Drug Discovery Today: Disease Mechanisms
`
`Vol. 3, No. 4 2006
`
`Editors-in-Chief
`
`Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA
`Charles Lowenstein – The John Hopkins School of Medicine, Baltimore, USA
`
`Gastrointestinal disorders
`
`DRUG DISCOVERY
`
`TODAY
`DISEASE
`MECHANISMS
`
`Propagation of hepatitis C virus
`infection: Elucidating targets for
`therapeutic intervention
`David E. Kaplan
`Division of Gastroenterology, University of Pennsylvania, 600 CRB, 415 Curie Blvd., Philadelphia, PA 19104, USA
`
`The hepatitis C virus affects 2–3% of the world’s popu-
`
`lation. The inability to efficiently propagate the virus in
`
`cell-culture greatly impaired the study of the viral life
`
`cycle. Recent advances have facilitated extensive study
`
`of viral entry, translation, proteolysis, and replication
`
`that will foster intelligent drug design for this infection,
`
`a major cause of cirrhosis and hepatocellular carci-
`
`noma worldwide.
`
`Introduction
`The hepatitis C virus (HCV), most commonly via injection
`drug use or blood transfusion, after a generally asymptomatic
`acute infection, persists in 50–75% of exposed subjects. Per-
`sistent infection is associated with chronic hepatitis, which
`progresses to cirrhosis at a rate of approximately 10% per
`decade [1]. Current first-line therapy for chronic hepatitis C
`combines two non-HCV specific medications, interferon-
`alpha (IFNa) injections and oral ribavirin, a nucleoside ana-
`logue. Sustained virological response (SVR), the absence of
`detectable viremia in peripheral blood 6 months after dis-
`continuation of a 24–48 week course of IFNa/ribavirin ther-
`apy, occurs in fewer than half of patients infected with the
`most common genotype of HCV (genotype 1) [2,3]. IFNa/
`ribavirin is associated with significant treatment-related fati-
`gue, depression, hemolytic anemia and other serious side
`effects. Thus, unsatisfactory response rates, poor tolerability
`and frequent adverse events have stirred strong interest in
`
`E-mail address: D.E. Kaplan (dakaplan@mail.med.upenn.edu)
`
`1740-6765/$ ß 2006 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddmec.2006.10.004
`
`Section Editor:
`Yu-Xiao Yang – University of Pennsylvania, Philadelphia, USA
`
`specific antiviral therapies, the development of which has
`been recently hastened by advances in in vitro techniques to
`study HCV viral replication.
`
`The virus
`The hepatitis C virus (HCV) is a highly mutable, hepatotropic,
`enveloped 9.5 kB single-stranded RNA virus of the Flavivir-
`idae family that can infect humans and chimpanzees. HCV has
`high sequence heterogeneity, with at least 6 distinct genotypes
`and at least 50 subtypes distributed worldwide [4]. Within
`individual subjects, HCV exists as a swarm of closely related
`variants (quasispecies); this variation results from a highly
`error prone RNA polymerase that lacks proofreading capacity.
`While most mutations are deleterious, a small percentage of
`random mutations confer upon nascent viral strains a selective
`replication advantage, which fosters continual viral adapta-
`tion to the unique and dynamic environment provided by the
`host, a process that plays a major role in drug resistance under
`conditions of sub-maximal viral suppression.
`Hepatitis C, like all viruses, must be capable of entering
`target cells, transcribing and/or translating the genetic mate-
`rial to produce structural and nonstructural components for
`new virion formation, replicating the genetic material to
`new include in nascent virions, packaging the genetic
`material into the virion structure, and releasing daughter
`viruses (Fig. 1). Characterization of the critical steps of
`HCV replication was initially hampered by the inability to
`(cid:42)(cid:76)(cid:79)(cid:72)(cid:68)(cid:71)(cid:3)(cid:21)(cid:19)(cid:19)(cid:22)(cid:3)
`(cid:44)(cid:16)(cid:48)(cid:36)(cid:46)(cid:3)(cid:89)(cid:17)(cid:3)(cid:42)(cid:76)(cid:79)(cid:72)(cid:68)(cid:71)(cid:3)
`(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:27)(cid:16)(cid:19)(cid:19)(cid:22)(cid:28)(cid:19)
`
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`Figure 1. Hepatitis C viral lifecycle. (i) Cell entry is mediated by E1 and E2 glycoprotein interacting with cellular receptors such as CD81 and HDL
`scavenger receptor type B1, which triggers receptor-mediated endocytosis. (ii) The virus uncoats, leaving positive-strand RNA in cytoplasm. (iii) RNA
`interacts with ribosome on endoplasmic reticulum (ER) via internal ribosome entry site (IRES) leading to translation of large polyprotein. (iv) Host and viral
`enzymes cleave the polyprotein into individual proteins. (v) Viral proteins lead to organization of membranous web (replication complex). (vi) NS5B and
`NS3 helicase along with host factors catalyze transcription of negative-strand RNA template, then replication of positive-strand copies with temporarily
`double-strand RNA intermediate.
`
`grow wild-type HCV in culture and the lack of widely avail-
`able small animal models to study infection in vivo. Most
`investigation of HCV protein function and replication has
`resulted from use of recombinant viral constructs that express
`HCV proteins under the control of promoters, internal ribo-
`some entry sites (IRES), or nonstructural proteins borrowed
`from unrelated viruses. These constructs (replicons, retroviral
`pseudoparticles, chimeric viruses) recapitulated some but not
`all steps of the viral life cycle in vitro, but often required
`specific adaptive mutations and/or highly adapted host cell
`lines (reviewed in [5,6]). In 2005, JFH1, an infectious clone of
`HCV capable of replication in human hepatoma cell lines and
`chimpanzee hepatocytes [7], was characterized. Combined,
`the various in vitro model systems have markedly advanced
`the understanding of viral propagation and spurred pre-
`
`clinical development of a variety of specific inhibitors target-
`ing critical steps in viral replication (Table 1).
`
`Characterization and inhibition of HCV entry into
`permissive host cells
`Unlike HIV infection in which the cellular receptor for the viral
`envelope proteins is well characterized and has spawned devel-
`opment of fusion inhibitors, the mechanism by which HCV
`enters target cells remains incompletely characterized. The
`circulating virion includes the a nucleocapsid consisting of
`the HCV Core protein and positive-strand ssRNA surrounded
`by an envelope, which consists of a lipid bilayer derived from
`the previous host cell’s membrane and HCV envelope glyco-
`proteins E1 and E2. E1 and E2 form heterogeneous mixtures of
`covalently- and noncovalently-associated heterodimers that
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`Vol. 3, No. 4 2006
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`Drug Discovery Today: Disease Mechanisms | Gastrointestinal disorders
`
`Table 1. Viral and host cellular protein targets for specific inhibitor therapya
`
`Therapy against
`target
`
`Stage of
`development
`
`Advantages and/or
`disadvantages
`
`NS3/4 protease
`
`Peptidomimetic
`
`Phase II
`
`Potent as monotherapy– 3–4 log10 drop;
`viral escape; strain-specificity
`
`NS5B polymerase
`
`Nucleotide analogue
`Non-nucleotide inhibitors
`(benzimidazole, benzothiadiazine,
`thiophene caroboxylic acids)
`
`Phase III
`
`Low potency as monotherapy?
`Adverse effects?
`Viral escape
`
`NS3 helicase
`
`IRESb
`
`Modified benzimidazoles,
`ribavirin esters,
`tropolone analogues
`
`Antisense
`shRNA
`siRNA
`Ribozyme
`Aptamers
`Peptide-nucleic acids
`Other small molecule
`
`Phase I?
`
`Unknown effects in vivo
`
`Phase II
`
`Delivery mechanism
`Toxicity?
`Potentially more resistant to
`viral escape due to conservation
`of IRES
`Cross-genotype activity
`
`Cell entry
`(CD81, SR-B1c,
`d
`DC-SIGN
`)
`
`Particle formation
`(lipids, glycoproteins)
`
`Blocking antibody
`(endogenous, exogenous)
`Small molecules
`
`Alpha-glucosidase inhibitors
`Prenylation inhibitors
`Sphingolipidation blockers
`
`Phase I
`
`Strain specificity
`
`Viral escape
`
`Pre-clinical
`
`Unknown
`
`a Published safety and efficacy data are very limited—advantages/disadvantages are highly speculative.
`b Internal ribosome entry site.
`c HDL scavenger receptor type B1.
`d Dendritic cell-specific ICAM3 grabbing nonintegrin.
`
`Who is working on
`the target (group and
`institute or company)
`
`Refs
`
`Vertex
`
`Schering-Plough
`Gilead
`Intermune
`Others
`
`Idenix
`Roche
`XTL
`Pharmasset
`Viropharma
`Others
`
`Vertex
`
`Viral Genomix
`AVI Biopharma
`Others
`
`[25,26]
`
`[30,31]
`
`[33,34]
`
`[18,19,23]
`
`Innogenetics
`XTL
`Others
`
`Migenix
`
`[13]
`
`[36,38–41]
`
`mediate viral entry. In cell culture systems, mainly utilizing
`viral pseudoparticles and more recently the JFH1 clone, viral
`entry occurs via low-pH-dependent, clathrin-mediated recep-
`tor-mediated endocytosis [8]. The E2 glycoprotein mediates
`binding to cellular receptors such as CD81, a tetraspannin
`receptor expressed on hepatocytes among other cell types,
`and the HDL receptor scavenger receptor class B type 1 (SR-
`B1), blockade of which inhibits E2-mediated viral entry in vitro
`[9,10]. Other putative co-receptors including the LDL-receptor,
`C-type lectins, L-SIGN and DC-SIGN [11], may be involved.
`Recent data identifies a conserved GWLAGLFY motif at posi-
`tion 436 of E2 as the critical residues required for CD81-
`dependent viral entry [12]. The accessibility of virus-cell sur-
`face interactions allows consideration for antibody-based, pep-
`tidomimetic or small molecule approaches to disrupt early
`stages of cellular infection.
`Some preliminary success has been reported with envelope
`protein-including vaccines [13] (INN-0101, Innogenetics,
`Gent, Belgium, http://www.innogenetics.com/) and with
`human monoclonal antibodies against E2 (XTL-6865, XTL,
`
`Rehovot, Israel, http://www.xtlbio.com/) to augment clear-
`ance of circulating viral particles and/or inhibit cellular re-
`infection. A polyclonal immune globulin is also in develop-
`ment (Civacir, Nabi Pharmaceuticals, Boca Raton, FL USA,
`http://www.nabi.com/). At this time, data regarding specific
`small molecule inhibitors of E2-CD81 or other envelope-cell
`receptor binding have yet to be published.
`
`Genome translation
`Once HCV has entered a target cell, translation of the viral
`positive strand RNA proceeds using host cell machinery. The
`9.6 kB RNA genome encodes a single open reading frame
`0
`0
`and 3
`non-coding regions. Three hundred
`(ORF) flanked by 5
`0
`untranslated region (and begin-
`thirty nucleotides of the 5
`ning of the core protein encoding nucleotides) of the virus
`contain the internal ribosome entry site (IRES), a highly
`structured domain critical for initiation of HCV polyprotein
`translation. The HCV IRES contains
`three stem-loops
`(domains II–IV) and a pseudoknot [14]. Translation mediated
`0
`by the HCV IRES eliminates the needs for a 5
`cap structure on
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`RNA, ATP-dependent scanning, and multiple canonical
`initiation factors (reviewed in [15]). The human La autoanti-
`gen appears required for the interaction of the IRES and the
`40S ribosome subunit [16]. Due to constraints on stem-loop
`base-pairing, only a minority of nucleotides can be substi-
`tuted without impairment IRES functionality and thus the
`region is highly conserved among HCV subtypes [17]. These
`constraints similarly make the IRES an attractive target for
`therapeutics.
`Initial approaches to inhibit IRES function have focused on
`0
`antisense oligonucleotides to block the interaction of the 5
`IRES with the 40S ribosome [18] and two agents are currently
`in phase I/II trials (VGX-410C, Viral Genomix Inc. Blue Bell,
`PA, USA, http://www.viralgenomix.com/; AVI-4065, AVI Bio-
`pharma, Portland, OR, USA, http://www.avibio.com/). ISIS
`14803 (ISIS Pharmaceuticals, Carlsbad, CA, USA, http://
`www.isispharm.com/) showed some promise but questions
`regarding toxicity in early development [19] and was sus-
`pended. Other RNA-based IRES inhibitors such as RNAi,
`shRNA, ribozymes, aptamers, and peptide nucleic acids that
`disrupt IRES function have been shown to inhibit in vitro
`replication of HCV RNA [20,21]. A caveat to this approach is
`that there is some evidence that HCV core protein interferes
`with siRNA activity [22]. A novel competitive La peptide
`variant [23] has been shown in vitro to impair HCV replication
`via IRES inhibition and may be a candidate for further devel-
`opment.
`
`Protein processing
`After IRES engagement with ribosomes, viral RNA is trans-
`lated as a single long polyprotein which requires proteolytic
`cleavage (Fig. 2). Host cell signal peptidases cleave at the
`Core/E1, E1/E2, E2/p7 and p7/NS2 junctions. The Core pro-
`tein forms the viral nucleocapsid which localizes replicated
`
`RNA via a highly conserved RNA binding domain, with the
`budding envelope studded with E1 and E2 heterodimers. The
`p7 protein encodes an ion channel critical for viral infectiv-
`ity. The NS2/NS3 cleavage is catalyzed by NS2, a transmem-
`brane zinc-dependent protease, a step critical to efficient
`function of the NS3 protein; NS2 may also regulate certain
`cellular factors involved with apoptosis and fibrosis. The
`remaining cleavages, those at NS3/NS4, NS4A/NS4B, NS4B/
`NS5A, and NS5A/NS5B, are catalyzed by the NS3 using NS4A
`as a cofactor, termed the NS3/4A serine protease. Addition-
`ally, NS3 has a separate NTP-dependent RNA helicase
`domain. NS4A is a cofactor for the NS3 protease and recent
`data indicate that the NS3/4A protease blocks signaling path-
`ways that trigger NF-kB nuclear translocation and interferon-
`response factor pathways by cleaving an adaptor protein
`necessary for RIG-I signaling, called Cardif (also known as
`MAVS, IPS-1 and VISA) [24]. The role of NS4B is unclear, but it
`may along with NS4A regulate viral translation and replica-
`tion. The NS5A protein contains a PKR binding region and
`may modulate sensitivity to interferon-alpha and NS5B is the
`viral RNA-dependent RNA polymerase.
`As described,
`the NS3 protein has three functional
`domains: an autoprotease that cleaves the NS2/3 junction
`using NS2 as a cofactor, an RNA helicase domain and the NS3/
`4A protease. The NS3/4A serine protease in addition to its
`proteolytic function also appears to regulate the NS3 helicase
`domain. Due to the critical role that the NS3/4 protease plays
`in various aspects of viral replication, development of a
`specific inhibitor has been a therapeutic priority. BILN
`2061 (Boehringer Ingelheim GmbH, Ingelheim, Germany,
`http://www.boehringer-ingelheim.com/), the first of these
`inhibitors to enter clinical trials achieved 2–3 log10 reduc-
`tions in HCV RNA titers [25] in genotype 1 infections. Both in
`vitro and in vivo BILN 2061 was significantly less effective
`
`0
`UTR (untranslated region) contains the
`Figure 2. Hepatitis C viral translation and proteolysis. Translation and proteolysis of the HCV polyprotein. 5
`internal ribosome entry site, which facilitates interaction of positive-strand RNA with the 40S ribosome resulting in translation of a single polyprotein of
`3011 amino acids. Numerals indicate amino acid positions for subsequent cleavages. Blue triangles indicate cleavages catalyzed by host cell signal peptide
`peptidases. The green triangle shows cleavage site of NS2/3 autoprotease. Red triangles indicate cleavage sites for the NS3/4A protease.
`
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`against genotype non-1 NS3/4A proteases. Development of
`BILN 2061 was suspended due to cardiotoxicity in non-
`human primates given the drug. VX-950 (Vertex Pharmaceu-
`ticals, Cambridge MA USA, [26], http://www.vpharm.com/)
`and SCH 503034 (Schering-Plough, Kenilworth NJ USA,
`http://www.schering-plough.com/) are currently in phase II
`and at least two more candidates (GS 3192/ACH806, Gilead,
`Foster City CA USA, http://www.gilead.com/; ITMN 191,
`Intermune, Brisbane CA, USA, http://www.intermune.com/)
`are in phase I testing with a host of other candidate drugs
`nearing clinical testing. Published safety, tolerability, and
`efficacy data for this class of drugs are limited, but VX-950
`administered orally three times daily led to median 3 log10
`reductions in HCV RNA after 3 days and 4.4 log10 reductions
`after 14 days [26] in a phase II trial.
`
`The NS3 helicase enzymatically unwinds duplex RNA
`structures by disrupting the hydrogen bonds that keep the
`two strands together, an activity that requires hydrolysis of
`nucleoside triphosphate (NTP). Potential specific inhibitors
`of NTPase/helicases theoretically could function by inhibit-
`ing NTP binding or NTPase activity, uncoupling NTP hydro-
`lysis from the unwinding reaction, competing with dsRNA
`binding and/or by blocking translocation of the NTPase/heli-
`case along the dsRNA and thus blocking propagation of
`unwinding [32]. Peptide libraries have identified aminophe-
`nylbenzimidazole moieties [32],
`imidazo[4,5-d]pyridazine
`nucleosides, halogenated benzimidazoles, halogenated ben-
`0
`-O-(4-fluorosulphonylbenzoyl)-esters of riba-
`zotriazoles, 5
`virin [33], and bromo- and morpholinomethyl-analogues
`of tropolone [34] as potential inhibitors of the NS3 helicase.
`
`Genomic replication
`The primary goal of the viral proteins is to coordinate the
`replication of viral RNA and the packaging new RNA strands
`into nascent structural units that can be extruded. As a
`positive-strand RNA virus, RNA replication depends on the
`creation of a negative-strand RNA template, from which new
`positive-strands can be duplicated. RNA replication occurs in
`the cytoplasm facilitated by the formation of a membrane-
`associated complex involving all of the nonstructural viral
`proteins as well as host factors [27]. Two viral enzymes
`play the dominant roles in this process: the NS5B RNA, the
`RNA-dependent RNA polymerase and the NS3 helicase.
`The NS5B polymerase is a right hand structured polymerase
`with thumb, palm and finger domains and a completely
`encircled active site [28], for which both nucleotide and
`non-nucleoside inhibitors have been identified. Nucleotide
`analogs compete with nucleotide binding at the polymerase
`active site and can cause chain termination upon incorpora-
`tion into the RNA molecules [29]. Non-nucleoside inhibitors of
`NS5B RNA synthesis activity mainly consist of a heterogeneous
`group of benzimidazole, benzothiadiazine, and thiophene
`carboxylic-acid-based compounds that bind to three non-over-
`lapping binding sites on NS5B [30] and work primarily by
`allosterically inhibiting formation of replicase complexes.
`There are at least five RNA polymerase inhibitors in phase I–
`III human clinical testing at present: NM283 (Idenix, Cam-
`bridge MA, USA, http://www.idenix.com/), R1626 (Roche,
`Nutley NJ USA, http://www.roche.com/), XTL-2125 (XTL,
`Rehovot, Israel), PSI-6130 (Pharmasset Inc., Princeton, NJ
`USA http://www.pharmasset.com/) and HCV-796 (Viro-
`pharma, Exton PA USA, http://www.viropharma.com/). Cur-
`rently, safety and tolerability data are limited, but preliminary
`0
`results from NM283, a prodrug of 2
`-C-methylcytidine, indi-
`cate a fairly modest reductions in HCV RNA titers (<1 log10
`decrease)
`for monotherapy but virologic response rates
`similar to interferon/ribavirin when used in combination
`with interferon [31].
`
`Inhibitors of viral particle formation
`In order for new virions to be completed, nascent positive
`strand HCV RNA must be packaged into structural units. The
`replication of HCV RNA occurs in membrane-associated
`complexes, and the structural units are assembled closely
`associated with membranes. This membrane association
`depends on glycosylation of proteins and lipophilic protein
`modifications, processes that could be considered for thera-
`peutic interference.
`The importance of lipids and HCV was first suggested by
`the interaction of HCV with the LDL receptor and/or HDL
`scavenger receptor B1. Evidence now suggests that HMG-coA
`(fluvastatin > atorvastatin,
`reductase
`inhibitors
`lovasta-
`tin > simvastatin) do indeed have anti-HCV activity [35],
`but surprisingly that this inhibition more likely occurs at
`the level of viral replication, not at the level of cell entry, and
`that these effects are reversed by the addition of cholesterol
`biosynthesis metabolites [35–37]. Studies in HCV replicon
`systems further demonstrate that HCV replication is intri-
`cately controlled by fatty acid and cholesterol metabolism;
`HCV RNA replication is inhibited in vitro with polyunsatu-
`rated fatty acids and acetyl-coA carboxylase inhibitors [37]. In
`culture, the combination of interferon-alpha and fluvastatin
`exhibited strong synergistic inhibitory effects on HCV RNA
`replication suggesting that fluvastatin in particular, but
`potentially other statins, could be potentially useful as an
`adjunct to interferon-alpha [35].
`An offshoot of cholesterol metabolism, the prenylation
`pathway, also appears to be important in viral particle assem-
`bly for the unrelated hepatitis D virus [38,39] and the findings
`from the HCV replicon system also suggest dependence of
`HCV particle production on a prenylated substrate [36].
`Geranylgeranyl diphosphate is derived from farnesyl dipho-
`sphate itself a product of the cholesterol biosynthesis path-
`way (Fig. 3). Farnesylation and geranylgeranylation (together
`termed prenylation) are catalyzed by specific enzymes (far-
`nesyl transferase, geranylgeranyl transferase I and II) for
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`Figure 3. Cholesterol biosynthesis pathways and potential enzymatic targets affecting HCV RNA replication. Farnesylation geranylgeranylation signal
`sequences consist of CXXK and CXXL motifs, respectively, resulting in lipidation of cysteine residues and cleavage of XXK and XXL residues. Lipidation
`fosters membrane localization and possibly interaction with the multiple-drug resistance (MDR) transporter.
`
`which specific small molecule inhibitors exist. Prenylation
`fosters membrane association of the modified target protein
`and might also facilitate extrusion from the cell via the
`multiple drug resistance transporter. Although the specific
`proteins of HCV that are dependent on prenylation for their
`function have yet to be determined, theoretically inhibition of
`prenylation might be another avenue for drug development in
`HCV. Recently, a sphingolipid modification of NS5B necessary
`for linking NS5B to lipid rafts, a step thought to be critical for
`the association of NS5B with other membrane bound viral
`proteins, was found to be sensitive to inhibition in vitro by a
`secondary fungal metabolite [40]. Human cellular alpha-glu-
`cosidase blockade has been shown to inhibit glycosylation of
`various HCV proteins and may inhibit viral particle assembly
`[41]; Celgosivir (MBI-3253, Migenix, Vancouver BC Canada,
`http://www.migenix.com/), an oral prodrug of castanosper-
`mine, a natural product derived from the Australian Black
`Bean chestnut tree, targets this host cell enzyme and may
`have efficacy. Thus, the interaction of viral proteins and
`subcellular membranes represents a relatively novel area
`for development.
`
`Conclusion
`Specific antiviral therapies for hepatitis C will fill a critical
`unmet demand for patients with chronic HCV that have
`either not responded to, are unable to receive, or are unable
`to tolerate interferon-based therapy. Other types of therapy
`in active development, but beyond the scope of this review,
`include novel or improved immunomodulators such as TLR7
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`agonists, thymosin, modified interferons, and therapeutic
`vaccines. Although invaluable for initial preclinical develop-
`ment, the remarkable advances in HCV in vitro culture sys-
`tems cannot foreshadow the effects of these agents in vivo due
`to various confounders such as unexpected toxicities, diffi-
`culties in drug delivery, alternative in vivo metabolism, and
`perhaps most importantly sensitivity to viral resistance. The
`rapid mutation rate of HCV will clearly necessitate combina-
`tions of complementary direct antivirals with or without
`concomitant interferon-alpha (or other immunomodula-
`tors). Due to limited availability of safety and efficacy data
`for most of the agents in development, predictions as to when
`specific antivirals will enter the clinical armamentarium
`against HCV remain difficult; a five-year horizon would be
`optimistic. However, the wide variety of potential targets
`elucidated by in vitro studies raises the likelihood that one
`or several of the strategies under investigation will yield
`significant clinical benefits for the huge populations of
`patients worldwide infected with the hepatitis C virus.
`
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