Cyclophilin Inhibitors Research Articles

Development of a Flow Cytometry Live Cell Assay for the Screening of Inhibitors of Hepatitis C Virus (HCV) Replication

In this study, we established a flow cytometry live cell-based assay that permits the screening of hepatitis C virus (HCV) inhibitors. Specifically, we created a stable cell line, which harbors a subgenomic replicon encoding an NS5A-YFP fusion protein. This system allows direct measurement of YFP fluorescence in live hepatoma cells in which the HCV replicon replicates. We demonstrated that this stable fluorescent system permits the rapid and sensitive quantification of HCV replication inhibition by direct-acting antiviral agents (DAA) including protease and NS5A inhibitors and host-targeting antiviral agents (HTA) including cyclophilin inhibitors. This flow cytometry-based live cell assay is well suited for multiple applications such as the evaluation of HCV replication as well as antiviral drug screening.

Subtype Specific Differences in NS5A Domain II Reveals Involvement of Proline at Position 310 in Cyclosporine Susceptibility of Hepatitis C Virus

Hepatitis C virus (HCV) is susceptible to cyclosporine (CsA) and other cyclophilin (CypA) inhibitors, but the genetic basis of susceptibility is controversial. Whether genetic variation in NS5A alters cell culture susceptibility of HCV to CypA inhibition is unclear. We constructed replicons containing NS5A chimeras from genotypes 1a, 2a and 4a to test how variation in carboxy terminal regions of NS5A altered the genotype 1b CsA susceptibility. All chimeric replicons including genotype 1b Con1LN-wt replicon exhibited some cell culture sensitivity to CsA with genotype 4a being most sensitive and 1a the least. The CypA binding pattern of truncated NS5A genotypes correlated with the susceptibility of these replicons to CsA. The Con1LN-wt replicon showed increased susceptibility towards CsA when proline at position 310P was mutated to either threonine or alanine. Furthermore, a 15 amino acid long peptide fused N terminally to GFP coding sequences confirmed involvement of proline at 310 in CypA binding. Our findings are consistent with CypA acting on multiple prolines outside of the previously identified CypA binding sites. These results suggest multiple specific genetic variants between genotype 1a and 1b in the C-terminus of NS5A alter the CsA susceptibility of replicons, and some variants may oppose the effects of others.

Cyclophilin Inhibitors Block Arterivirus Replication by Interfering with Viral RNA Synthesis

Virus replication strongly depends on cellular factors, in particular, on host proteins. Here we report that the replication of the arteriviruses equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) is strongly affected by low-micromolar concentrations of cyclosporine A (CsA), an inhibitor of members of the cyclophilin (Cyp) family. In infected cells, the expression of a green fluorescent protein (GFP) reporter gene inserted into the PRRSV genome was inhibited with a half-maximal inhibitory concentration (IC(50)) of 5.2 μM, whereas the GFP expression of an EAV-GFP reporter virus was inhibited with an IC(50) of 0.95 μM. Debio-064, a CsA analog that lacks its undesirable immunosuppressive properties, inhibited EAV replication with an IC(50) that was 3-fold lower than that of CsA, whereas PRRSV-GFP replication was inhibited with an IC(50) similar to that of CsA. The addition of 4 μM CsA after infection prevented viral RNA and protein synthesis in EAV-infected cells, and CsA treatment resulted in a 2.5- to 4-log-unit reduction of PRRSV or EAV infectious progeny. A complete block of EAV RNA synthesis was also observed in an in vitro assay using isolated viral replication structures. The small interfering RNA-mediated knockdown of Cyp family members revealed that EAV replication strongly depends on the expression of CypA but not CypB. Furthermore, upon fractionation of intracellular membranes in density gradients, CypA was found to cosediment with membranous EAV replication structures, which could be prevented by CsA treatment. This suggests that CypA is an essential component of the viral RNA-synthesizing machinery.

New agents for hepatitis C and the challenges in treating co‐infected patients

More recently, the improved understanding of the hepatitis C viral life cycle has led to the identification of numerous potential targets for novel, direct‐acting antiviral compounds. Two NS3/4A protease inhibitors, telaprevir and boceprevir, were the first oral HCV protease inhibitors which were approved in Europe and the United States in 2011 in combination with pegylated interferon (IFN)‐α and ribavirin for the treatment of chronic hepatitis C related to HCV genotype 1, in both treatment‐naïve and treatment‐experienced patients. Sustained virological response rates in the range of 66–75% and 59–66% have been achieved in these two patient populations, respectively, with treatment durations of 24 to 48 weeks. Despite these significant advances the high rate of adverse events and the high costs of these regimens have limited the uptake of these new regimens. In particular, the quest for removal of interferon from HCV therapy remains one of the great unmet medical needs in HCV therapy development. With an increasing number of other DAAs in clinical development including second‐wave, first‐generation, and second‐generation NS3/4A protease inhibitors, nucleoside/nucleotide analogue inhibitors and non‐nucleoside inhibitors of HCV‐RNA‐dependent RNA polymerase, inhibitors of nonstructural protein 5A (NS5A) and host‐targeted compounds such as cyclophilin inhibitors and silibinin, the hope for better tolerated HCV therapies and potentially interferon‐free HCV combination therapies has grown considerably. The proof of concept that IFN‐free regimens may lead to HCV eradication has recently been brought in GT2/3 patients treated with PSI‐7977 and ribavirin for 12 weeks as well as in GT1b patients treated with BMS‐790052 + BMS‐650032 for 24 weeks. Although some patients with GT1a infection were also cured under this regimen, the majority of patients relapsed after treatment discontinuation, suggesting that at least for the 1a HCV genotype more effective interferon‐free regimens still need to be developed. Interestingly, also GT1 non‐responders treated with GS‐7977+ribavirin showed a high relapse rate, again underlining that more challenging patient populations will still exist which either need broader oral combinations or longer treatment durations. Overall, the dramatic increase in efficacy has also been seen in DAA‐based HCV therapy in HIV/HCV‐coinfected patients. However, complex drug‐drug interactions and tolerability issues remain a concern for all HCV patients, but particularly for HIV patients receiving antiretroviral therapy with a high potential for drug interactions.

Hepatitis B and C

Hepatitis B and C

Cyclophilin Inhibitors: An Emerging Class of Therapeutics for the Treatment of Chronic Hepatitis C Infection

The advent of the replicon system together with advances in cell culture have contributed significantly to our understanding of the function of virally-encoded structural and nonstructural proteins in the replication cycle of the hepatitis C virus. In addition, in vitro systems have been used to identify several host proteins whose expression is critical for supporting such diverse activities as viral entry, RNA replication, particle assembly, and the release of infectious virions. Among all known host proteins that participate in the HCV replication cycle, cyclophilins are unique because they constitute the only host target that has formed the basis of pharmaceutical drug discovery and drug development programs. The introduction of the nonimmunosuppressive cyclophilin inhibitors into clinical testing has confirmed the clinical utility of CsA-based inhibitors for the treatment of individuals with chronic hepatitis C infection and has yielded new insights into their mechanism(s) of action. This review describes the biochemical evidence for the potential roles played by cyclophilins in supporting HCV RNA replication and summarizes clinical trial results obtained with the first generation of nonimmunosuppressive cyclophilin inhibitors.

Lentiviral Gene Therapy Against Human Immunodeficiency Virus Type 1, Using a Novel Human TRIM21-Cyclophilin A Restriction Factor

TRIM5α (tripartite motif-containing protein-5, isoform α)-cyclophilin A fusion proteins are anti-human immunodeficiency virus (HIV) restriction factors that have evolved in certain nonhuman primates over millions of years and protect against HIV and related viruses. Restriction by TRIM5αCypA is potent and highly resistant to viral escape by mutation and, in combination with a suitable gene delivery platform, offers the possibility of novel therapeutic approaches against HIV. Here we report that lentiviral vector delivery of human mimics of TRIM5α-cyclophilin A (TRIM5CypA) fusion proteins afforded robust and durable protection against HIV-1, but resulted in downregulation of host cell antiviral responses mediated by endogenous TRIM5α. We found that substitution of TRIM5α RING, B-box, and coiled-coil domains with similar domains from a related TRIM protein, TRIM21, produced a novel and equally potent inhibitor of HIV-1. Both TRIM5CypA and TRIM21CypA inhibited transduction by HIV-1-derived viral vectors and prevented propagation of replication-competent HIV-1 in human cell lines and in primary human T cells. Restriction factor-modified T cells exhibited preferential survival in the presence of wild-type HIV. Restriction was dependent on proteasomal degradation and was reversed in the presence of the cyclophilin inhibitor cyclosporin. Importantly, TRIM21CypA did not disturb endogenous TRIM5α-mediated restriction of gammaretroviral infection. Furthermore, endogenous TRIM21 antiviral activity was assessed by measuring inhibition of adenovirus-antibody complexes and was found to be preserved in all TRIMCypA-modified groups. We conclude that lentivirus-mediated expression of the novel chimeric restriction factor TRIM21CypA provides highly potent protection against HIV-1 without loss of normal innate immune TRIM activity.

Suppression of Viral RNA Binding and the Assembly of Infectious Hepatitis C Virus Particles In Vitro by Cyclophilin Inhibitors

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both in vitro and in vivo. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors.

Mitochondrial modulation of store-operated Ca2+ entry in model cells of Alzheimer’s disease

Mitochondrial malfunction and calcium dyshomeostasis are early pathological events considered as important features of the Alzheimer’s disease (AD) brain. Recent studies have suggested mitochondrion as an active regulator of Ca2+ signaling based on its calcium buffering capacity. Herein, we investigated the mitochondrial involvement in the modulation of store-operated calcium entry (SOCE) in neural 2a (N2a) transgenic AD model cells. Results showed that SOCE was significantly depressed in N2a cells transfected with wild-type human APP695 (N2a APPwt) compared with empty vector control (N2a WT) cells. Pharmacological manipulation with mitochondrial function blockers, such as FCCP, RuR, or antimycin A/oligomycin, could inhibit mitochondrial calcium handling, and then impair SOCE pathway in N2a WT cells. Furthermore, mitochondria of N2a APPwt cells exhibited more severe swelling in response to Ca2+, which is an indication of mitochondrial membrane permeability transition (MPT), than the wild-type controls. Additionally, treatment with cyclosporin A, a potent inhibitor of cyclophilin D, which can block MPT, could significantly restore the attenuated SOCE in N2a APPwt cells. Therefore, inhibition of cyclophilin D might be a therapeutic strategy for Alzheimer’s disease.

Resistance to Direct-Acting Antivirals

Viral resistance corresponds to the selection, during treatment, of pre-existing viral variants less susceptible to the drug’s inhibitory activity because they bear amino acid substitutions altering the drug target. Hepatitis C virus (HCV) drugs in development can be split into two groups according to their barrier to resistance. Direct-acting antiviral drugs with a low barrier to resistance include first-generation NS3-4A protease inhibitors, non-nucleoside inhibitors of HCV RNA-dependent RNA polymerase and first-generation NS5A inhibitors. HCV drugs with a high barrier to resistance include nucleoside/nucleotide analogues, possibly second-generation protease and NS5A inhibitors, and host-targeted agents, such as cyclophilin inhibitors or microRNA-122 antagonists. This article reviews recent findings that add to our knowledge and understanding of HCV resistance to direct-acting antiviral drugs and discusses them in the context of new therapeutic strategies, with and without pegylated interferon-α and/or ribavirin.

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

Hepatitis C virus (HCV) is the main agent of acute and chronic liver diseases leading to cirrhosis and hepatocellular carcinoma. The current standard therapy has limited efficacy and serious side effects. Thus, the development of alternate therapies is of tremendous importance. HCV NS5A (nonstructural 5A protein) is a pleiotropic protein with key roles in HCV replication and cellular signaling pathways. Here we demonstrate that NS5A dimerization occurs through Domain I (amino acids 1-240). This interaction is not mediated by nucleic acids because benzonase, RNase, and DNase treatments do not prevent NS5A-NS5A interactions. Importantly, DTT abrogates NS5A-NS5A interactions but does not affect NS5A-cyclophilin A interactions. Other reducing agents such as tris(2-carboxyethyl)phosphine and 2-mercaptoethanol also abrogate NS5A-NS5A interactions, implying that disulfide bridges may play a role in this interaction. Cyclophilin inhibitors, cyclosporine A, and alisporivir and NS5A inhibitor BMS-790052 do not block NS5A dimerization, suggesting that their antiviral effects do not involve the disruption of NS5A-NS5A interactions. Four cysteines, Cys-39, Cys-57, Cys-59, and Cys-80, are critical for dimerization. Interestingly, the four cysteines have been proposed to form a zinc-binding motif. Supporting this notion, NS5A dimerization is greatly facilitated by Zn(2+) but not by Mg(2+) or Mn(2+). Importantly, the four cysteines are vital not only for viral replication but also critical for NS5A binding to RNA, revealing a correlation between NS5A dimerization, RNA binding, and HCV replication. Altogether our data suggest that NS5A-NS5A dimerization and/or multimerization could represent a novel target for the development of HCV therapies.

TAL Effectors Target the C-Terminal Domain of RNA Polymerase II (CTD) by Inhibiting the Prolyl-Isomerase Activity of a CTD-Associated Cyclophilin

Transcriptional activator-like (TAL) effectors of plant pathogenic bacteria function as transcription factors in plant cells. However, how TAL effectors control transcription in the host is presently unknown. Previously, we showed that TAL effectors of the citrus canker pathogen Xanthomonas citri, named PthAs, targeted the citrus protein complex comprising the thioredoxin CsTdx, ubiquitin-conjugating enzymes CsUev/Ubc13 and cyclophilin CsCyp. Here we show that CsCyp complements the function of Cpr1 and Ess1, two yeast cyclophilins that regulate transcription by the isomerization of proline residues of the regulatory C-terminal domain (CTD) of RNA polymerase II. We also demonstrate that CsCyp, CsTdx, CsUev and four PthA variants interact with the citrus CTD and that CsCyp co-immunoprecipitate with the CTD in citrus cell extracts and with PthA2 transiently expressed in sweet orange epicotyls. The interactions of CsCyp with the CTD and PthA2 were inhibited by cyclosporin A (CsA), a cyclophilin inhibitor. Moreover, we present evidence that PthA2 inhibits the peptidyl-prolyl cis-trans isomerase (PPIase) activity of CsCyp in a similar fashion as CsA, and that silencing of CsCyp, as well as treatments with CsA, enhance canker lesions in X. citri-infected leaves. Given that CsCyp appears to function as a negative regulator of cell growth and that Ess1 negatively regulates transcription elongation in yeast, we propose that PthAs activate host transcription by inhibiting the PPIase activity of CsCyp on the CTD.

Multiple Mutations in Hepatitis C Virus NS5A Domain II Are Required To Confer a Significant Level of Resistance to Alisporivir

Alisporivir is the most advanced host-targeting antiviral cyclophilin (Cyp) inhibitor in phase III studies and has demonstrated a great deal of promise in decreasing hepatitis C virus (HCV) viremia in infected patients. In an attempt to further elucidate the mechanism of action of alisporivir, HCV replicons resistant to the drug were selected. Interestingly, mutations constantly arose in domain II of NS5A. To demonstrate that these mutations are responsible for drug resistance, they were reintroduced into the parental HCV genome, and the resulting mutant viruses were tested for replication in the presence of alisporivir or in the absence of the alisporivir target, CypA. We also examined the effect of the mutations on NS5A binding to itself (oligomerization), CypA, RNA, and NS5B. Importantly, the mutations did not affect any of these interactions. Moreover, the mutations did not preserve NS5A-CypA interactions from alisporivir rupture. NS5A mutations alone render HCV only slightly resistant to alisporivir. In sharp contrast, when multiple NS5A mutations are combined, significant resistance was observed. The introduction of multiple mutations in NS5A significantly restored viral replication in CypA knockdown cells. Interestingly, the combination of NS5A mutations renders HCV resistant to all classes of Cyp inhibitors. This study suggests that a combination of multiple mutations in domain II of NS5A rather than a single mutation is required to render HCV significantly and universally resistant to Cyp inhibitors. This in accordance with in vivo data that suggest that alisporivir is associated with a low potential for development of viral resistance.

Antiviral drug advances in the treatment of human immunodeficiency virus (HIV) and chronic hepatitis C virus (HCV)

Antiviral therapy is recommended for all HIV infected individuals. Therefore, there is a continuous need for improvement and optimization of current therapy and the development of novel agents and drug classes. Fixed dose combinations (FDC) with the advantage of once daily dosing and improved tolerability and toxicity profiles are attractive options. The non-nucleoside reverse transcriptase inhibitor (NNRTI) based FDC of rilpivirine plus tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) (Complera(®)) and an extended release version of the NNRTI nevirapine, (Viramune XR(®)) were recent additions to the HIV armamentarium. In addition, the approval of the second integrase inhibitor, elvitegravir, and a novel pharmacoenhancer cobicistat is anticipated in 2012. These agents have been co-formulated with TDF and FTC as a single tablet and represent the first integrase inhibitor based complete FDC regimen. A new standard of care for the treatment of Chronic Hepatitis C (HCV) emerged in 2011 with the approval of the first antiviral drugs to directly inhibit HCV NS3/4A protease, telaprevir (Incivik(®)) and boceprevir (Victrelis(®)). Combined with peginterferon-alfa plus ribavirin they offer genotype-1 infected patients significantly improved sustained virologic response rates and the potential for shorter durations of therapy. HCV therapeutics will continue to evolve as there are several drugs in the protease inhibitor class and other classes such as NS5B polymerase inhibitors, NS5A inhibitors and cyclophilin inhibitors currently in development.

Hepatitis C therapy in non‐genotype 1 patients: the near future

Worldwide, 50-70 million subjects are infected with an hepatitis C virus (HCV) genotype 2, 3, 4, 5 or 6. In these patients, the combination of PEG-INF-α and ribavirin remains the currently approved standard-of-care treatment. The identification of different potential therapeutic targets in the HCV life cycle has led to the development of both direct antiviral agents (DAAs) and reagents targeting host functions essential for viral replication. DAAs comprise so far first-generation, second-wave and second-generation NS3/4A protease inhibitors (PIs), nucleos(t)ide (NIs) and non-nucleoside inhibitors of the NS5B RNA polymerase and NS5A complex inhibitors. The main host-protein-directed antiviral agents are cyclophilin inhibitors and silibinin. Whereas the launch of first-generation PIs was a major landmark in the management of genotype 1 (GT-1)-infected patients, these drugs are inactive in most non-GT-1-infected patients. Several of these and other drugs have now reached phase II and even phase III clinical stage development. The purpose of this article is to provide an overview of the clinical results recently reported for the treatment for non-GT-1 HCV infection with a focus on the most promising new compounds and combinations.

New Antiviral Therapies in the Management of HCV Infection

Improved knowledge of the HCV life cycle and of structural features of HCV proteins have led to the discovery of numerous potential targets for antiviral therapy. Viral replication and polyprotein processing have been tagged as promising viral targets. Clathrin-mediated endocytosis, fusion of HCV with cellular membranes, translation of viral RNA, virus production and release as well as several host cell factors may provide alternative targets for future anti-HCV therapies. Several compounds are currently under investigation in clinical trials and showed high antiviral activity in patients with chronic hepatitis C. Recently, Phase III studies for two protease inhibitors, telaprevir and boceprevir, each given in combination with pegylated interferon (standard of care [SOC]), were completed. In HCV-genotype-1-infected patients, the addition of telaprevir or boceprevir to SOC increased sustained virological response rates from <50% to >70%. Nucleoside/nucleotide inhibitors of the HCV NS5B polymerase have shown antiviral activity against different HCV genotypes, and have a higher barrier to resistance than protease inhibitors. In addition, several allosteric binding sites have been identified for non-nucleoside inhibitors of the NS5B polymerase. Inhibitors of NS5A are potentially active against all HCV genotypes. Among the different host cell-targeting compounds, cyclophilin inhibitors have shown promising results. Future hope lies in the combination of direct-acting antiviral agents with the possibility of interferon-free treatment regimens.

Cyclophilin involvement in the replication of hepatitis C virus and other viruses

In recent months, there has been a wealth of promising clinical data suggesting that a more effective treatment regimen, and potentially a cure, for hepatitis C virus (HCV) infection is close at hand. Leading this push are direct-acting antivirals (DAAs), currently comprising inhibitors that target the HCV protease NS3, the viral polymerase NS5B, and the non-structural protein NS5A. In combination with one another, along with the traditional standard-of-care ribavirin and PEGylated-IFNα, these compounds have proven to afford tremendous efficacy to treatment-naíve patients, as well as to prior non-responders. Nevertheless, by targeting viral components, the possibility of selecting for breakthrough and treatment-resistant virus strains remains a concern. Host-targeting antivirals are a distinct class of anti-HCV compounds that is emerging as a complementary set of tools to combat the disease. Cyclophilin (Cyp) inhibitors are one such group in this category. In contrast to DAAs, Cyp inhibitors target a host protein, CypA, and have also demonstrated remarkable antiviral efficiency in clinical trials, without the generation of viral escape mutants. This review serves to summarize the current literature on Cyps and their relation to the HCV viral life cycle, as well as other viruses.

What happens after hepatitis C eradication?

The treatment of hepatitis C virus infection (HCV) by a combination of pegylated interferon and ribavirin, according to early viral kinetics, leads to a sustained virological response (SVR) in more than 50% of patients with chronic infection. This SVR is a complete recovery of the infection but more than 50% of genotype 1-infected patients do not achieve SVR. A better understanding of the viral cycle, and the characterization of viral enzymes which are potential targets, resulted in the development of new molecules, direct acting antiviral drugs (DAA) targeted against HCV, either specific of genotype 1 (NS3/NS4A protease inhibitors and NS5B polymerase inhibitors) or with a wider spectrum (NS5A or entry inhibitors), and non-specific antivirals (new interferons, cyclophilin inhibitors). The results of these phase II and III trials which clearly demonstrated a 20 to 30% increase in the SVR rate of genotype 1-infected patients, either naive or treatment experienced. These new drugs has now been approved by the end of 2011, after a temporary approval for compassionate use in cirrhotic genotype 1 patients with previous relapse or partial response to the combination therapy and a new turn appears with the “interferon free regimens” which combine different direct acting antivirals. The complete virologic recovery which is achieved with all the antiviral treatments clearly reduces the rates of liver-related morbidity and mortality but, in the absence of biopsy-proven cirrhosis reversal, the risk of occurrence of hepatocellular carcinoma is still present and requires a regular US follow-up in those patients with extensive fibrosis or cirrhosis for an early screening. In the experienced patients with active replication, new antivirals are mandatory to reduce the risks of complications, including liver transplantation, which is high, especially in patients with rapid fibrosis. In the future, the main limitations of triple therapy will be safety (cutaneous rash or anemia which may be controlled), cost, compliance, viral resistance, and drug-drug interactions that must be avoided by educating patients and physicians.

The Cyclophilin Inhibitor SCY-635 Disrupts Hepatitis C Virus NS5A-Cyclophilin A Complexes

The nonimmunosuppressive cyclophilin (Cyp) inhibitor SCY-635 blocks hepatitis C virus (HCV) replication both in vitro and in vivo and represents a novel potent anti-HCV agent. However, its mechanism of action remains to be fully elucidated. A growing body of evidence suggests that cyclophilin A (CypA) is absolutely necessary for HCV replication and that the HCV nonstructural 5A (NS5A) protein serves as a main viral ligand for CypA. In this study, we examined the effect of SCY-635 on HCV replication. Specifically, we asked whether SCY-635 blocks HCV replication by targeting CypA-NS5A interactions. We also investigated the possibility that HCV can escape SCY-635 selection pressure and whether this resistance influences either CypA-NS5A interactions or the dependence of HCV on CypA. We found not only that SCY-635 efficiently inhibits HCV replication, but it is sufficient alone to clear HCV replicon-containing cells. We found that SCY-635 prevents CypA-NS5A interactions in a dose-dependent manner. SCY-635 prevents the contact between CypA and NS5A derived from genotypes 1 to 3. Together, these data suggest that NS5A-CypA interactions control HCV replication and that SCY-635 blocks viral replication by preventing the formation of these complexes. We also found that NS5A mutant proteins found in SCY-635-resistant HCV replicons behave similarly to wild-type NS5A in terms of both CypA binding and SCY-635-mediated dissociation and inhibition of CypA binding. However, the NS5A mutations found in SCY-635-resistant HCV replicons rescued viral replication in CypA-knockdown cells, suggesting that the NS5A mutations, which arose in vitro under SCY-635 selection, do not alter the binding affinity of CypA for NS5A. These specific mutations in NS5A eliminate the dependence of HCV RNA replication on the expression of host CypA.

Anti−Hepatitis C Virus Drugs in Development

Development of robust cell culture models for hepatitis C viral infection has greatly increased our understanding of this virus and its life cycle. This knowledge has led to the development of many drugs that target specific elements of viral replication, including viral proteins and host factors required for replication. The NS3/4A serine protease inhibitors were the first of these to be used in the clinic, and reagents that target other elements of the viral lifecycle are in advanced stages of clinical development. These include new NS3/4A protease inhibitors, NS5B RNA-dependent RNA polymerase inhibitors, NS5A inhibitors, and host-directed antivirals, such as cyclophilin inhibitors. Alternative interferons with possibly improved tolerability, specifically interferon-λ1 (interleukin-29), are also under development. These new reagents against hepatitis C virus should lead to highly effective, well-tolerated, and likely interferon-sparing therapies in the next several years.