Hepatitis C Virus NS5B RNA-Dependent RNA Polymerase Inhibitor

C H A P T E R

8 Hepatitis C Virus NS5B RNA-Dependent RNA Polymerase Inhibitor: An Integral Part of HCV Antiviral Therapy Srikanta Dash, Yucel Aydin and Christopher M. Stephens Department of Pathology and Laboratory Medicine, Tulane University Health Sciences Center, New Orleans, LA, United States

8.1 INTRODUCTION Hepatitis C is a blood-borne viral pathogen that specifically infects the liver (Stanaway et al., 2016; Gower et al., 2014; Shepard et al., 2005; Mohd Hanafiah et al., 2013). Most individuals (75%
85%) infected with HCV fail to clear infection naturally, leading to chronic infection (Shepard et al., 2005). Chronic HCV infection triggers long-lasting inflammation that can promote onset of liver diseases, such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), frequently resulting in death (El-Serag et al., 2012; El-Serag, 2012). HCV infection is the leading cause of liver transplantation in many parts of the world. In fact, a study published in 2013 reported that more than 184 million people have been infected with HCV worldwide, representing approximately 2.8% of the global population (Mohd Hanafiah et al., 2013). The incidence of HCV infection varies in different parts of the world, ranging from ,1% in Western countries to rates .3.5% in Central Asia, East Asia, North Africa, and the Middle East.

Viral Polymerases DOI: https://doi.org/10.1016/B978-0-12-815422-9.00008-5

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Accumulated evidence from clinical studies has shown that the disease course and treatment response varies widely based on the genotype of HCV responsible for the infection. Global HCV strains can be classified into seven major genotypes that exhibit approximately 30% variation between nucleotide sequences, as well as a number of subtypes that exhibit .10% genomic variation (Messina et al., 2015; Smith et al., 2016). Approximately, 46.2% of the global HCV-infected population is infected with genotype 1 of the virus, 30.1% with genotype 3, and 22.8% with genotypes 2, 4, and 6. The prevalence of genotype 5 is low, making up only 1% of all HCV infections (Messina et al., 2015). Several decades have passed since HCV was identified as a causative agent of non-A, non-B hepatitis. Cloning and sequencing of the HCV genome has enabled swift progress in many areas of HCV research, as shown in Fig. 8.1, offering encouragement for patients infected with HCV. However, research has been hampered by ongoing technical difficulties in culturing HCV. Although HCV replicates well in the human

FIGURE 8.1 Overview of rapid progress made in hepatitis C virus (HCV) research that leads to major breakthroughs in antiviral development and viral cure.

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liver, researchers have struggled to find ways to grow the virus in the laboratory. It took more than 10 years to develop a cell culture model for HCV minigenomes (the replicon model), which was later followed by development of an infectious cell-culture system using an HCV strain derived from Japanese patients with fulminant hepatitis (JFH). This system uses a Huh-7.5-based liver cell culture model to facilitate replication and assembly of the whole virus (Scheel and Rice, 2013). In vivo, after initial infection, HCV replicates in hepatocytes, the predominant cell type in the liver, producing millions of new virus particles. In chronic HCV infection, approximately 1012 HCV virions are secreted into circulation every 24 hours (Dubuisson and Cosset, 2014; Lindenbach and Rice, 2013; Bartenschlager et al., 2011). The continuous cycle of intracellular virus replication, virus release, and reinfection is central to the mechanisms of HCV persistence and pathogenesis. The observation that HCV establishes a chronic infection in most infected individuals suggests that HCV infection promotes cell survival after triggering an initial stress response (Dash et al., 2016). The molecular mechanisms by which HCV balances the negative effects of the virusinduced stress response with cell survival in order to support persistent infection in hepatocytes are not well understood and represent an important ongoing area of research in our laboratory. Elucidation of these mechanisms may increase our understanding of the interplay between long-lasting chronic HCV infection and development of hepatocellular cancer. At present, there is no vaccine available that can prevent new HCV infection by priming the immune system, and interferon-alpha (IFN-α) plus ribavirin (RBV) combination antiviral therapy has been used as the standard-of-care for patients with chronic HCV infection for many years. This treatment eliminates the virus in only a little more than half of all patients (Hoofnagle, 2009), and low responsiveness to IFN/RBV therapy for chronic HCV infection has been associated with specific interleukin-28B (IL-28B) genotypes (Lange and Zeuzem, 2011). However, recent development of a combination of direct-acting antivirals (DAAs) targeting the NS3/4A protease, NS5B polymerase, and NS5A has rapidly changed the therapeutic landscape for curing HCV infection (Pawlotsky et al., 2015). Treatments for chronic HCV infection incorporating second-generation DAA drugs are expected to soon be available in many developing countries with high infection rates, providing hope that HCV infection can be eradicated globally. However, HCV eradication will require that all infected patients receive early diagnosis and access to antiviral treatments. In the following sections, we highlight studies that led to the discovery of DAA drugs for HCV by increasing our understanding of HCV

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replication mechanisms and promoting the development of relevant model systems. Because sorting through all of the literature available in the rapidly developing field of HCV research can be daunting, we chose to focus on several key fronts in the development of drugs targeting the viral RNA polymerase.

8.2 THE HCV GENOME AND VIRAL REPLICATION HCV is an enveloped, positive-strand RNA virus belonging to the Flaviviridae family. HCV infection is initiated by the attachment and entry of viral particles into hepatocytes via receptor-mediated endocytosis (Fig. 8.2). Multiple host cell-surface proteins have been implicated in the attachment of the HCV particle to hepatocytes and entry into the cell through clathrin-mediated endocytosis (Lindenbach and Rice, 2013). During internalization, fusion of the viral envelope with the host

FIGURE 8.2 Infection cycle of hepatitis C virus (HCV). The infection of HCV is initiated by the attachment and entry of virus particles through a number of cell-surface receptors. The HCV RNA binds to ribosome and translates a single large polyprotein, which is processed into structural and nonstructural proteins. Accumulation of viral proteins induces proliferation of endoplasmic reticulum (ER)-membranes and formation of membranous web structure. HCV replication produces many new genomic positive- and negative-strand RNA. Genomic HCV positive-strand RNA packages into complete infectious virus particles that release through secretory pathway.

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endosomal membrane results in pH-dependent uncoating, releasing the HCV genomic RNA into the cytoplasm. The positive-strand RNA genome binds directly to ribosomes through an internal ribosome entry site (IRES) present in its 50 untranslated region (UTR) (Niepmann, 2013). Translation of the HCV genome leads to the production of a single large polyprotein of 3000 amino acids. This protein is subsequently cleaved by cellular and viral proteases into the structural proteins, core and envelope proteins E1 and E2, and the nonstructural (NS) proteins, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (Moradpour and Penin, 2013). The structural proteins are necessary for the formation and release of infectious virus particles, whereas the NS proteins are required for HCV RNA replication (Paul et al., 2014). HCV is an RNA virus that replicates exclusively in the cytoplasm without integrating into the host cell genome. Sustained viral replication in hepatocytes leads to increased production of genome-length, positive-strand RNA; replicative intermediate (negative-strand RNA); and viral proteins in the endoplasmic reticulum (ER). As a result, replication of HCV in hepatocytes leads to the proliferation and remodeling of the ER membranes into a structure referred to as the membranous web (Fig. 8.2). Intrahepatic HCV replication stimulates lipid metabolism, leading to the accumulation of lipid droplets in multilayer membrane vesicles, which in turn facilitates virus assembly and maturation (Aizawa et al., 2015). These ER-derived membranous vesicles support HCV replication and production of progeny viral RNA. Virus particle assembly and release occurs in the membranous web and is closely linked to very low-density lipoprotein (VLDL) synthesis and secretion (Suzuki et al., 2013). HCV RNA replication is catalyzed by the viral RNA-dependent RNA polymerase (RdRP), NS5B, which synthesizes negative-strand RNA. The NS5A protein and the protease and helicase domains of NS3 play important roles in the viral replication cycle. Additional details regarding the precise mechanisms associated with HCV replication, assembly, and secretion have been summarized in several reviews (Lindenbach and Rice, 2013; Niepmann, 2013; Moradpour and Penin, 2013; Paul et al., 2014; Aizawa et al., 2015; Suzuki et al., 2013). In principle, HCV drug development can target either viral components (e.g., HCV-specific RNA structures, such as the IRES element, viral proteins) or host factors that support virus replication. Several viral proteins and host factors essential for HCV replication have been demonstrated to be promising targets for antiviral therapy. Among these, the most successful agents developed over the last few years have targeted the HCV protease NS3/4A, NS5A, and the NS5B RNA polymerase. In this review, we focus on new DAA drugs in development targeting the protease and polymerase.

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8.3 DEVELOPMENT OF HCV-SPECIFIC DAA DRUGS TARGETING THE NS3 PROTEASE In 1992, IFN-α was approved in the United States for the treatment of hepatitis C; IFN-α monotherapy had a success rate of 20%. In 1998, the combination of IFN-α plus RBV was approved for the treatment of chronic HCV infection. Pegylated IFN-α (PEG-IFN-α) was introduced in 2011 to enhance the antiviral effects of the combination therapy. For almost a decade, the standard treatment for chronic HCV infection in the United States was a combination of PEG-IFN-α and RBV, although this combination was ineffective for around 50% of patients infected with HCV genotype 1 and 20% of patients with genotype 2 or 3 (Hadziyannis et al., 2004; Manns et al., 2001; Fried et al., 2002). Subsequent studies found that the success of clearing HCV infection with IFN/RBV therapy depended on the IL-28B genotype. An unfavorable IL-28B genotype, encoding a novel IFN-λ4 protein, was associated with poor responses to IFN-based antiviral therapy (Hayes et al., 2012). This discovery led to renewed pharmaceutical industry interest in the development of new antiviral drugs targeting NS3, NS5A, and NS5B to improve viral clearance in this set of patients (Fig. 8.3). Based on the success of HIV protease inhibitors, many studies were originally designed with the aim of developing small molecule inhibitors targeting the HCV protease (NS3/NS4A). The HCV NS3 protein has both serine protease and helicase activities that are essential for HCV replication. The catalytic activity of NS3 depends on the formation of a noncovalent complex with HCV NS4A, a protein cofactor required

FIGURE 8.3 Structure of hepatitis C virus (HCV) RNA genome. Ten different mature proteins are produced from the single large open reading frame (ORF). Type I, Type II, Type III interferons, and ribavirin specifically inhibit the internal ribosome entry site (IRES) (50 UTR) function. Core, E1, and E2 are structural proteins and NS2
NS5 are nonstructural proteins. The nonstructural proteins (NS3/4A, NS5A, and NS5B) are the targets of antiviral drug discovery. The 30 -UTR is important for HCV genome replication. Approved DAA drugs targeted to the NS3, NS5A, and NS5B are shown.

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for the proper folding and stabilization of the protease in the cellular lipid membrane. The NS3/4A protease cleaves the junctions between NS3/NS4A, NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B, whereas the helicase activity of NS3 is required for unwinding of double-strand RNA (dsRNA) during genome replication. Both the protease and helicase activities of NS3/NS4A were major targets in the development of new pharmaceuticals targeting HCV infection. In principle, inhibiting NS3 protease activity should inhibit viral replication by blocking the cleavage and maturation of the viral polyprotein. The first NS3/4A protease inhibitor developed against HCV was ciluprevir (BILN 2061), an orally bioavailable, peptidomimetic, macrocyclic drug that binds noncovalently to the active site of the protease (Lamarre et al., 2003). In a proof-of-concept study, ciluprevir monotherapy rapidly decreased HCV RNA serum levels in HCV genotype 1 patients, but severe cardiotoxicity prevented this agent from being adopted for clinical use. However, this effort led to the development of two additional protease inhibitors known as boceprevir and telaprevir. These two drugs received US Food and Drug Administration (FDA) approval in 2011 to become the first DAA drugs for treatment of chronic HCV infection. Triple therapy with PEG-IFN-α plus RBV and one of these protease inhibitors significantly improved the sustained virologic response (SVR) rate among HCV patients (Bacon et al., 2011; McHutchison et al., 2009). Although the first-generation protease inhibitor DAA drugs resulted in improved therapeutic responses, many patients still failed therapy, and 40%
60% of patients were classified as partial or null responders. These drugs have since been abandoned due to unwanted side effects and a low barrier to resistance. As shown in Fig. 8.3, a new generation of protease inhibitors, including simeprevir, paritaprevir, grazoprevir, glecaprevir, and voxilaprevir has shown increased antiviral potency, improved pharmacokinetics, broader coverage of viral genotypes, and a higher barrier to resistance (Bartenschlager et al., 2013).

8.4 DEVELOPMENT OF HCV-SPECIFIC DAA DRUGS TARGETING NS5A Progress in the realm of HCV protease inhibitors encouraged the development of antiviral drugs targeting other NS proteins of HCV. NS4B and NS5A are NS proteins essential for formation of the membranous web needed to support HCV replication and assembly in the ER (Zayas et al., 2016; Appel et al., 2008; Masaki et al, 2008; Tellinghuisen et al., 2008). NS5A also plays an important role in HCV replication by directly binding the HCV RNA at cellular lipid membranes. This interaction is essential for sustained viral replication. As a result of its critical role in viral assembly and replication, NS5A was recognized as a VIRAL POLYMERASES

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potential antiviral target by many pharmaceutical companies. Recent advancements have yielded many small molecule NS5A inhibitors that effectively block HCV replication. A notable example is daclatasvir (BMS-790052), the first NS5A inhibitor that binds to domain I of NS5A (Gao et al., 2010). Subsequent studies have shown that very low doses of daclatasvir can suppress the replication of all major HCV genotypes without any major adverse effects (Fridell et al., 2011). Consistent with these outcomes, the safety profile of many NS5A inhibitors appears to be excellent. These promising results have encouraged the development of additional NS5A inhibitors (ledipasvir, ombitasvir, velpatasvir, elbasvir, pibrentasvir) with broad antiviral activities against all HCV genotypes (Fig. 8.3), placing the NS5A inhibitors in a prominent position in the current arsenal of DAA-based antiviral therapies.

8.5 STRUCTURAL AND FUNCTIONAL STUDIES OF HCV RNA POLYMERASE Viral polymerases represent key targets for antiviral drug discovery because they are absolutely required to replicate viral genomes. These polymerases can be classified into four general types based on the nature of the viral genetic material: RdRPs, RNA-dependent DNA polymerases (RdDPs), DNA-dependent RNA polymerases (DdRPs), and DNA-dependent DNA polymerases (DdDPs) (Choi, 2012). DdRPs and DdDPs are primarily used in the replication and transcription of DNA viruses, whereas RdRPs and RdDPs are used by RNA viruses. Retroviruses, such as the human immunodeficiency virus (HIV), use RdDPs to synthesize DNA from mRNA by reverse transcription. RdRPs are employed by viruses that do not produce DNA replicative intermediates, such as the Flaviviruses, of which HCV is a member, and the Polioviruses. The Flavivirus RdRP, NS5B, also has an additional methyltransferase domain that catalyzes the methylation of the genomic 50 RNA cap (Zhou et al., 2007). In this section, we will review recent advances in the structural and functional analysis of HCV RdRP (NS5B RNA polymerase), as well as the studies that led to the development of its small molecule inhibitor sofosbuvir. The cloning and sequencing of the HCV genome in 1989 allowed for the identification of HCV NS5B as an RdRP based on the presence of the highly conserved GDD (Gly-Asp-Asp) sequence motif characteristic of RdRPs from many RNA viruses (Miller and Purcell, 1990). The gene encoding NS5B was cloned into an expression vector, and recombinant NS5B was expressed in insect cells and in Escherichia coli. The protein was purified using a nickel-nitrilotriacetic acid (Ni-NTA) affinity column and assayed for RdRP activity (Behrens et al., 1996; Lohmann et al., 1997). The full-length NS5B polymerase was not very soluble due VIRAL POLYMERASES

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to a hydrophobic, C-terminal, 21-amino-acid domain that anchors the polymerase to the ER membrane during replication (Hwang et al., 1997). Interestingly, deletion of this domain increased the protein’s solubility without compromising its enzymatic activity. These advances led to the successful expression and isolation of highly purified, soluble, recombinant NS5B (Ferrari et al., 1999; Yamashita et al., 1998). An in vitro assay showed that the affinity purified NS5B is able to copy a complete in vitro synthesized HCV-genome without any additional viral and cellular factors. It was found that HCV NS5B has binding affinities to RNA template in the order: poly (U). poly (G) . Poly (A). poly (C). The RdRp activity of HCV NS5B has a strong preference for GTP (guanosine triphosphate) as compared to GDP (guanosine 50 -diphosphate) and GMP (guanosine 50 -monophosphate). The enzyme was also found to be more selective toward nucleotides lacking 30 -OH groups than nucleotides lacking 20 -OH groups, since stronger inhibition of RdRp activity was seen with dGTP (20 -deoxy-guanosine-50 -triphosphate than with ddGTP (20 ,30 -dideoxy-guanosine-50 -triphosphate) (Lohmann et al., 2000). Importantly, high concentrations of GTP can support initiation of HCV RNA synthesis irrespective of the RNA template, leading to speculation that GTP may be required for structural support of the interaction between the 30 end of the template and the priming nucleotide (Luo et al., 2000; Kao et al., 2001). The HCV polymerase lacks proofreading activity, increasing the genetic diversity of the virus and promoting development of the resistant variants found in clinical HCV samples. Purification of recombinant NS5B enabled its structural analysis by X-ray crystallography (Bressanelli et al., 1999; Lesburg et al., 1999; Ago et al., 1999). NS5B resembles the shape of an encircled right hand and can be divided into three domains, termed the palm, fingers, and thumb (Fig. 8.4). This nomenclature aligns with that used to describe the domain structure of the Klenow fragment of DNA polymerase (Ollis et al., 1985). HCV NS5B is 591 amino acids long. The C-terminal 21 hydrophobic amino acids anchor the polymerase to the ER membrane during viral replication. The remaining part of the polymerase is organized into the finger domain (1
187 and 228
286 amino acids), the palm domain (188
227 and 287
370 amino acids), and the thumb domain (371
563 amino acids). The palm domain contains the active site and is the most highly conserved domain (Fig. 8.4). In contrast, the fingers and thumb domains vary significantly in both size and secondary structure organization among polymerases. The thumb domain and finger domains are bridged by loop structures that maintain the closed and open conformations required for RNA binding and movement of the enzyme along the RNA template during elongation. The HCV RdRP shares structural homology with other viral RdRPs and reverse transcriptases (RT), including RdRPs from the RNA bacteriophage phi6 (Butcher et al., 2001).

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FIGURE 8.4

Small molecules that target NS5B RNA polymerase. (A) Represents the crystal structure of the hepatitis C virus (HCV) RNA polymerase. The secondary structure of the HCV GT1b polymerase (Protein Data Bank accession number 3FQL) is shown. The enzyme has right-hand structure with fingers (red), thumb (blue), and palm (green) domains. (B) Represents an encircled “right-hand” configuration showing the movement of HCV RNA along with NS5B polymerase during initiation of viral replication. (C) Structures of FDA approved nucleoside and nonnucleoside NS5B inhibitors.

Changes in the positions of the fingers and thumb domains are associated with conformational changes in the polymerase at different stages of viral genome replication (Shatskaya, 2013). Three well-defined channels have been identified within the polymerase. The template and NTP channels serve as entry points for the template strand and nucleoside phosphates (NTPs), respectively, while the duplex channel allows the dsRNA produced during viral replication to exit the enzyme (McDonald, 2013). The thumb domain of HCV NS5B contains a β-hairpin loop that protrudes into the active site cavity. This loop is thought to influence the orientation of the newly synthesized RNA, and its position regulates the polymerase activity of the enzyme (Mosley et al., 2005). NS5B oligomerizes to catalyze the synthesis of both positive- and negative-strand RNAs during viral replication.

8.6 INITIATION OF HCV REPLICATION BY NS5B POLYMERASE Structural and biochemical analysis of purified recombinant RdRP has increased our understanding of the mechanism of HCV RNA replication,

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initiation, and elongation. Replication of the HCV positive-strand RNA occurs via negative-strand RNA synthesis, and the process can be divided into the initiation, elongation, and termination phases. Like all polymerases, NS5B synthesizes nucleic acids in the 50
30 direction, so formation of a new phosphodiester bond at a 30 -OH is required for initiation of nucleic acid synthesis. The 30 -OH group can be supplied through primer-dependent or primer-independent mechanisms, depending on the type of polymerase. Some viruses, such as those belonging to the Picornaviridae and Caliciviridae families, employ a primer-dependent mechanism. However, in the case of HCV and BVDV (bovine viral diarrhea virus), a high concentration of GTP is required for primerindependent initiation of RNA synthesis irrespective of the template. Studies carried out by a number of laboratories have demonstrated that HCV polymerase initiates negative-strand synthesis via a two-step mechanism involving distinct polymerase conformations. In the first step, the polymerase initiates negative-strand RNA synthesis from the 30 end in the absence of a primer (de novo mechanism) by adding a dinucleotide (dGG) complementary to the 30 end of the template (Luo et al., 2000; Zhong et al., 2000; Kao et al., 2001). This mechanism does not affect the virus genome, and only a single dinucleotide (dGG) primer is required for genome replication. In the second step, the polymerase dramatically changes its conformation to switch from initiation to elongation mode for rapid synthesis of the full-length RNA. The closed conformation of the HCV RdRP is thought to be associated with de novo formation of the dinucleotide complementary to the 30 end of the template RNA, whereas the open conformation is thought to promote the primer-extension activity of the polymerase (Harrus et al., 2010; Scrima et al., 2012; Jin et al., 2012). The open conformation displaces the β-hairpin loop of the thumb domain. This structure is thought to facilitate the initiation reaction, but also blocks the active site in the closed conformation, preventing the binding of duplex RNA (Appleby et al., 2015). A breakthrough in our understanding of the polymerization reaction was achieved through biochemical studies showing that 1
2 hour incubation of purified NS5B with the RNA template, 50 -monophosphorylated pGG dinucleotide primer, and 2 of the 4 nucleoside triphosphates (NTPs) leads to the formation of a stalled stable complex lead of 3
6 nucleotides in length that catalyzed the subsequent elongation step of RNA replication (Jin et al., 2012; Appleby et al., 2015; Jin et al., 2013). A study by Li et al. showed that this initiation reaction generated abortive intermediates, three to five nucleotides in length, prior to formation of the stable elongation complex (Li and Johnson, 2016). Their results demonstrated that initiation is the rate-limiting step of replication and showed that transition from the initiation to the elongation mode is required for replication of the full-length RNA. Structural studies also

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support preferential use of the dinucleotide (pGG), since an allosteric GTP-binding site has been identified in the interface between the fingers and thumb subdomains (Bressanelli et al., 2002). Binding of GTP to this site has been implicated in driving the conformational changes required for formation of the processive RdRP complex (Dutartre et al., 2005), suggesting that high concentrations of GTP promote initiation of HCV viral RNA synthesis. The mechanism of HCV RNA replication at the level of initiation and elongation was also confirmed using atomicresolution ternary structures of NS5B (Appleby et al., 2015). According to this study, the β-loop and the C-terminal membrane anchoring linker are buried within the active site of the polymerase that is positioned close to the 30 terminus of viral RNA. During the initiation step, the 30 end of the viral RNA template and the incoming nucleotides enter the active site of the polymerase, that leading to the generation of dinucleotide primer through an initial phosphoryl transfer reaction. This initiation step generates large quantities of two to six nucleotide-long abortive transcripts. The accumulation of abortive transcripts displaces the β-loop and C-terminal residues, opening the polymerase that to allows for the second step of viral RNA synthesis (Harrus et al., 2010). The two-step mechanism is thought to be the predominant mechanism used in vivo by HCV to synthesize the negative strand beginning from the 30 UTR. These newly synthesized negative-strand RNAs then serve as templates for the production of new positive-strand HCV RNA genomes.

8.7 DIRECT-ACTING ANTIVIRALS TARGETING THE HCV RNA POLYMERASE Two classes of small molecule inhibitors have been developed to target HCV NS5B: nucleos(t)ide inhibitors (NIs) and nonnucleos(t)ide inhibitors (NNIs) (Table 8.1). We describe the development of both classes of inhibitors and define their distinct mechanisms of action in the following sections.

8.7.1 Nucleos(t)ide Inhibitors NIs are small molecules that terminate elongation of the HCV genomic RNA. NIs bind to the active site of the polymerase and are incorporated into the growing HCV strand during RNA synthesis, resulting in chain termination. The advantage of NIs over NNIs is that they have been shown to have stronger antiviral activity with a high barrier to drug resistance. However, one disadvantage is that NIs can also inhibit host

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TABLE 8.1 Nucleoside and Nonnucleoside Inhibitors That Are Approved and Under Investigation Inhibitors

Site of action

NUCLEOSIDE INHIBITORS Sofosbuvir*, VX-135, ACH-3432, uprifosbuvir, GS6620

Active site

NONNUCLEOSIDE INHIBITORS Beclabuvir, TMC647055, deleobuvir

Thumb 1 site

Lomibuvur, radalbuvir, filibuvir, VCH-759

Thumb 2 site

Dasabuvir*, setrobuvir, ABT-072

Palm 1 site

Nesbuvir, GSK-5852, tegobuvir, PPI-383

Palm 2 site

* FDA approved.

RNA polymerases since the active sites are quite similar between different types of RNA polymerases. The intracellular delivery of naturally occurring nucleotide triphosphates (NTPs) is not efficient due to the presence of the ionic phosphate group. Therefore, most antiviral NIs are administered in an unphosphorylated form and they get phosphorylated by the cellular kinases (Poijarvi-Virta, 2006; Lavie and Konrad, 2004). Initial experiments confirmed that 30 -deoxy-modified nucleosides are highly effective chain terminators; however, these NIs are ineffective in vivo because they are poorly phosphorylated by the host cell (Shim et al., 2003). Prior studies also found that 20 -deoxyribonucleotide chain terminators such as azidothymidine are not effective against HCV, whereas 20 modified nucleotides are effective HCV antivirals (Sofia, 2013). As a result, most of the NIs in development retain the 30 -hydroxyl group and instead exhibit modifications at the 20 -position of the ribose moiety. These modifications terminate RNA synthesis through steric hindrance. For example, modified nucleosides containing a 20 -methyl group or 20 -fluoro group have been developed as chain terminators (Delang et al., 2013). Several related classes of these NIs have been developed and assessed for their ability to inhibit HCV replication (reviewed extensively in Delang et al., 2013). Valopicitabine (NM283, prodrug 20 -C-methylcytosine) was the first nucleoside inhibitor of HCV to be investigated in a clinical trial and was administered in combination with PEG-IFN-α and RBV (LePogam et al., 2010). Unfortunately, the low clinical efficacy of this drug prevented its further development. Mericitabine (RG7128) is another cytidine nucleoside analog (β-D-2-deoxy-2-fluoro-C-methylcytidine) that showed antiviral activity against HCV genotypes 1
4 (Guedj et al., 2012). Mericitabine therapy resulted in early virologic responses in more than

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80% of patients, with evidence of resistance. Treatment with mericitabine in combination with PEG-IFN-α for 24 weeks was superior to PEGIFN-α alone in clearing HCV infection. Like many NIs, mericitabine exhibited a favorable safety profile. Sofosbuvir is a 20 -F-20 -C-methyluridine monophosphate that received FDA approval (Sovaldi; GS-7977; Gilead Sciences, Inc.) as the first nucleotide analog NS5B inhibitor to be used for the treatment of chronic HCV infection (Sofia et al., 2010). This drug, when used in combination, has revolutionized the treatment of HCV infection. Addition of sofosbuvir to PEG-IFN-α and RBV for 12 weeks resulted in a high rate of viral clearance in .90% of naı¨ve, HCV genotype 1 infected patients. Furthermore, striking results showed that treatment with sofosbuvir in combination with an NS5A inhibitor (daclatasvir, GS-5885) and RBV resulted in 100% success among treatment-naı¨ve patients. Viral resistance was rarely observed in clinical studies of sofosbuvir, consistent with a high genetic barrier to resistance. Sofosbuvir is currently used as an IFN-free therapy in combination with a number of other DAA drugs, including NS3/4A protease inhibitors (GS939, simeprevir) and an NS5A inhibitor (daclatasvir), to treat chronic HCV infection. This drug shows very high efficacy against almost all HCV genotypes in combinations without PEG-IFN-α and RBV (Lawitz et al., 2013a,b; Gane et al., 2013). Other NIs in clinical development include VX-135, MK-3682 (Uprifosbuvir) (Wyles et al., 2017), GS6620 (Cho et al., 2014; Murakami et al., 2014), and ACH-3422. Clinical trials with VX-135 in combination with daclatasvir reported an SVR rate .90% among patients infected with HCV genotype 1. In these studies, HCV relapse was associated with an S282T substitution in NS5B. This mutation is also the primary amino acid substitution associated with rare resistance to sofosbuvir. Although many other NIs targeting NS5B polymerase have been developed, the trials for most of these drugs have been halted due to toxicity. The mechanism of cytotoxicity appears to be due to inhibition of mitochondrial RNA polymerase activity. At present, many combination therapies include the use of NS3 protease inhibitors. NS5A inhibitors with an NS5B inhibitor (sofosbuvir) were approved by the Food and Drug Administration (FDA) for the treatment of chronic HCV infection (Table 8.2).

8.7.2 Nonnucleos(t)ide Inhibitors NNIs are allosteric, noncompetitive inhibitors that induce structural changes in the enzyme that block the initiation and the elongation stages of viral RNA replication (Caillet-Saguy et al., 2011; Eltahla et al., 2014, 2015). There is substantial interest in the development of NNIs because these inhibitors are likely to have fewer off-target effects on the

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TABLE 8.2

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FDA Approved Nucleoside Inhibitors for Treatment of HCV Infection

Year/Brand name

Active ingredients

Manufacturer

2014/Sovaldi/Olysio

Sofosobuvir/simeprevir

Gilead Sciences/ Janssen

2014/Harvoni

Ledipasvir and sofosobuvir

Gilead Sciences

2014/ Viekira Pak

Dasbuvir, obitasvir, paritaprevir, and ritonavir

Abbvie

2015/Dalklinza/ Sovaldi

Daclatasvir/sofosbuvir

Bristol-Myers Squibb/ Gilead Sciences

2015/Technivie

Ombitasvir, paritaprevir, and ritonavir

Abbvie

2016/Epclusa

Sofosbuvir and velpatasvir

Gilead Sciences

2016/Zepatier

Elbasvir/grazoprevir

Merck Sharp Dohme

host cell compared to NIs. To date, five allosteric binding pockets within NS5B polymerase have been identified, two in the thumb domain (termed thumb 1 (T1) and thumb 2 (T2)), two in the palm region (termed palm 1 (P1) and palm 2 (P2)), and one that blocks the interaction between the β-hairpin and the palm domain (Vliegen et al., 2009; Shih et al., 2011) (Fig. 8.4). Recent progress in development of NNIs that bind to these sites is summarized in the following sections. 8.7.2.1 Thumb 1 inhibitors T1 inhibitors target the upper section of the thumb domain and induce conformational changes in the polymerase. A number of compounds have been reported to bind to the T1 pocket and thereby inhibit HCV replication. Crystallographic studies of NS5B bound to several T1 inhibitors show that T1 inhibitors block the formation of intramolecular contacts between the thumb and fingers domains, thus inhibiting the enzyme activity during viral RNA synthesis in an NTP noncompetitive manner (Di Marco et al., 2005). Examples of T1 NNIs include benzimidazole and indole derivatives such as deleobuvir (207127), TMC 647055, beclabuvir (BMS-791325), MBX-700, and MBX-701 (Eltahla et al., 2015; Zeuzem et al., 2011). Despite their inhibitory activity, resistance to T1targeted NNIs is common. Initial cell culture studies showed that HCV develops resistance to deleobuvir through amino acid substitutions in the NS5B protein at P495, P496, and V499 (Beaulieu et al., 2014). In agreement with these results, clinical development of this drug was halted due to frequent viral breakthrough and relapse associated with P495L/S/T amino acid substitutions in NS5B (Zeuzem et al., 2013).

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TMC-647055 is a related indole derivative that has been shown to exhibit cross-genotypic antiviral activity against all strains except genotype 2; however, HCV clones with amino acid substitutions in the NS5B protein at residues L392 and P495 frequently cause resistance to this drug (Devogelaere et al., 2012). Beclabuvir (BMS-791325) is another indole derivative that has strong antiviral activity against genotype 2 and variable activity against genotype 6. In phase 3 trials, beclabuvir in combination with a protease inhibitor and an NS5A inhibitor showed frequent virologic failure due to P495 substitutions in NS5B (Lemm et al., 2014; Rigat et al., 2014). 8.7.2.2 Thumb 2 Inhibitors The binding site of T2 inhibitors is at the base of the thumb domain. ˚ The T2 site lies at the border between thumb and palm domains 35 A away from the active site. Compounds that bind to the T2 region include thiophene-2-carboxylic acids (e.g., lomibuvir, VX-222), GS-9669 (radalbuvir), and dihydropyranones (e.g., filibuvir). These agents have been tested against genotype 1 viruses, and HCV resistance to lomibuvir and filibuvir has been found to be associated with amino acid substitutions in NS5B at L419, M423, and M426 (Shi et al., 2009; Yi et al., 2012; Jacobson et al., 2009; Le Pogam et al., 2006). Similarly, clinical studies with these two compounds revealed the presence of resistant variants, hindering further clinical development. Mechanistic studies showed that amino acid substitutions in NS5B present in mutant viruses reduce the binding affinity of these molecules for NS5B RdRP (Jacobson, et al., 2009). Of the T2 inhibitors, GS-9669 has shown the most promising results in clinical trials in combination with two other agents with no detectable resistant viruses (Gane et al., 2014). Monotherapy with VCH759 progressed to phase 2 trials in HCV genotype 1 infected patients. This was found to be well tolerated but showed gastrointestinal side effects and developed resistant viruses (Cooper et al., 2009). 8.7.2.3 Palm 1 Inhibitors These inhibitors block HCV replication initiation by interfering with the interaction between the inner thumb/palm domain near the active site of the polymerase. Compounds targeting the P1 pockets of NS5B are the most advanced NNIs in clinical development to date (Dhanak et al., 2002). These include dasabuvir (ABT-333), setrobuvir (RG-7790), and ABT-072. Dasabuvir is the first NNI approved for treatment of HCV genotype 1 infection (Feld et al., 2014). A clinical trial with a combination regimen containing dasabuvir reported .95% SVR rates after 12 weeks of treatment. Viral breakthrough and relapse occurred among a small fraction of patients and was associated with amino acid substitutions at the P1 pocket (S556G) and at RdRP residues C316, M414, G554, S556, and

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D559 (Feld et al., 2014; Kowdley et al., 2014). Setrobuvir, in combination with PEG-IFN-α/RBV, shows strong antiviral activity against genotype 1 patients (Lawitz et al., 2010). In addition, setrobuvir in combination with IFN-free regimens exhibits high antiviral activity against genotype 1b, but lower activity against genotype 1a. Resistance to these regimens is associated with the development of escape mutants with amino acid substitutions at M414, G554, S556, and D559 (Jensen et al., 2013; Thompson et al., 2008; Le Pogam et al., 2008). Proline and benzodiazepine also target the P1 pocket of NS5B and inhibit HCV replication (Gopalsamy et al., 2006; Nyanguile et al., 2008). In a phase 3 trial, dasabuvir (ABT-333) in combination with ABT-450, a protease inhibitor, ombitasvir, and RBV for 12-week treatment resulted high rate viral clearance and showed improved survival of HCV genotype 1b with liver cirrhosis (Poordad et al., 2014). Another phase IIa study of triple combination therapy of two DAA drugs (ABT-450, a potent NS3 protease inhibitor; ABT-072, a nonnucleoside NS5B polymerase inhibitor) and RBV showed increased viral clearance among 9 out of 11 patients infected with HCV genotype during 12-week treatment (Lawitz et al., 2013a,b). 8.7.2.4 Palm 2 Inhibitors Benzofuran-C3-carboxamide (Nesbuvir) belongs to a class of inhibitors that binds to P2 pocket of NS5B and inhibit RdRP activity (Howe et al., 2008 ). This drug binds to residues in the primer grip site of the polymerase. Resistance was associated with amino acid substitutions at C316Y, S365T, and M414T in NS5B that resulted in reduced binding of the drug. Nesbuvir demonstrated promising results in cell culture and progressed to a phase II trial. However, clinical development was halted, as many HCV patients showed elevated liver enzymes during the study. Another potent inhibitor of this class is a boronic acid derivative, GSK5852, that shows good anti-HCV activity against HCV genotype 1 and 2 with an excellent resistance profile. This molecule is under clinical evaluation (Maynard et al., 2014; Voitenleitner et al., 2013). Tegobuvir (GS-9190) is a novel nonnucleoside inhibitor that inhibits NS5B polymerase activity through a completely different mechanism used by other P2 inhibitors. Tegobuvir undergoes metabolic activation to glutathione adducts that specifically interact with NS5B and inhibit HCV replication (Hebner et al., 2012). Resistance analysis of treatmentnaı¨ve patients receiving GS-9190 showed a mutation associated with Y448H in the NS5B region reduced clinical efficacy (Mo et al., 2016). A recent study reported that tegobuvir inhibits HCV replication by altering the interactions between the palm and the β-hairpin in the thumb domain (P-β-hairpin site). This P-β-hairpin site represents a novel NNIbinding site that could be used for future antiviral drug development. Tegobuvir binds HCV RdRP at the palm domain and interacts with the

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β-hairpin in the thumb domain (Vliegen et al., 2009; Shih et al., 2011). Resistance profiling using a cell culture HCV replicon model revealed that tegobuvir resistance was associated with mutations in the palm domain (C316Y, Y448H, Y452H, C445F), as well as mutations in the β-hairpin motif. Unfortunately, a clinical trial of tegobuvir with PEGIFN-α therapy did not show effective clearance of HCV infection with treatment (Lawitz et al., 2011). The combination of tegobuvir and the protease inhibitor GS-9256 triggered resistance via the NS5B Y448H substitution in genotype 1a patients, whereas different mutations (C316Y, C445F) were observed in genotype 1b patients (Zeuzem et al., 2012). Another nonnucleoside inhibitor targeting P2 region has been developed by Presidio pharmaceuticals, Inc. called PPI-383. This compound has an EC50 of 8.3 nM and 2.2 mM against genotypes 1a and 1b in the HCV replicon assay and it is active against HCV genotype 2a, 3a, and 4a. This compound is currently in clinical development. Development of resistant mutants prevented further clinical development of tegobuvir. In summary, the search for more effective NNIs that inhibit HCV replication in the absence of resistance is ongoing.

8.8 CONCLUSIONS Cloning and sequencing of the HCV genome and use of in vitro cell culture models have greatly improved our understanding of the critical roles of the NS3, NS5A, and NS5B proteins in HCV replication. In particular, advances in our understanding of the structure and function of the NS5B RNA polymerase has led to the development of nucleoside/nucleotide analogs that directly inhibit the activity of this polymerase. For example, a combination of the polymerase inhibitor sofosbuvir with NS3 and NS5A inhibitors shows great promise in clearing HCV within a short-time frame. Structural analysis of NS5B has also contributed to the development of numerous nonnucleoside analogs that target different allosteric pockets of the viral RNA polymerase to alter its native conformations. With these advances, the pace of DAA development against HCV is increasing rapidly, and thus more potent DAA drugs will likely be available in the future. In addition to sofosbuvir, several novel NIs and NNIs are currently in clinical development. Hopefully, combination DAA therapy will soon be a feasible treatment modality to combat HCV infection worldwide.

Acknowledgments The authors thank Samantha Hoekstra, Department of Pathology and Laboratory Medicine, Tulane Medical School, for critical review of this manuscript. This work was supported by NIH grants CA089121 and AI103106 and Louisiana Clinical and Translational Science (LACaTS) Center Grant U54 GM104940.

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REFERENCES

229

References Ago, H., Adachi, T., Yoshida, A., Yamamoto, M., Habuka, N., Yatsunami, K., et al., 1999. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure 7, 1417
1426. Aizawa, Y., Seki, N., Nagano, T., Abe, H., 2015. Chronic hepatitis C virus infection and lipoprotein metabolism. World J. Gastroenterol. 21 (36), 10299
10313. Appel, N., Zayas, M., Miller, S., Krijnse-Locker, J., Schaller, T., Friebe, P., et al., 2008. Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLOS Pathog. 4, e1000035. Appleby, T.C., Perry, J.K., Murakami, E., Barauskas, O., Feng, J., Cho, A., et al., 2015. Viral replication: structural basis of RNA replication by the hepatitis C virus polymerase. Science 347, 771
775. Bacon, B.R., Gordon, S.C., Lawitz, Z., Marcellin, P., Vierling, J.M., Zeuzem, S., et al., 2011. Boceprevir for previousl, y treated chronic HCV genotype 1 infection. N. Engl. J. Med. 364, 1207
1217. Bartenschlager, R., Penin, F., Lohmann, V., Andre, P., 2011. Assembly of infectious hepatitis C virus particles. Trends. Microbiol. 19 (2), 95
103. Bartenschlager, R., Lohmann, V., Pennin, F., 2013. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat. Rev. Microbiol. 11, 482
496. Beaulieu, P., et al., 2014. Discovery of BI 207127, an indole diamide NS5B thumb pocket 1 inhibitor with improved potency for potential treatment of chronic HCV infection. J. Med. Chem. 57 (23), 10130
10143. Behrens, S.E., Tomei, L., De Francesco, R., 1996. Identification and properties of the RNAdependent RNA polymerase of hepatitis C virus. EMBO J. 15, 12
22. Bressanelli, S., Tomei, L., Roussel, A., Incitti, I., Vitale, R.L., Mathieu, M., et al., 1999. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc. Natl. Acad. Sci. 96, 13034
13039. Bressanelli, S., Tomei, L., Rey, F.A., De Francesco, R., 2002. Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides. J. Virol. 76, 3482
3492. Butcher, S.J., Grimes, J.M., Makeyev, E.V., Bamford, D.H., Stuart, D.I., 2001. A mechanism for initiating RNA-dependent RNA polymerization. Nature 410, 235
240. Caillet-Saguy, C., Simister, P.C., Bressanelli, S., 2011. An objective assessment of conformational variability in complexes of hepatitis C virus polymerase with non-nucleoside inhibitors. J. Mol. Biol. 414, 370
384. Choi, K.H., 2012. Viral polymerases. Viral Molecular Machines. Springer Science, New York, NY. Cho, A., et al., 2014. Discovery of the first C-nucleoside HCV polymerase inhibitor [GS6620] with demonstrated antiviral response in HCV-infected patients. J. Med. Chem. 57, 1812
1825. Cooper, C., Lawitz, E.J., Ghall, P., Rodriguez-Torres, M., Anderson, F.H., Lee, S.S., et al., 2009. Evaluation of VCH-759 monotherapy in hepatitis C infection. J. Hepatol. 51 (1), 39
46. Dash, S., Chava, S., Aydin, Y., Chandra, P.K., Ferraris, P., Chen, W., et al., 2016. Hepatitis C virus infection induces autophagy as a pro-survival mechanism to alleviate hepatic ER-stress response. Viruses 8 (5), 150. Delang, L., Neyts, J., Vliegen, I., Abrigani, S., Neddetmann, P., De Francesco, R., 2013. Hepatitis C virus specific directly acting antiviral drugs. Curr. Top. Microbial. Immunol. 369, 320. Devogelaere, B., Berke, J.M., Vijgen, L., Dehertogh, P., Fransen, E., Cleiren, E., et al., 2012. TMC647055, a potent nonnucleoside hepatitis C virus NS5B polymerase inhibitor with cross-genotypic coverage. Antimicrob. Agents. Chemother. 56 (9), 4676
4684.

VIRAL POLYMERASES

230

8. HEPATITIS C VIRUS NS5B RNA-DEPENDENT RNA POLYMERASE INHIBITOR

Dhanak, D., Duffy, K.J., Johnston, V.K., Lin-Goerke, J., Darcy, M., Shaw, A.N., et al., 2002. Identification and biological characterization of heterocyclic inhibitors of the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 277, 38322
38327. Di Marco, S., Volpari, C., Tomei, L., Altamura, S., Harper, S., Narjes, E., et al., 2005. Interdomain communication in hepatitis C virus polymerase abolished by small molecule inhibitors bound to a novel allosteric site. J Biol Chem 280 (33), 29765
29770. Dubuisson, J., Cosset, F.L., 2014. Virology and cell biology of the hepatitis C virus life cycle: an update. J. Hepatol. 61 (Suppl. 1), S3
S13. Dutartre, H., Boretto, J., Guillemot, J.C., Canard, B., 2005. A relaxed discrimination of 20 -Omethyl-GTP relative to GTP between de novo and elongative RNA synthesis by the hepatitis C RNA-dependent RNA polymerase NS5B. J. Biol. Chem. 280, 6359
6368. El-Serag, H.B., 2012. Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology 142, 1264
1273. El-Serag, H.B., Kanwal, F., Davila, J.A., Kramer, J., Richardson, P., 2012. A new laboratorybased algorithm to predict development of hepatocellular carcinoma in patients with hepatitis C and cirrhosis. Gastroenterology 146, 1249
1255. Eltahla, A., Luciani, F., White, P.A., Lioyd, A.R., Bull, R.A., 2015. Inhibitors of the hepatitis C virus polymerase; mode of action and resistance. Viruses 7, 5206
5224. Eltahla, A.A., Lim, K.L., Eden, J.S., Kelly, A.G., Mackenzie, J.M., White, P.A., 2014. Nonnucleoside inhibitors of norovirus RNA polymerase: scaffolds for rational drug design. Antimicrob. Agents. Chemother. 58, 3115
3123. Feld, J.J., Kowdley, K.V., Coakley, E., Sigal, S., Nelson, D.R., Crawford, D., et al., 2014. Treatment of HCV with ABT-450/r-ombitasvir and dasabuvir with ribavirin. N. Engl. J. Med. 370, 1594
1603. Ferrari, E., Wright-Minogue, J., Fang, J.W., Baroudy, B.M., Lau, J.Y., Hong, Z., 1999. Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli. J. Virol. 73, 1649
1654. Fridell, R.A., Wang, C., Sun, J.-H., O’Boyle, D.R., Nower, P., Valera, L., et al., 2011. Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replicon complex inhibitor BMS-790052 in humans: in vitro and in vivo correlations. Hepatology 54, 1924
1955. Fried, M.W., Shiffman, M.L., Reddy, K.R., Smith, C., Marinos, G., Goncales Jr., F.L., et al., 2002. Peginterferon α-2a plus ribavirin for chronic hepatitis C virus infection. N. Engl. J. Med. 347, 975
982. Gane, E.J., Stedman, C.A., Hyland, R.H., Ding, X., Svarovskaia, E., Symonds, T., et al., 2013. Nucleotide polymerase inhibitor sofosbuvir plus ribavirin for hepatitis C. N. Engl. J. Med. 368 (3), 4
44. Gane, E.J., Stedman, C.A., Hyland, R.H., Ding, X., Svarovskaia, E., Subramanian, G.M., et al., 2014. Efficacy of nucleotide polymerase inhibitor sofosbuvir plus the NS5A inhibitor ledipasvir or the NS5B non-nucleoside inhibitor GS-9669 against HCV genotype 1 infection. Gastroenterology 146, 736
743. Gao, M., et al., 2010. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465 (7294), 96
100. Gopalsamy, A., Chopra, R., Lim, K., Ciszewski, G., Shi, M., Curran, K.J., et al., 2006. Discovery of proline sulfonamides as potent and selective hepatitis C virus NS5b polymerase inhibitors. Evidence for a new NS5b polymerase binding site. J. Med. Chem. 49 (11), 3052
3055. Gower, E., Estes, C., Blach, S., Razavi-Shearer, K., Razavi, H., 2014. Gobal epidemiology and genotype distribution of the hepatitis C virus infection. J. Hepatol. 61 (1 Suppl.), S45
S57. Guedj, J., Dahari, H., Shudo, E., Smith, P., Perelson, A.S., 2012. Hepatitis C viral kinetics with the nucleoside polymerase inhibitor mericitabine (RG7128)*. Hepatology 55, 1030
1037.

VIRAL POLYMERASES

REFERENCES

231

Hadziyannis, S.J., Sette Jr., H., Morgan, T.R., Balan, V., Diago, M., Marcelin, P., et al., 2004. Peginterferon-alpha 2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose. Ann. Intern. Med. 140, 346
355. Harrus, D., Ahmed-El-Sayed, N., Simister, P.C., Miller, S., Triconnet, M., Hagedorn, C.H., et al., 2010. Further insights into the roles of GTP and the C terminus of the hepatitis C virus polymerase in the initiation of RNA synthesis. J. Biol. Chem. 285, 32906
32918. Hayes, C.N., Imamura, M., Aikata, H., Chayama, K., 2012. Genetics of IL28B and HCVresponse to infection and treatment. Nat. Rev. Gastroenterol. Hepatol. 9, 406
417. Hebner, C.M., Han, B., Brendza, K.M., Nash, M., Sulfab, M., Tian, Y., et al., 2012. The HCV non-nucleoside inhibitor tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function. PLOS ONE 7, e39163. Hoofnagle, J.H., 2009. A step forward in therapy for hepatitis C. N. Eng. J. Med. 360, 1899
1901. Howe, A.Y.M., et al., 2008. Molecular mechanism of hepatitis C virus replicon variants with reduced susceptibility to benzofuran inhibitor, HCV-796. Antimicrob. Agents. Chemother. 52, 3327
3338. Hwang, S.B., Park, K.J., Kim, Y.S., Sung, Y.C., Lai, M., 1997. Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization. Virology 227, 439
446. Jacobson, I., Pockros, P., Lalezari, J., Lawitz, E., Rodriguez-Torres, M., DeJesus, E., et al., 2009. Antiviral activity of filibuvir in combination with pegylated interferon alfa-2a and ribavirin for 28 days in treatment naive patients chronically infected with HCV genotype 1. J Hepatol. 50, S382
S383. Jensen, D., Brunda, M., Elston, R., 2013. Interferon-free regimen containing setrobuvir in combination with ritonavir-boosted danoprevir and ribavirin with or without mericitabine in HCV genotype 1 treatment-naive patients: Interim results from the ANNAPURNA study. Hepatology 58 (Suppl. 4), 849A. Jin, Z., Leveque, V., Ma, H., Johnson, K.A., Klumpp, K., 2012. Assembly, purification, and pre-steady-state kinetic analysis of active RNA dependent RNA polymerase elongation complex. J. Biol. Chem. 287, 10674
10683. Jin, Z., Leveque, V., Ma, H., Johnson, K.A., Klumpp, K., 2013. NTP-mediated nucleotide excision activity of hepatitis C virus RNA-dependent RNA polymerase. Proc. Natl. Acad. Sci. 110, E348
E357. Kao, C.C., Singh, P., Ecker, D.J., 2001. De novo initiation of viral RNA-dependent RNA synthesis. Virology 287, 251
260. Kowdley, K.V., Lawitz, E., Poordad, F., Cohen, D.E., Nelson, D.R., Zeuzem, S., et al., 2014. Phase 2b trial of interferon-free therapy for hepatitis C virus genotype 1. N. Eng. J. Med. 370 (3), 222
232. Lamarre, D., et al., 2003. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature 426, 186
189. Lange, C.M., Zeuzem, S., 2011. IL-28B single nucleotide polymorphisms in the treatment of hepatitis C. J. Hepatology 55, 692
701. Lawitz, E., Rodriquez-Torres, M., Rustgi, V.K., Hassanein, T., Rahimy, M.H., Crowley, C. A., et al., 2010. Safety and antiviral activity of ANA598 in combination with pegylated interferon α2a plus ribavirin in treatment-naive genotype-1 chronic HCV patients. J. Hepatol. 52 (Suppl. 1), 334A
335A. Lawitz, E., Jacobson, I., Godofsky, E., Foster, G.R., Filisiak, R., 2011. A Phase 2b Trial comparing 24 to 48 weeks treatment with tegobuvir (GS-9190)/PEG/RBV to 48 weeks treatment with PEG/RBV for chronic genotype 1 HCV infection. J. Hepatol. 54, S181-S181. Lawitz, E., Mangia, A., Wyles, D., Rodriguez-Torres, M., Hassanein, T., Gordon, S.C., et al., 2013a. Sofosbuvir for previously untreated chronic hepatitis C infection. N. Engl. J. Med. 368, 1878
1887.

VIRAL POLYMERASES

232

8. HEPATITIS C VIRUS NS5B RNA-DEPENDENT RNA POLYMERASE INHIBITOR

Lawitz, E., Poordad, F., Kowdley, K.V., Cohen, D.E., Podsadecki, T., Siggelkow, S., et al., 2013b. A phase 2a trial of 12-week interferon-free therapy with two direct-acting antivirals (ABT-450/r, ABT-072) and ribavirin in IL-28B C/C patients with chronic hepatitis C genotype1. J Hepatol. 59, 18
23. Le Pogam, S., Kang, H., Harris, S.F., Leveque, V., Giannetti, A.M., Ali, S., et al., 2006. Selection and characterization of replicon variants dually resistant to thumb- and palmbinding nonnucleoside polymerase inhibitors of the hepatitis C virus. J. Virol. 80 (12), 6146
6154. Le Pogam, S., Seshaadri, A., Kosaka, A., Chiu, S., Kang, H., Hu, S., et al., 2008. Existence of hepatitis C virus NS5B variants naturally resistant to non-nucleoside, but not to nucleoside, polymerase inhibitors among untreated patients. J. Antimicrob. Chemother. 61 (6), 1205
1216. Le Pogam, S., Seshaadri, A., Ewing, A., Kang, H., Kosaka, A., Yan, J.-M., et al., 2010. RG7128 alone or in combination with PEGylated interferon-alpha 2a and ribavirin prevents hepatitis C virus (HCV) replication and selection of resistant variants in HCVinfected patients. J. Infect. Dis. 202 (10), 1510
1519. Lemm, J.A., Liu, M., Gentles, R.G., Ding, M., Voss, S., Pelosi, L.A., et al., 2014. Preclinical characterization of BMS-791325, an allosteric inhibitor of hepatitis C virus NS5B polymerase. Antimicrob. Agents. Chemother. 58 (6), 3485
3495. Lesburg, C.A., Cable, M.B., Ferrari, E., Hong, Z., Mannarino, A.F., Weber, P.C., 1999. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat. Struct. Biol. 6, 937
943. Lavie, A., Konrad, M., 2004. Structural requirements for efficient phosphorylation of nucleotide analogs by human thymidine kinases. Mini Rev. Med. Chem. 4, 351
359. Li, J., Johnson, K.A., 2016. Thumb site 2 inhibitors of hepatitis C viral RNA-dependent RNA polymerase allosterically block the transition from initiation to elongation. J. Biol. Chem. 291, 10067
10077. Lindenbach, B.D., Rice, C.M., 2013. The ins and outs of hepatitis C virus entry and assembly. Nat. Rev. Microbiol. 11 (10), 688
700. Lohmann, V., Ko¨rner, F., Herian, U., Bartenschlager, R., 1997. Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J. Virol. 71, 8416
8428. Lohmann, V., Korner, R.F., Koch, J.O., Bartenschalager, R., 2000. Biochemical and structural analysis of the NS5B RNA-dependent RNA polymerase of the hepatitis C virus. J. Viral Hepatitis 7, 167
174. Luo, G., Hamatake, R.K., Mathis, D.M., Racela, J., Rigat, K.L., Lemm, J., et al., 2000. De novo initiation of RNA synthesis by the RNA-dependent RNA polymerase (NS5B) of hepatitis C virus. J. Virol. 74, 851
863. Manns, M.P., McHutchison, J.G., Gordon, S.C., Rustgi, V.K., Shiffman, M., Reindollar, R., et al., 2001. Peginterferon α-2b plus ribavirin compared with interferon α-2b plus ribavirin for initial treatment of chronic hepatitis C: A randomized trial. Lancet 358, 958
965. Masaki, T., Suzuki, R., Murakami, K., Aizaki, H., Ishii, K., Murayama, A., et al., 2008. Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles. J Virol. 82, 7964
7976. Maynard, A., Crosby, R., Walker, J., Remlinger, K., Vamathevan, J., Wang, A., et al., 2014. Discovery of a potent boronic acid derived inhibitor of the HCV RNA-dependent RNA polymerase. J. Med. Chem. 57, 1902
1913. McDonald, S.M., 2013. RNA synthetic mechanisms employed by diverse families of RNA viruses. WIREs RNA 4, 351
367. McHutchison, J.G., Everson, G.T., Gordon, S.C., Jacobson, I.M., Sulkowski, M., Kauffman, R., et al., 2009. Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection. N. Engl. J. Med. 360, 1827
1838.

VIRAL POLYMERASES

REFERENCES

233

Messina, J.P., Humphreys, I., Flaxman, A., Brown, A., Cooke, G.S., Pybus, O.G., et al., 2015. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology 61 (1), 77
87. Miller, R.H., Purcell, R.H., 1990. Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc. Natl. Acad. Sci. 87, 2057
2061. Mohd Hanafiah, K., Groeger, J., Flaxman, A.D., Wiersma, S.T., 2013. Global epidemiology of hepatitis C virus infection: new estimates of age specific antibody to HCV seroprevalence. Hepatology 57 (4), 1333
1342. Moradpour, D., Penin, F., 2013. Hepatitis C virus proteins: from structure to function. Curr. Top. Microbiol. Immunol. 369, 113
142. Mosley, J.W., Operskalski, E.A., Tobler, L.H., Andrews, W.W., Phelps, B., Dockter, J., et al., 2005. Viral and host factors in early hepatitis C virus infection. Hepatology 42, 86
92. Mo, H., Hedskog, C., Svarovskaia, E., Sun, S.C., Jacobson, I.M., Brainard, D.M., et al., 2016. Antiviral response and resistance analysis of treatment-naı¨ve HCV infected patients recue 5 iving single and multiple doses of GS-9190. J. Viral Hepat. 23 (98), 644
651. Murakami, E., et al., 2014. Metabolism and pharmacokinetics of the anti-hepatitis C virus nucleotide prodrug GS-6620. Antimicrob. Agents Chemother. 58, 1943
1951. Niepmann, M., 2013. Hepatitis C virus RNA translation. Curr. Top. Microbiol. Immunol 369, 143
166. Nyanguile, O., Pauwels, F., Van den Broeck, W., Boutton, C.W., Quirynen, L., Ivens, T., et al., 2008. 1,5-benzodiazepines, a novel class of hepatitis C virus polymerase nonnucleoside inhibitors. Antimicrob. Agents Chemother. 52 (12), 4420
4431. Ollis, D.L., Brick, P., Hamlin, R., Xuong, N.G., Steltz, T.A., 1985. Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP. Nature 313 (6005), 762
766. Paul, D., Madan, V., Bartenschlager, R., 2014. Hepatitis C virus RNA replication and assembly: living on the fat of the land. Cell Host Microbe. 16 (5), 569
579. Pawlotsky, J.M., Feld, J.J., Zeuzem, S., Hoofnagle, J.H., 2015. From non-A, non-B hepatitis to hepatitis C virus cure. J Hepatology 62 (1 Suppl.), S87
S99. Poijarvi-Virta, P., 2006. Prodrug approaches of nucleotides and oligonucleotides. Curr. Med. Chem. 13, 3441
3465. Poordad, F., Hezode, C., Trinh, R., Kowdley, K.V., Zeuzem, S., Agarwal, K., et al., 2014. ABT-450/r-ombitasvir and dasabuvir with ribavirin for hepatitis C with cirrhosis. N. Eng. J. Med. 370, 1973
1982. Rigat, K.L., Lu, H., Wang, Y.-K., Argyrou, A., Fanslau, C., Beno, B., et al., 2014. Mechanism of inhibition for BMS-791325, a novel non-nucleoside inhibitor of hepatitis C virus NS5B polymerase. J. Biol. Chem. 289 (48), 33456
33289. Scheel, T.K., Rice, C.M., 2013. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Nat. Med. 19 (7), 837
849. Scrima, N., Caillet-Saguy, C., Ventura, M., Harrus, D., Astier-Gin, T., Bressanelli, S., 2012. Two crucial early steps in RNA synthesis by the hepatitis C virus polymerase involve a dual role of residue 405. J. Virol. 86, 7107
7117. Shatskaya, G.S., 2013. Structural organization of viral RNA-dependent RNA polymerase. Biochemistry 78, 231
235. Shepard, C.W., Finelli, L., Alter, M.J., 2005. Global epidemiology of hepatitis C virus infection. Lancet Infect. Dis. 5, 558
567. Shi, S.T., Herlihy, K.J., Graham, J.P., Nonomiya, J., Rahavendran, S.V., Skor, H., et al., 2009. Preclinical characterization of PF-00868554, a potent nonnucleoside inhibitor of the hepatitis C virus RNA-dependent RNA polymerase. Antimicrob. Agents. Chemother. 53 (6), 2544
2552.

VIRAL POLYMERASES

234

8. HEPATITIS C VIRUS NS5B RNA-DEPENDENT RNA POLYMERASE INHIBITOR

Shih, I.H., Vliegen, I., Peng, B., Yang, H., Hebner, C., Paeshuyse, J., et al., 2011. Mechanistic characterization of GS-9190 (tegobuvir), a novel nonnucleoside inhibitor of hepatitis C virus NS5B polymerase. Antimicrob. Agents Chemother. 55, 4196
4203. Shim, J., Larson, G., Lai, V., Naim, S., Wu, J.Z., 2003. Canonical 30 -deoxyribonucleotides as chain terminator of HCV NS5B RNA-dependent RNA polymerase. Antiviral Res. 58, 243
251. Smith, D.B., Becher, P., Bukh, J., Gould, E.A., Meyers, G., Monath, T., et al., 2016. Proposed update to the taxonomy of the genera hepacivirus and pegivirus within the Flaviviridae family. J. Gen. Virol. 97, 2894
2907. Sofia, M.J., et al., 2010. Discovery of a β-D-20 deoxy-20 -α-fluoro-20 -β -C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus. J. Med. Chem. 53 (19), 7202
7218. Sofia, M.J., 2013. Nucleotide prodrugs for the treatment of HCV infection. Adv. Pharmacol. 67, 39
73. Stanaway, J.D., et al., 2016. The global burden of viral hepatitis from 1990-2013: Findings from the global burden of disease study. Lancet 388, 1981-1088. Suzuki, R., Matsuda, M., Watashi, K., Aizaki, H., Matsuura, Y., Wakita, T., et al., 2013. Signal peptidase complex subunit 1 participates in the assembly of hepatitis C virus through an interaction with E2 and NS2. PLOS Pathog. 9 (8), e1003589. Tellinghuisen, T.L., Foss, K.L., Treadaway, J.C., Rice, C.M., 2008. Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein. J Virol. 82, 1073
1083. Thompson, P.A., Patel, R., Showalter, R.E., Li, C., Applemon, J.R., Steffy, K., 2008. In vitro studies demonstrate that combinations of antiviral agents that include HCV polymerase inhibitor ANA598 have the potential to overcome viral resistance. Hepatology 48, 1164A. Vliegen, I., Paeshuyse, J., De Burghgraeve, T., Lehman, L.S., Paulson, M., Shih, I.H., et al., 2009. Substituted imidazopyridines as potent inhibitors of HCV replication. J. Hepatol. 50 (5), 999
1009. Voitenleitner, C., Crosby, R., Walker, J., Remlinger, K., Vamathevan, J., Wang, A., et al., 2013. In vitro characterization of GSK2485852, a novel hepatitis C virus polymerase inhibitor. Antimicrob. Agents Chemother. 57, 5216
5224. Wyles, D., et al., 2017. Grazoprevir, ruzasvir, and uprifosbuvir for hepatitis C virus after NS5A treatment failure. Hepatology 66 (6), 1794
1804. Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K., et al., 1998. RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region. J. Biol. Chem. 273, 15479
15486. Yi, G., Deval, J., Fan, B., Cai, H., Soulard, C., Ranjith-Kumar, C.T., et al., 2012. Biochemical study of the comparative inhibition of hepatitis C virus RNA polymerase by VX-222 and filibuvir. Antimicrob Agents Chemother. 56 (2), 830
837. Zayas, M., Long, G., Madan, V., Bartenschlager, R., 2016. Combination of hepatitis C virus assembly by distinct regulatory regions in nonstructural protein 5A. PLOS Pathog. 12, e1005376. Zeuzem, S., Asselah, T., Angus, P., Zarski, J.P., Larrey, D., Mu¨llhaupt, B., et al., 2011. Efficacy of the protease inhibitor BI 201335, polymerase inhibitor BI 207127, and ribavirin in patients with chronic HCV infection. Gastroenterology 141, 2047
2055. Zeuzem, S., Buggisch, P., Agarwal, K., Marcellin, P., Sereni, D., Klinker, H., et al., 2012. The protease inhibitor, GS-9256, and non-nucleoside polymerase inhibitor tegobuvir alone, with ribavirin, or PEGylated interferon plus ribavirin in hepatitis C. Hepatology 55 (3), 749
758.

VIRAL POLYMERASES

FURTHER READING

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Zeuzem, S., Soriano, V., Asselah, T., Bronowicki, J.P., Lohse, A.W., Mu¨llhaupt, B., et al., 2013. Faldaprevir and deleobuvir for HCV genotype 1 infection. N. Engl. J. Med. 369, 630
639. Zhong, W., Uss, A.S., Ferrari, E., Lau, J.Y., Hong, Z., 2000. De novo initiation of RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase. J. Virol. 74, 2017
2022. Zhou, Y., Ray, D., Zhao, Y., Dong, H., Ren, S., Li, Z., et al., 2007. Structure and function of flavivirus NS5 methyltransferase. J Virol. 81 (8), 3891
3903.

Further Reading Haudecoeur, R., Peuchmaur, M., Ahmed-belkacem, A., Pawlotsky, J.M., Boumendjel, A., 2013. Structure-activity relationships in the development of allosteric hepatitis C virus RNA polymerase inhibitors: ten years of research. Med. Res. Rev. 33, 934
984. Lindenbach, B.D., 2013. Virion assembly and release. Curr. Top. Microbiol. Immunol. 369, 199
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VIRAL POLYMERASES