Most novel drugs directed against the hepatitis C virus (HCV) including the recently approved NS3/4A protease inhibitors boceprevir and telaprevir are inhibitors of viral enzymes. Since HCV is an RNA virus with a short and highly variable genome, these direct-acting antivirals (DAAs) are prone to rapidly inducing the emergence of resistant HCV variants. This problem could be mitigated by the development of drugs that target host factors that the virus depends on during the various stages of its replication cycle. An increasing understanding of the molecular interactions between the virus and its host cell has allowed the identification of promising host targets for anti-HCV therapy and several host-targeting agents (HTAs) are currently under development. The most advanced compounds as yet may be inhibitors of cyclophilin A, a host factor known to be critical for viral RNA replication and possible virion assembly or release. One such compound, alisporivir, has demonstrated in vivo efficacy and is now in a phase 3 trial. Several other HTAs with very different host targets are further upstream in the development pipeline. This paper reviews promising host targets, their role in the viral replication cycle, and the HTAs that target them.
According to WHO data, about 160 million people globally are chronically infected with hepatitis C virus (HCV), representing 2.3% of the human population (Lavanchy, 2011). HCV prevalence rates show marked geographic variation ranging between 0.1% and 15-20% of the population. Chronic infection with HCV eventually results in liver damage including fibrosis, cirrhosis, or hepatocellular carcinoma in a substantial percentage of patients. Accordingly, the complications of HCV infection are a common indication for liver transplantations worldwide.
The majority of patients with acute HCV infection have relatively mild or no symptoms. This is also the reason why most patients are diagnosed with an HCV infection in its chronic stage several years after the infection occurred. The mainstay of therapy is a combination of pegylated interferon alpha (peginterferon) and ribavirin (RBV) (Zeuzem et al., 2009). Both drugs are not specifically directed against HCV, but rather act by inducing an antiviral state in the host. The combination (peginterferon/RBV) achieves a sustained virologic response (SVR), i.e., HCV-RNA negativity 6 months after completion of therapy or, put more simply, cure, in just over half of cases in selected clinical trial populations (Fried et al., 2002; Manns et al., 2001). However, outcomes are worse in real life settings since peginterferon/RBV therapy is associated with significant side effects that preclude therapy in many individuals with advanced liver disease or concomitant other conditions.
Now in 2011, some 22 years after the molecular identification of HCV, the first directly acting antiviral drugs (DAA) against this pathogen are expected to reach the market in North America and Western Europe. These are two specific inhibitors of the viral protease NS3/4A, boceprevir and telaprevir. These first DAAs have been approved for clinical use by the U.S. FDA in May 2011 based on phase III trial results. Both drugs have achieved SVR rates of 70-80% in difficult-to-treat genotype 1 infected patients — at least in carefully selected trial populations — and offer real hope of achieving SVR in patients who have failed previous treatments with peginterferon/RBV (Bacon et al., 2011; McHutchison et al., 2009; 2010; Poordad et al., 2011). However, these drugs will complement rather than replace current treatment regimens and will not allow HCV treatment to be interferon-free.
Another major shortcoming of these new drugs will certainly be emerging viral resistance, since HCV is a highly variable RNA-virus with an enormous genetic flexibility (Sarrazin et al., 2007). It can be divided into seven different genotypes (1-7), which differ from each other by 31-33% at the nucleotide level (Simmonds, 2004). Among the different genotypes further subtypes (a, b, c, etc.) have been described. The high level of diversity is a result of both the error prone RNA dependent RNA polymerase (RdRp), which is lacking a proof-reading function, and the high replication rate of HCV, together leading to a very high mutation rate (Lindenbach et al., 2006). This remarkable degree of variability manifests itself in that within an infected host HCV exists not as a single genetically homogenous virus but rather a population of closely related but yet distinct virions, referred to as the “quasispecies swarm” (Pawlotsky, 2006). Within the swarm exist many preformed variants that are resistant to DAAs and these often emerge when the drugs are present. Resistance to DAAs will likely be the major challenge of HCV therapy in years to come.
Besides the unspecific peginterferon and RBV that make up the current standard of care (SOC) and the DAAs that target viral enzymes and are thus prone to resistance development, there is a third group of therapeutics in various stages of development: these compounds target host factors that the virus needs to replicate and have hence been designated host-targeting agents (HTAs) (Table 1). The development of these compounds is based on our increasing understanding of the molecular biology of HCV and its interaction with the host cell. Theoretically, targeting host cell factors instead of viral gene products should combine a specific anti-viral action with a higher barrier to resistance and broader genotype specificity. This is because, different from viral targets, host cell factors are encoded in the host genome and hence not subject to the high genetic variability of the viral genome. While this may be a major advantage of HTAs compared to DAAs, it may come at the price of an increased potential for toxic side effects. Moreover, although much less frequently compared to viral genomes, sequence variation in host factors such as single nucleotide polymorphisms exist and in some cases strongly influence the course of the disease or the response to therapy (Balagopal et al., 2010). In this paper we describe current anti-HCV approaches that target host factors as well as their molecular mechanism of action and current stage of development.
Molecular Biology of HCV
HCV’s 9.6 kb RNA-genome encodes, in a single open reading frame, a polyprotein that is post-translationally cleaved by cellular and viral proteases, releasing at least ten individual viral proteins (Lindenbach et al., 2006). Polyprotein translation is driven by an internal ribosomal entry site (IRES) located in the highly conserved 5′ N-terminal region of the viral RNA genome. The IRES mediates the initial interaction of the viral genome released from the invading viral particle and a ribosome. The viral proteins are divided into the structural proteins (core, E1, and E2), the small ion channel protein p7, and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Cleavages from the N-terminus through p7 are mediated by host-encoded proteases, cleavage between NS2 and NS3 is mediated by the autoprotease NS2/NS3, and the remaining cleavages in the C-terminal part of the polyprotein are mediated by the NS3/NS4A serine protease.
The viral replicase complex that generates copies of the RNA genome is made up of the non-structural proteins NS3, NS4A, NS4B, NS5A, NS5B, and a number of host factors. The central RNA-dependent RNA-polymerase activity is contributed by NS5B. The structural proteins, i.e., core, E1, and E2, form the virus particle: the nucleocapsid is built out of copies of the core protein, and the envelope glycoprotein E1 and E2 form heterodimers embedded in the host-derived membrane envelope. The ion channel p7 has a partly understood function in assembly and release of infectious particles.
Host Factors Involved in the HCV Replication Cycle as Antiviral Targets
Broadly, the viral replication cycle (Figure 1) has three stages: entry, replication, and assembly/release (Lindenbach et al., 2006). All stages depend on various host encoded factors (Bode et al., 2009; Georgel et al., 2010). Targeting these host factors is an attractive option for future HCV therapies. The intention is to minimize development of HCV resistance and to enable efficient treatment of all HCV genotypes. This approach has been pursued successfully in the HIV field, where maraviroc (Selzentry, Celsentri), a small molecule compound that binds to the essential HIV co-receptor chemokine receptor type 5 (CCR5) and prevents its interaction with HIV, is now in clinical use (Gilliam et al., 2011).
For HCV entry, four essential cellular entry factors have been described: the tetraspanin CD81, the scavenger receptor class B member I (SR-BI), and the tight junction components claudin-1 (CLDN1) and occludin (OCLN) (Zeisel et al., 2009). Out of these four, SR-BI and CLDN1 are highly expressed in liver and may thus contribute to the hepatotropism of HCV. In the case of CD81 and OCLN, the human homologue can support HCV entry much more efficiently than the murine form which could explain host restriction and is important information with a view to developing a small animal model of HCV infection (Flint et al., 2006; Ploss et al., 2009). The above set of four essential entry factors is believed to be complete in the sense that their presence is sufficient to allow HCV glycoprotein (HCVgp)-mediated entry in all cell types tested so far (Ploss et al., 2009). Whether the high-density lipoprotein (LDL) receptor has a role in the in vivo setting is still a matter of debate (Owen et al., 2009). Very recently, epidermal growth factor receptor (EGFR) has also been described as a potential additional HCV entry factor (Lupberger et al., 2011). Following internalization by receptor-mediated endocytosis the nucleocapsid is released by a pH-dependent fusion event of the glycoproteins with the endosome.
It has been shown that neutralizing antibodies against CD81 can block HCV infection in vitro and in vivo and are currently in preclinical development (Meuleman et al., 2008). Prophylactic injection of monoclonal anti-CD81 antibodies prevented infection of human liver-uPA-SCID mice. However once infection had been established, no significant difference in viral load was observed between anti-CD81-treated and control animals (irrelevant antibody). Another approach to inhibiting HCV entry is to develop small molecules targeting one of the entry factors. ITX 5061 is an orally bioavailable small molecule compound that enhances HDL levels in animals and patients by targeting the SR-BI. ITX 5061 has a good safety profile in animal toxicology studies and in clinical studies involving over 250 subjects. This compound exhibits picomolar potency in inhibiting both genotype 1 and genotype 2 HCV presumably by blocking the interaction between the HCV glycoprotein E2 and SR-BI (Syder et al., 2011). Currently, the safety of ITX 5061 is being evaluated in HCV-treatment naive patients (phase 1b). In March 2011, the company iTherx announced that it has also commenced patient recruitment in an open-label, proof-of-concept phase 1b study of ITX 5061 in liver transplant patients with HCV. For CLDN1 and OCLN, efficient neutralizing antibodies have not yet been developed but due to their essential function in HCV entry both are potential targets analogous to CD81 antibodies.
Such entry blockers are especially interesting in the context of liver transplant where an efficient means of blocking cell entry might help to prevent re-infection of the liver graft after transplantation.
Very recently, it has been reported that erlotinib can block HCV entry at post-binding steps at similar timepoints as anti-CD81 antibodies by inhibition of the activity of EGFR (Lupberger et al., 2011). Using FRET analyses and tagged entry factors expressed in polarized HepG2 cells it was suggested that EGFR activity is required for the formation of CD81-claudin-1 co-receptor associations in liver-derived cell lines. Furthermore, erlotinib also could control viral spread and dissemination in cell culture. Importantly, treatment of animals with the EGFR inhibitor erlotinib, which is in clinical use for the treatment of non-small cell lung cancer and pancreatic cancer, resulted in a somewhat delayed HCV infection and decreased viral load. However, whether erlotinib has a clinical benefit in the setting of HCV infection that outweighs its side effects has to be evaluated in the future.
HCV RNA replication
After fusion of the viral envelope with the endosomal membrane, the nucleocapsid is released into the cytosol and the genomic RNA is then released and serves as an mRNA template for polyprotein translation and subsequently as a template for RNA replication (Lindenbach et al., 2006). Translation is initiated by the association of ribosomes with the HCV IRES. After co- and post-translational modification, mature viral proteins can then form replication complexes and assemble into new virions.
By the use of siRNA-based and other screening approaches, several dozens of host factors involved in or, in some cases, even essential for HCV RNA replication have been identified in recent years. A representative factor that generated a lot of interest is cyclophilin A (CypA), a cellular cis-trans-prolyl isomerase that is required for HCV RNA replication and probably also assembly (Watashi et al., 2005; Yang et al., 2008). It has been shown in vitro that CypA binds to HCV NS5A and can facilitate replication through an unknown mechanism. Moreover, it has been shown that HCV is much more sensitive to CypA inhibitors in the presence of an intact NS2/3 junction, indicating that important interactions between HCV and CypA may occur outside NS5A (Ciesek et al., 2009; Kaul et al., 2009). Importantly, CypA seems to be important for all HCV genotypes. Interestingly, the first compound exerting an inhibitory effect on HCV replication by targeting CypA was the immunosuppressant cyclosporin A (Inoue et al., 2007; Watashi et al., 2005). Subsequently, cyclosporin A derivatives without immunosuppressive properties were developed as potential antivirals. The CypA inhibitor alisporivir, formerly known as Debio-025 (Flisiak et al., 2009), is currently in a phase III trial for the treatment of treatment-naive HCV genotype 1 patients; patient recruitment for this study is ongoing. At the EASL International Liver Congress in April 2011, results of a phase II study were presented: a triple regimen consisting of peginterferon/RBV plus alisporivir for 48 weeks achieved SVR in 76% of the patients compared to 55% in the control arm treated with peginterferon/RBV alone. The study involved nearly 300 previously untreated patients infected with HCV genotype 1. Treatment with alisporivir resulted in a low incidence of adverse events, with discontinuation rates comparable between intervention and control groups. As all HCV genotypes seem to be similarly dependent on CypA, alisporivir may offer an effective treatment option for a broad range of HCV genotypes. In fact, alisporivir has also shown antiviral activity against other common HCV genotypes (2-4) in clinical studies. Interestingly, this drug might also have the potential to be key to an interferon-free regimen since cases of SVR after 29 days of alisporivir monotherapy were reported in HCV genotype 3 infected individuals. Moreover, the genetic barrier for development of viral resistance seems to be very high in comparison to HCV polymerase or protease inhibitors both in vitro and in vivo. Thus alisporivir arguably represents the most advanced and most promising anti-HCV drug targeting a host factor at this point.
Another host factor critical for HCV genome replication has recently been described by several groups: the enzyme phosphatidyinositol-4-kinase III alpha (PI4KIII-a) binds to HCV NS5A and its enzymatic activity is required for efficient HCV RNA replication (Berger et al., 2009; Borawski et al., 2009; Tai et al., 2009; Trotard et al., 2009). Data by Reiss et al. (2011) suggest that the direct activation of this lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex. Whether this kinase is also a suitable antiviral target for the treatment of HCV and if it is possible to design a potent inhibitor with tolerable side effects in vivo remain to be determined.
An unusual and scientifically intriguing host encoded antiviral target is the microRNA 122 (miR-122) (Jopling et al., 2005). miR-122 is an abundant liver-specific miRNA which is crucial for efficient HCV RNA replication in cultured Huh7 cells stably expressing HCV replicons (Esau et al., 2006). It stimulates HCV RNA replication and translation through interaction with two adjacent sites downstream of stem loop I within the HCV 5′ untranslated region (Chang et al., 2008; Henke et al., 2008). Moreover, a recent study found that among chronically HCV-infected individuals pre-treatment intrahepatic miR-122 levels were significantly lower among patients who responded poorly to interferon therapy (Sarasin-Filipowicz et al., 2009). Santaris Pharma has developed a locked nucleic acid-modified oligonucleotide (miravirsen or SPC3649) complementary to the 5′-end of miR-122 that resulted in functional inactivation of miRNA-122. Miravirsen was shown to be active in HCV positive chimpanzees, markedly reducing HCV RNA replication and showing no significant side effects except for a profound decrease in serum cholesterol levels (Lanford et al., 2010). Moreover, miravirsen-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1-6 in vitro (Li et al., 2011). Thus miravirsen holds promise as a new antiviral therapy with a high barrier to resistance and a tolerable side effect profile. Moreover, functional inactivation of a microRNA to treat an infectious disease represents a truly novel therapeutic paradigm far beyond the HCV or virology field. A phase II trial of miravirsen in chronically HCV infected individuals is currently recruiting patients.
Finally, HCV RNA replication seems to be intricately linked to the cholesterol and fatty acid biosynthesis pathways. Statins, a widely used group of drugs that target cholesterol metabolism by inhibition of 3-hydroxyl-3-methylglutary coenzyme A (HMG CoA) reductase, have been reported to inhibit HCV RNA replication in vitro, albeit the exact mechanism is still under investigation (Ikeda et al., 2006). Interestingly, the addition of fluvastatin to peginterferon/RBV has been reported to improve rapid viral response rates (RVR) but not SVR in HIV/HCV genotype 1 co-infected patients (Milazzo et al., 2010) and SVR rates in diabetic patients with chronic hepatitis C (Rao and Pandya, 2011). However, in another study atorvastatin showed no effect on HCV RNA and statins are currently not considered part of the standard of care (O’Leary et al., 2007).
Assembly and release
The final stages, i.e., assembly and release of progeny virions, are as yet the least understood part of the HCV replication cycle. Several laboratories worldwide are involved in the hunt for the critical host factors required for HCV assembly and release. Any factor identified would then become a potential target for future antiviral strategies. An early example of a compound targeting the final replication cycle stages was celgosivir, an oral prodrug of the natural product castanospermine which is derived from the Australian chestnut. Celgosivir is a potent inhibitor of alpha-glucosidase I, a host enzyme required for viral assembly, release, and infectivity (Durantel, 2009). Alpha-glucosidase I inhibitors have been shown to inhibit the replication of a broad range of enveloped viruses by preventing the correct folding of their envelope glycoproteins. The drug demonstrated broad antiviral activity in vitro and although the agent was not active as a monotherapy for HCV infection, it demonstrated a synergistic effect in combination with the current standard of care, both in vitro and in phase II clinical trials. However, development of celgosivir has recently been terminated due to safety concerns. Another cellular system emerging as central to HCV virion production is the LDL and VLDL pathways with apoE and microsomal triglyceride transfer protein being thought to have central roles (Chang et al., 2007; Gastaminza et al., 2008; Huang et al., 2007). However, this interesting and rapidly moving research field has yet to produce promising therapeutic footholds.
Conclusions and Future Perspectives
The growing understanding of the molecular interactions between HCV and the host cell has revealed numerous cellular factors that the virus is dependent on in all stages of its replication cycle. Many of these are promising therapeutic targets, enabling a higher genetic barrier to resistance development, although side effects are certainly a concern when therapeutics have host instead of viral components as their targets. Nonetheless, several HTAs that target entry (CD81, SR-BI) and RNA replication (miR-122, CypA) factors are in clinical development. At this point in time the CypA inhibitor alisporivir may be the most advanced in development. For anti-HCV HTAs, as for any promising compound, it is difficult to predict which ones, if any, will eventually make it to market approval. Nonetheless, HTAs may become an important addition to the toolbox as we brace for the emergence of resistant HCV isolates as a result of the introduction of DAAs into the clinical practice. Moreover, they may become an essential part of interferon free treatment regimens as we learn more about the limitations of purely DAA-based regimens. This would be a major contribution since an interferon-free regimen is the ultimate goal of HCV therapy.
M.P.M. has been a speaker for Bristol-Myers Squibb, Gilead, GlaxoSmithKline, Merck, and Roche. He has been a consultant for Boehringer Ingelheim, Bristol-Myers Squibb, Gilead, GlaxoSmithKline, Merck, Novartis, Roche, Tibotec, and Vertex. He has received grant/research support from Boehringer Ingelheim, Bristol Myers Squibb, Gilead, Roche, and Novartis.
S.C. has received grant support from Novartis.
Prof. Dr. med. Michael P. Manns, Medizinische Hochschule Hannover, Klinik für Gastroenterologie, Hepatologie und Endokrinologie, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
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[Discovery Medicine; ISSN: 1539-6509; Discov Med 12(64):237-244, September 2011. Copyright © Discovery Medicine. All rights reserved.]