hopefully results in IFN and/or ribavirin sparing regimen. The first dual -- combination clinical trial with oral antiviral is still ongoing. Patients receiving this combination for 14 days had undetectable HCV RNA in 63% of naive genotype1 patients. This early success has moved IFN-free regimen a step closer to reality for patients. In the future, there might be combinations of antivirals having additive potency, lacking cross resistance and with a good safety profile.

Abstract 3564: Anti-breast cancer activity of NIM811, a nonimmunosuppressive cyclosporine A analogue

Abstract Cyclophilin A (CypA) is a member of the immunophilin family of peptidyl prolyl isomerases (PPI) that catalyze the cis-trans interconversion of proline imide bonds of peptides. In this role, cyclophilins have been found to function as signaling switches, regulating the activity of receptors, kinases and transcription factors. The PPI activity of CypA is inhibited by the immunosuppressive drug cyclosporine A (CsA), and in turn the CypA-CsA complex inhibits calcineurin-mediated NFAT activation. Our laboratory has recently demonstrated that CypA is necessary for the prolactin (PRL)-induced activation of Jak2/Stat5 signaling and gene expression (Ca Res 68:7769, 2008). In addition, we have also shown that CsA inhibits the in vitro and in vivo growth and progression of both ER+ and ER- breast cancer cell lines. Here, we show that the non-immunosuppressive CsA analog NIM811 blocked PRL-stimulated activation of the Jak2/Stat5 pathway, and the kinases AKT and MAPK in T47D cells. NIM811 also inhibited ER+, ER-, and Her2+ breast cancer cell proliferation, survival and anchorage independent growth in a dose-dependent manner. NIM811 also blocked the PRL-induced association between Stat5 and cMyb, and at higher doses, triggered PARP cleavage. In summary, these results indicate that non-immunosuppressive cyclophilin inhibitor NIM811 has significant potential as a novel for breast cancer therapeutic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3564.

HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A–cyclophilin A interaction to cyclophilin inhibitors

The cyclophilin (Cyp) inhibitors - cyclosporine A (CsA), NIM811, Debio 025, and SCY 635 - block HCV replication both in vitro and in vivo, and represent a novel class of potent anti-HCV agents. We and others showed that HCV relies on cyclophilin A (CypA) to replicate. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which controls the isomerase activity of CypA, is critical for HCV replication. Recent studies showed that under Cyp inhibitor selection, mutations arose in the HCV nonstructural 5A (NS5A) protein. This led us to postulate that CypA assists HCV by acting on NS5A. We tested this hypothesis by developing several interaction assays including GST pull-down assays, ELISA, and mammalian two-hybrid binding assays. We demonstrated that full-length NS5A and CypA form a stable complex. Remarkably, CsA prevents the CypA-NS5A interaction in a dose-dependent manner. Importantly, the CypA-NS5A interaction is conserved among genotypes and is interrupted by CsA. Surprisingly, the NS5A mutant protein, which arose in CsA-resistant HCV variants, behaves similarly to wild-type NS5A in terms of both CypA binding and CsA-mediated release from CypA. This latter finding suggests that HCV resistance to CsA does not correlate with a resistance of the CypA-NS5A interaction to Cyp inhibitors. Moreover, we found that CypA, devoid of its isomerase activity, fails to bind NS5A. Altogether these data suggest that CypA, via its isomerase pocket, binds directly to NS5A, and most importantly, that disrupting this interaction stops HCV replication.

666 THE EFFECTS OF SCY-635 A NON-IMMUNOSUPPRESSIVE CYCLOSPORIN ANALOG ON STELLATE CELL PROLIFERATION, COLLAGEN SYNTHESIS, TIMP-1 AND COLLAGENASE PRODUCTION

Aims: The non-immunosuppressive cyclophilin inhibitor SCY-635 is a potent inhibitor of HCV-specific RNA replication in genotype 1 derived HCV replicons. Clinical studies indicate that acute administration of SCY-635 significantly reduces plasma viremia in patients with chronic hepatitis C virus infection. Recent data have shown that cyclosporin and nonimmunosuppressive analogues such as NIM-811 can suppress the proliferation of, and collagen production in, hepatic stellate cells (HSC) (Kohjima et al. Liver Int., 2007). To examine the potential effects of SCY-635 on liver fibrosis and apoptosis, we have investigated the effects of SCY-635 on cell growth, collagen synthesis, TIMP-1 and collagenase levels and TGF-b signaling in HSCs. Methods: Human HSC-derived LI-90 cells either resting or stimulated with TGFb1 were used for these studies. The levels of collagen, matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1) and levels of phosphorylation of p38 and Smad2 were evaluated by ELISA. Cell proliferation (BrdU incorporation) and apoptosis (ApoGlow) were measured. Apoptosis was evaluated using primary rat hepatocytes after treatment with Ad5IkB adenovirus followed by overnight incubation with TNF-α. Results: SCY-635 and cyclosporin (CsA) decreased the synthesis of collagen. At a clinically relevant concentration of 0.25 μM in non-stimulated or in TGFβ1 stimulated LI-90 cells, SCY-635 significantly reduced the collagen production by more than 70%. There was a parallel decrease in TIMP-1 secretion into the HSC medium and an increase in the production of MMP-1. SCY-635 inhibited proliferation without induction of apoptosis. Concentrations of SCY-635 ranging from 0.1 to 5 μM enhanced the phosphorylation of p38 and suppressed the phosphorylation of Smad2. SCY-635 inhibition of liver apoptosis was equipotent with CsA exhibiting IC50 values of 2.8 ± 0.3 and 3.6 ± 0.3 μM, respectively. Conclusions: These findings demonstrated that SCY-635 suppressed cell growth, reduced type I collagen synthesis and TIMP-1 secretion, and enhanced MMP-1 production suggesting that SCY-635 may exert an anti-fibrogenic effect. These effects may be mediated through the TGF-β signaling pathway. Taken together, these data suggest that SCY-635 may not only reduce the replication of HCV but also reduce the fibrosis associated with chronic hepatitis C virus infection.

New direct-acting antivirals in the development for hepatitis C virus infection.

A large number of new therapies are in development for chronic hepatitis C including direct-acting antiviral drugs (DAA), which target specific hepatitis C virus enzymes. Two of these compounds have already advanced into phase 3 development in the USA and EU, and many more are in phase 2 trials and likely to advance. In this review, the results of recent studies on ribavirin analogues, nonstructural (NS) 3/4 serine protease inhibitors, NS5B polymerase inhibitors, cyclophilin inhibitors, silimarin components, and thiazolides have been updated. Each compound includes a brief summary of its proposed mechanism of action, results of early clinical trials, and more advanced trial data where available. These compounds are likely to be the first approved in the USA and EU and will initially be used in combination with the current standard of care. It is possible that future treatment paradigms with these agents will offer the potential of interferon-free regimens. It is most likely that patients for these new therapies will be selected carefully by identifying and treating first those who have excellent sustained virologic response rates with 24 weeks of pegylated interferon and ribavirin, the current standard of care. It is also likely that there will be a need to identify those patients who are not likely to have a sustained virologic response with the addition of a protease inhibitor to the current standard of care and delaying their therapy until combination viral suppression therapy becomes an option. The cost and side effects of the DAA will be important considerations for treating physicians. This review is current through 2009; however, data are rapidly changing.

Cyclosporine Inhibits a Direct Interaction between Cyclophilins and Hepatitis C NS5A

BackgroundHepatitis C Virus (HCV) infection is a leading indication for liver transplantation. HCV infection reoccurs almost universally post transplant, decreasing both graft longevity and patient survival. The immunosuppressant, cyclosporine A (CsA) has potent anti-HCV activity towards both HCV replicons and the genotype 2a cell culture infectious virus. Previously, we isolated mutations in the 1bN replicon with less sensitivity to CsA that mapped to both NS5A and NS5B regions of the virus. Mutations in NS5A alone conferred decreased CsA susceptibility regardless of NS5B mutations.Methodology/Principal FindingsWe examined the mechanisms by which NS5A mutations contribute to CsA resistance and if they are strain dependent. Using in vitro mutagenesis, the amino acid position 321 mutation of NS5A was restored to the wild-type tyrosine residue conferring partial CsA susceptibility on the mutant replicon. The 321 mutation also alters CsA susceptibility of the JFH cell culture virus. Additionally, we demonstrated a novel CsA-sensitive interaction between NS5A and both cyclophilin A and B. Both the mutant NS5A and wild type NS5A bind cyclophilin in vitro. The NS5A: cyclophilin interaction requires both the NS5A region identified by the resistance mutants and cyclophilin catalytic residues. In cell culture, NS5A from CsA resistant mutant has an enhanced interaction with cyclophilin B. Additionally; NS5B facilitates a stronger binding of mutant NS5A to endogenous cyclophilin B than wild-type in cell culture.Conclusions/SignificanceCollectively, this data suggests direct interactions between cyclophilins and NS5A are critical to understand for optimal use of cyclophilin inhibitors in anti-HCV therapy.

Mechanism of Resistance of Hepatitis C Virus Replicons to Structurally Distinct Cyclophilin Inhibitors

The current standard of care for hepatitis C virus (HCV) infection, pegylated alpha interferon in combination with ribavirin, has a limited response rate and adverse side effects. Drugs targeting viral proteins are in clinical development, but they suffer from the development of high viral resistance. The inhibition of cellular proteins that are essential for viral amplification is thought to have a higher barrier to the emergence of resistance. Three cyclophilin inhibitors, the cyclosporine analogs DEBIO-025, SCY635, and NIM811, have shown promising results for the treatment of HCV infection in early clinical trials. In this study, we investigated the frequency and mechanism of resistance to cyclosporine (CsA), NIM811, and a structurally unrelated cyclophilin inhibitor, SFA-1, in replicon-containing Huh7 cells. Cross-resistance between all clones was observed. NIM811-resistant clones were selected only after obtaining initial resistance to either CsA or SFA-1. The time required to select resistance against cyclophilin inhibitors was significantly longer than that required for resistance selection against viral protein inhibitors, and the achievable resistance level was substantially lower. Resistance to cyclophilin inhibitors was mediated by amino acid substitutions in NS3, NS5A, and NS5B, with NS5A mutations conferring the majority of resistance. Mutation D320E in NS5A mediated most of the resistance conferred by NS5A. Taken together, the results indicate that there is a very low frequency and level of resistance to cyclophilin-binding drugs mediated by amino acid substitutions in three viral proteins. The interaction of cyclophilin with NS5A seems to be the most critical, since the NS5A mutations have the largest impact on resistance.

A Cyclosporin Derivative Discriminates between Extracellular and Intracellular Cyclophilins

Trimesic acid amide serves as a scaffold for a lipophilic cyclophilin inhibitor, a fluorescent rhodamine dye (TAMRA), and a (D-Glu)6 oligopeptide residue. Although the affinity of 1 for intracellular cyclophilin A (CypA) is very high, fluorescence measurements indicate complete exclusion from the cell. CypA-induced chemotaxis of lymphocytes is inhibited by 1 since extracellular cyclophilins are responsible for the physiological signal.

Multiple cyclophilins involved in different cellular pathways mediate HCV replication

Three cyclophilin inhibitors (DEBIO-025, SCY635, and NIM811) are currently in clinical trials for hepatitis C therapy. The mechanism of action of these, however, is not completely understood. There are at least 16 cyclophilins expressed in human cells which are involved in a diverse set of cellular processes. Large-scale siRNA experiments, chemoproteomic assays with cyclophilin binding compounds, and mRNA profiling of HCV replicon containing cells were used to identify the cyclophilins that are instrumental to HCV replication. The previously reported cyclophilin A was confirmed and additional cyclophilin containing pathways were identified. Together, the experiments provide strong evidence that NIM811 reduces viral replication by inhibition of multiple cyclophilins and pathways with protein trafficking as the most strongly and persistently affected pathway.

Platelet Senescence and Phosphatidylserine Exposure.

Abstract 2117Poster Board II-94The transbilayer movement of phosphatidylserine from the inner to the outer leaflet of membrane bilayer occurs during platelet activation and is essential for platelet procoagulant activity. Expression of phosphatidylserine also occurs in platelets stored more than 5 days in the blood bank. We have examined the role of mitochondrial pathways in activation-induced platelet procoagulant activity and during in vitro senescence. Collagen and thrombin-induced exposure of phosphatidylserine is associated with a decrease in mitochondrial membrane potential (MMP). Cyclosporin A, a known inhibitor of cyclophilin D, inhibited phosphatidylserine exposure and the loss of MMP. Consistent with this, platelets from CypD deficient mice had decreased loss of MMP and impaired phosphatidylserine exposure when treated with a combination of thrombin and collagen. However, there is no release of cytochrome c into the cytosol. Also there is no activation of caspase-3 or Rho associated kinase I (ROCK1). In sharp contrast to this, in platelets stored for more than 5 days, phosphatidylserine exposure is found to be associated with caspase-3 and ROCK1 activation. To delineate the mechanism we used ABT737, a BH3 mimetic that induces mitochondrial pathway of apoptosis via Bcl-2 family of proteins. ABT737-induced phosphatidylserine exposure was also associated with cytochrome c release, caspase-3 and ROCKI activation and it is unaffected in platelets isolated from CypD knock out mice or in platelets treated with cyclosporine A prior to activation. Caspase-3 inhibitor (z-DVED-fmk and ROCK1 inhibitor Y-27632 reduced ABT-737 induced phosphatidylserine expression.Our results also show that activation-induced phosphatidylserine exposure is dependent on mitochondrial membrane depolarization and CypD but is independent of cytochrome c release or caspase-3 or ROCK1 activation. In contrast, the phosphatidylserine exposure in stored platelets occurs via mitochondrial cytochrome c release, caspase-3 activation and ROCKI activation and does not depend on mitochondrial membrane depolarization or CypD. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Debio-025 inhibits HIV-1 by interfering with an early event in the replication cycle

Cyclophilin A is a peptidyl-propyl isomerase that binds the capsid (p24) protein of HIV-1 and facilitates replication. We report a cyclophilin inhibitor, a non-immunosuppressive cyclosporine analogue, Debio-025, that is about 15-times more potent than cyclosporine A and less toxic resulting in a selectivity index of more than 300. It was equally active against virus strains that were resistant toward inhibitors of the viral entry, fusion, or reverse transcription while it was not inhibitory to HIV-2 or SIV MAC. Mechanism of action studies demonstrate that Debio-025 inhibits the HIV-1 replication by interfering with an early stage of the viral replication cycle. Indeed, addition of Debio-025 could be postponed for 2 h before loosing its antiviral activity. The compound proved inactive against mutant viruses that are independent of cyclophilin A binding suggesting Debio-025 targets the cyclophilin A–capsid interaction.

Cyclosporine A inhibits in vitro replication of betaretrovirus associated with primary biliary cirrhosis

Up to one-third of patients with primary biliary cirrhosis (PBC) experience recurrent disease following liver transplantation, which is associated with earlier and more severe recurrence in patients treated with tacrolimus as compared with cyclosporine A (CsA). As the latter has known antiviral activity, we hypothesized that CsA has the ability to inhibit the betaretrovirus characterized from patients with PBC. We investigated whether CsA, the cyclosporine analogue NIM811, tacrolimus and other compounds can modulate the mouse mammary tumour virus production from Mm5MT cells. Viral load was evaluated in the cell supernatants by quantifying reverse transcriptase (RT) levels and betaretrovirus RNA. A significant correlation was observed with increasing concentrations of CsA and NIM811, and decreasing of RT levels (rho-0.59, P=0.04 and rho-0.74, P=0.006 respectively), whereas tacrolimus had no significant effect (rho-0.27, P=0.4). At a dose of 3 microg/ml, CsA, NIM811 and the human immunodeficiency virus aspartyl protease inhibitor, lopinavir, were all associated with greater than three-fold reduction in the betaretrovirus RNA production from Mm5MT cells as compared with tacrolimus (P<0.005). These studies demonstrate that the cyclophilin inhibitors CsA and NIM811 have antiviral activity against betaretrovirus production in vitro.

Identification of cellular and viral factors related to anti‐hepatitis C virus activity of cyclophilin inhibitor

We have so far reported that an immunosuppressant cyclosporin A (CsA), a well-known cyclophilin (CyP) inhibitor (CPI), strongly suppressed hepatitis C virus (HCV) replication in cell culture, and that CyPB was a cellular cofactor for viral replication. To further investigate antiviral mechanisms of CPI, we here developed cells carrying CsA-resistant HCV replicons, by culturing the HCV subgenomic replicon cells for 4 weeks in the presence of CsA with G418. Transfection of total RNA from the isolated CsA-resistant cells to naïve Huh7 cells conferred CsA resistance, suggesting that the replicon RNA itself was responsible for the resistant phenotype. Of the identified amino acid mutations, D320E in NS5A conferred the CsA resistance. The replicon carrying the D320E mutation was sensitive to interferon-alpha, but was resistant to CsA and other CPIs including NIM811 and sanglifehrin A. Knockdown of individual CyP subtypes revealed CyP40, in addition to CyPA and CyPB, contributed to viral replication, and CsA-resistant replicons acquired independence from CyPA for efficient replication. These data provide important evidence on the mechanisms underlying the regulation of HCV replication by CyP and for designing novel and specific anti-HCV strategies with CPIs.

EM.P.5.03 The cyclophilin inhibitor Debio 025 normalizes mitochondrial function, muscle apoptosis and ultrastructural defects in Col6a1[formula omitted] myopathic mice

Background and purpose: We have investigated the therapeutic effects of the selective cyclophilin inhibitor D MeAla3 EtVal4 cyclosporin (Debio 025) in myopathic Col6a1−/− mice, a model of muscular dystrophies due to defects of collagen VI. Experimental approach: We have studied calcineurin activity, T cell activation, propensity to open of the permeability transition pore in mitochondria and skeletal muscle fibres, muscle ultrastructure and apoptotic rates in Col6a1−/− mice before after treatment with Debio 025.

The cyclophilin inhibitor Debio 025 normalizes mitochondrial function, muscle apoptosis and ultrastructural defects in Col6a1−/− myopathic mice

We have investigated the therapeutic effects of the selective cyclophilin inhibitor D-MeAla(3)-EtVal(4)-cyclosporin (Debio 025) in myopathic Col6a1(-/-) mice, a model of muscular dystrophies due to defects of collagen VI. We studied calcineurin activity based on NFAT translocation; T cell activation based on expression of CD69 and CD25; propensity to open the permeability transition pore in mitochondria and skeletal muscle fibres based on the ability to retain Ca(2+) and on membrane potential, respectively; muscle ultrastructure by electronmicroscopy; and apoptotic rates by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays in Col6a1(-/-) mice before after treatment with Debio 025. Debio 025 did not inhibit calcineurin activity, yet it desensitizes the mitochondrial permeability transition pore in vivo. Treatment with Debio 025 prevented the mitochondrial dysfunction and normalized the apoptotic rates and ultrastructural lesions of myopathic Col6a1(-/-) mice. Desensitization of the mitochondrial permeability transition pore can be achieved by selective inhibition of matrix cyclophilin D without inhibition of calcineurin, resulting in an effective therapy of Col6a1(-/-) myopathic mice. These findings provide an important proof of principle that collagen VI muscular dystrophies can be treated with Debio 025. They represent an essential step towards an effective therapy for Ullrich Congenital Muscular Dystrophy and Bethlem Myopathy, because Debio 025 does not expose patients to the potentially harmful effects of immunosuppression.

The Isomerase Active Site of Cyclophilin A Is Critical for Hepatitis C Virus Replication

Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies.

Novel drugs for hepatitis C virus

Hepatitis C virus is a major public health issue and a leading cause of chronic liver disease. The current treatment—pegylated interferon and ribavirin—is associated with significant adverse events and sustained virologic response rates of only approximately 50% in genotype-1 patients. New drugs in development include novel interferons, ribavirin analogs, NS3 protease and NS5B polymerase inhibitors, and cyclophilin inhibitors. The primary goal of drug development is to improve efficacy; secondary goals include shortening the duration and increasing the tolerability of treatment. Viral resistance and toxicity must be overcome before acceptance of new drugs. Future therapy may likely be approved in the United States and European Union by 2011, including a combination of these novel agents, with interferon and ribavirin remaining key components.