VIROLOGICA SINICA, August 2010, 25 (4):246-266 DOI 10.1007/s12250-010-3140-2

© Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg 2010

Development of Novel Antiviral Therapies for Virus Kai Lin** (Novartis Institutes for BioMedical Research, Inc. Cambridge, Massachusetts 02139, USA)

Abstract: Over 170 million people worldwide are infected with hepatitis C virus (HCV), a major cause of liver diseases. Current -based therapy is of limited efficacy and has significant side effects and more effective and better tolerated therapies are urgently needed. HCV is a positive, single-stranded RNA virus with a 9.6 kb genome that encodes ten viral proteins. Among them, the NS3 protease and the NS5B polymerase are essential for viral replication and have been the main focus of drug discovery efforts. Aided by structure-based drug design, potent and specific inhibitors of NS3 and NS5B have been identified, some of which are in late stage clinical trials and may significantly improve current HCV treatment. Inhibitors of other viral targets such as NS5A are also being pursued. However, HCV is an RNA virus characterized by high replication and mutation rates and consequently, resistance emerges quickly in patients treated with specific antivirals as monotherapy. A complementary approach is to target host factors such as cyclophilins that are also essential for viral replication and may present a higher genetic barrier to resistance. Combinations of these inhibitors of different mechanism are likely to become the essential components of future HCV therapies in order to maximize antiviral efficacy and prevent the emergence of resistance.

Key words: HCV; Hepatitis; Antiviral; Polymerase; Cyclophilin

Hepatitis C virus (HCV) is a single stranded implemented in the 1990s. Over 170 million people or enveloped RNA virus that belongs to the flaviviridae 3% of the world population are chronically infected family. It was first discovered in 1989 as the causative with HCV, with an additional 3 to 4 million new agent for non-A and non-B hepatitis [20]. However, the each year (WHO). It is estimated that in virus had spread unknowingly for decades through China alone there are 20-40 million people chronically blood transfusion, unsafe injection or other blood-to- infected, representing perhaps one of the most under- blood contacts before sensitive viral diagnosis kits appreciated health issues. Although only 25% of new were developed. As a result, a huge population had infections are symptomatic, 60%-80% of patients will been infected before mandatory blood screening was develop chronic liver disease, of whom an estimated 20% will progress to cirrhosis with a 1%-4% annual Received: 2010-03-22, Accepted:2010-05-29 risk of developing hepatocellular carcinoma. Overall, ** Corresponding author. Phone: +1- 617-871-7579, E-mail: [email protected] HCV is responsible for 50%-76% of all liver cancer

Virol. Sin. (2010) 25: 246-266 247 cases and two thirds of all liver transplants in treatment response. The current standard of care (SoC) developed countries. Ultimately, 5%-7% of infected for HCV is pegylated interferon alpha (PEG- patients will die from the consequences of HCV IFN-α) in combination with for 48 weeks in infection. patients with genotypes 1 and 4 virus and 24 weeks in There are 7 genotypes and over 50 subtypes of patients with genotypes 2 and 3 virus. Unfortunately HCV based on the genetic make-up of the virus. genotype 1 virus, the predominant HCV genotype in Among them genotype 1 is the most prevalent in the developed countries and China, is also the most difficult US, Europe, Japan and China. Unlike HIV-1, HCV to treat with IFN-based therapy. In patients with does not integrate into the host genome and genotypes 1 and 4 virus the SVR rate was 41%-52% vs. theoretically can be eradicated. The goal of HCV 76%-82% with genotypes 2 and 3 virus [39,94]. Moreover, therapy is to achieve sustained virologic response both interferon and ribavirin induce significant (SVR), defined as HCV RNA undetectable (<10 adverse effects, including flu-like symptoms (fever IU/mL) in plasma 6 months after the end of therapy. and fatigue), hematologic complications (leukopenia, There has been long-term follow-up of patients >5 thrombocytopenia), and neuropsychiatric issues years after SVR, suggesting that re-infection rarely (depression, insomnia) associated with interferon and occurs (<1%). In other words, patients achieving SVR significant hemolytic anemia associated with ribavirin. are essentially “cured”. Also, studies have shown that Also, ribavirin is teratogenic and cannot be given to the elimination of HCV infection leads to a reduction pregnant women. Therefore, the majority of HCV of fibrosis and the risk of developing cirrhosis and patients are not being treated with current SoC. More liver disease-related death. effective and better tolerated therapies are therefore Significant progress has been made over the past 20 urgently needed, which is the subject of this review. years in treating hepatitis C. In the mid-1980’s interferon alpha (IFN-α) was shown to reduce the HCV LIFE CYCLE AND ANTIVIRAL STRATEGY levels of serum aminotransferase (ALT) and HCV The life cycle of HCV has been well studied and RNA. However, only 6%-15% of patients achieved SVR has revealed many potential targets for novel therapies. after 6 months of IFN-α monotherapy and 13%-25% HCV, an enveloped RNA virus, first enters the cells after 12 months. The addition of an oral nucleoside through specific interactions of viral glycoproteins analog ribavirin to IFN-α in 1998 improved the (E1 and E2) with cell surface receptors CD81 [112] response rate to 30%-40%. The mechanism of action human scavenger receptor class B type I (SR-B1) [131], of ribavirin is not entirely clear. It does not have a tight junction proteins Claudin-1 [30] occludin [113], and significant antiviral effect on its own but can reduce likely other cell surface proteins. Following attachment, the relapse rate of IFN-α treatment. The introduction the HCV nucleocapsid is released in the cytoplasm as of long-acting IFN-α in 2002 not only reduced the a result of a fusion process between viral and cellular frequency of IFN injection from three times weekly to membranes, which is pH-dependent and is mediated once per week but also significantly improved by clathrin-dependent endocytosis. Decapsidation of

248 Virol. Sin. (2010) 25: 246-266 viral nucleocapsids releases positive-strand genomic 5 to 10-fold excess positive-strand RNA that will be RNA, which serves as the template for the synthesis used for polyprotein translation or packaging into new of the HCV polyprotein in the cytoplasm. The 5’-UTR virus particles. The virions are assembled on lipid of HCV contains an internal ribosomal entry site droplets (LD), which are located at endoplasmic (IRES), which mediates cap-independent initiation of reticulum (ER)-derived bilayer membranes. The Core HCV polyprotein translation by recruiting cellular protein of HCV localizes on the monolayer membrane proteins eukaryotic initiation factors eIF-2 and 3. The that surrounds the LD. It recruits non-structural (NS) 9.6 kb HCV RNA genome encodes a single large open proteins to the LD-associated membrane. E2 also reading frame corresponding to a polyprotein localizes around the LD. The positive strand HCV precursor of about 3,000 amino acids, which is RNA genome is encapsidated with the structural proteolytically cleaved into ten individual proteins, in proteins. The viral particle is probably enveloped the order of C-E1-E2-p7-NS2-NS3-NS4A-NS4B- through budding into the ER lumen and then trans- NS5A-NS5B (Fig. 1). Host signal peptidase and signal ported through Golgi to be released. peptide peptidase are responsible for the cleavage at As shown in Fig. 2, every step of the HCV life the junctions of core-E1, E1-E2, E2-p7 and p7-NS2. cycle could potentially be intervened with antiviral The zinc-dependent NS2-3 autoprotease ensures agents. All ten HCV proteins have been pursued as cis-cleavage of NS3 from NS2. The NS3 serine antiviral targets. Among them, drug discovery efforts protease, together with its cofactor NS4A, catalyzes have been mainly focusing on the NS3-4A serine cis-cleavage at the NS3-NS4A junction and trans- protease and the NS5B RNA-dependent RNA poly- cleavage at all downstream junctions including NS4A- merase, both of which have enzymatic activities NS4B, NS4B-NS5A, and NS5A-NS5B. The viral non- essential for viral replication and are considered structural proteins then form the replication complex highly druggable targets partly because of the success with cellular components and nascent RNA strands on of antiretroviral therapy targeting HIV-1 protease and an ER membrane derived structure named mem- polymerase. In addition, cellular proteins are involved branous web. The positive-strand genome RNA serves in every step of the viral life cycle and can also be as the template for the synthesis of a negativestrand considered as potential antiviral targets. Host factors RNA, which in turn serves as the template to produce not only provide a complementary antiviral strategy

Fig. 1. HCV genome.

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Fig. 2. HCV life cycle and antiviral targets. (HF=host factor, Cyp=cyclophilin) but also may have the advantage of creating higher and E2, provides a useful tool to study viral entry [5,58]. genetic barriers to resistance. Cyclophilins, a family of However, only the newly discovered genotype 2a cellular PPIase required for viral replication, represent HCV (JFH-1 strain) recapitulates the complete viral such a strategy. A number of other potential host life cycle [80,146,159]. A genotype 1 virus (H77 strain) targets have been identified through siRNA screens can also infect Huh-7 cells in vitro, albeit at much [9,77,104,145,153]. lower infectivity compared to that of the JFH-1 strain Drug discovery effort on HCV has long been [156]. Numerous attempts have been made to culture hampered by the lack of an in vitro virus culture HCV isolated from patient serum using primary system and suitable animal models. The establishment human hepatocytes, however no robust, reproducible of a subgenomic HCV replicon system in 2000 greatly method has been established to date. The development facilitated basic research studying viral replication in of animal models has been equally challenging. vitro as well as HCV drug discovery efforts. The Chimpanzees are the only immunocompetent animals subgenomic replicon contains all the non-structural that can be chronically infected with HCV, but their proteins of HCV that are required for autonomous use is restricted by ethical concerns, limited replication of viral RNA in a human hepatoma cell availability and prohibitively high cost [14]. SCID mice line, Huh-7 [8,84]. However, it lacks viral structural with human hepatocytes repopulated in the mouse proteins and therefore does not produce infectious liver can be infected with HCV and provide a useful virus. Another in vitro culture system, HCV pseu- tool for compound efficacy testing and possibly PK dotyped viral particle (HCVpp), generated from and toxicology studies [98]. However, these mice are lentivirus replacing native glycoproteins with HCV E1 also of limited availability and substantial variability

250 Virol. Sin. (2010) 25: 246-266 and cannot be used to study pathology or immunology N-terminal one third of the protein contains a aspects of infection. Despite these constraints, more chymotrypsin-like serine protease domain whereas the than two dozen novel HCV inhibitors have progressed C-terminal portion of the protein is a helicase/NTPase. beyond preclinical development and demonstrated Together with the NS4A cofactor the NS3 protease is clinical efficacy in HCV patients. Key classes of HCV responsible for proteolytic cleavage of the HCV inhibitors in development are listed in Table 1, some polyprotein at four junctions, NS3-4A (cis or self- of which are discussed in detail below. cleavage), 4A-4B, 4B-5A and 5A-5B, and thus is essential for viral replication [4,45,143]. The substrate NS3 PROTEASE INHIBITORS specificity of NS3 protease has been well The NS3 protein of HCV has dual functions: the characterized. The cleavage sites recognized by the

Table 1. Key classes of HCV inhibitors in development. (NI=nucleoside inhibitor, NNI=non-nucleoside inhibitor) Target Mechanism Compound Status References NS3 Reversible covalent inhibitor (VX-950) Phase 3 [38,52,78,93,97,110,119] (SCH503034) Phase 3 [67,90,129] (SCH900518) Phase 1 [2] Non-covalent inhibitor ITMN-191/R7227 Phase 2 [132] TMC435 Phase 2 [79,118] (MK-7009) Phase 2 [82] BI201335 Phase 2 [91,92] BMS-650032 Phase 1 [108] PHX1766 Phase 1 [22] GS-9256 Phase 1 [22] ABT-450 Phase 1 [7] (BILN2061) Terminated [53,69,120] NS5B NI active site R7128 Phase 2 [124] IDX184 Phase 1 [68] PSI-7851 Phase 1 [125] (NM283) Terminated [111] R1626 Terminated [114,122] NNI site 1 BI207127 Phase 2 [71] MK-3281 Phase 1 [12] BILB1941 Terminated [29] NNI site 2 (PF-00868554) Phase 2 [135] VX-222 (VCH-222) Phase 2 [126] VCH-759 Phase 1 [25] VCH-916 Phase 1 [73] NNI site 3 ANA598 Phase 2 [74] NNI site 4 GS-9190 Phase 1 [6] ABT-333/ABT-072 Phase 1 [22] HCV-796 Terminated [31,57] NS5A BMS-790052 Phase 2 [102] AZD7295/A-689 Phase 1 [22] PPI-461 Preclinical [24] EDP-239 Preclinical - Cyclophilin Alisporivir (Debio-025) Phase 2 [23,36,37,107] NIM811 Phase 2 [40,72,89,95,116] SCY-635 Phase 1 [54,55]

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NS3-NS4A protease have the following sequence in have greatly improved the potency and drug-like common: Asp/GluXXXXCys/Thr-Ser/Ala, with trans- properties of the inhibitors. Now there are at least a cleavages occurring downstream of a cysteine residue dozen NS3 protease inhibitors in development, which and the cis-cleavage occurring downstream of a can be generally divided into two groups based on threonine residue. Both HPLC and FRET-based NS3 differences in chemical structure as well as protease assays have been developed, which measure mechanism of inhibition. One group comprises the cleavage of a peptide substrate by either the substrate based inhibitors including telaprevir (VX- protease domain or the full-length NS3 protein 950) and boceprevir (SCH 503034), which contain an coupled with an NS4A peptide [70,140]. The assays are electrophile warhead (instead of the scissile bond) that quite amenable for high-throughput screening of small engages the catalytic serine of NS3 active site in a molecule inhibitors. However, none of the screening covalent but reversible interaction, so called “serine efforts led to any promising leads for NS3 inhibitors. trap”. The others are non-covalent product based Thus, the discovery of specific inhibitors of NS3 inhibitors which are either carboxylic acid such as protease has been largely depending on structure- BILN2061 or have an acylsulfonamide at P1’ like based drug design. ITMN-191. The crystal structure of the NS3 protease was first BILN2061 (ciluprevir) was the first NS3 protease solved in 1996 [64,86], which revealed a shallow, inhibitor demonstrating clinical efficacy. Treatment featureless substrate binding pocket, suggesting that with 200 mg bid (twice daily) of the compound in the design of small inhibitors could be challenging. genotype 1 HCV patients resulted in a rapid viral load [53,69] The initial peptidomimetic inhibitors were based on reduction of 3 log10 copies/mL after only 2 days . the decapeptide (P6-P4’) natural substrate of NS3, Unfortunately the development of the compound was which was the minimum required length of peptide terminated due to cardiotoxicity findings in animal substrate for efficient cleavage. Using non-cleavable studies [120]. active-site analogs, the substrate-based inhibitor can Currently, the most advanced protease inhibitors in be further truncated to P4-P1’, a more drug-like development are telaprevir and boceprevir in Phase III dimension [70]. In parallel, it was discovered that the clinical trials. Telaprevir is a ketoamide inhibitor of products of the natural substrates cleavage were NS3 protease with moderate in vitro potency (NS3_Ki [78,110] themselves inhibitors of NS3 protease and therefore = 44 nmol/L, HCV replicon EC50=354 nmol/L) . can also be used as the starting point for inhibitor In a Phase Ib trial, telaprevir monotherapy for 14 days [83,137] design . Initial combinatorial screen of the reduced HCV viral load by 3.5 to 4.8 log10, with the natural cleavage product sequences led to a series of optimal dosing determined to be 750 mg every 8 h very potent hexapeptide inhibitors. Over the past 15 (q8h) because of the relatively short half-life of the years, tremendous effort has gone into optimizing compound [119]. Subsequent Phase II trials in treatment these peptidomimetic inhibitors through extensive naive patients showed that the combination of substitutions at every position of the molecule, which telaprevir with PEG-IFN-α and ribavirin increased

252 Virol. Sin. (2010) 25: 246-266 sustained virologic response (SVR) by about 20% inhibitors are being evaluated in further clinical trials. compared to current standard of care [52,97]. However, adverse events (AEs) were more common in the NS5B POLYMERASE INHIBITORS telaprevir treatment group, including skin rash, The NS5B protein of HCV is an RNA-dependent gastrointestinal events and anemia, which resulted in RNA polymerase. It is responsible for synthesis of higher discontinuation rates compared to PEG-IFN-α/ both positive and negative strands of HCV RNA and ribavirin alone. In patients previously failed PEG- thus is essential for viral replication. NS5B is IFN-α/ribavirin therapy, the triple combination also anchored to the ER membrane through its C-terminal demonstrated significant improvement 51%-52% SVR 21 amino acids and catalyzes the polymerase reaction vs. 14% with SoC [93]. Three large Phase III trials are on the cytosolic side of ER. There is no mammalian currently on-going. New Drug Application (NDA) homolog of NS5B in terms of subcellular localization filing is expected in 2011. Boceprevir is another α- and template specificity, suggesting it may be possible ketoamide inhibitor of similar potency (replicon to identify selective inhibitors. NS5B that lacks the C- [90] EC50=200 nmol/L) . In genotype 1 HCV patients terminal membrane-anchoring domain can be efficiently previously not responding to IFN and ribavirin expressed in E. coli, which allows the establishment of therapy, 400 mg q8h of boceprevir monotherapy a robust polymerase assay in vitro measuring the resulted in a 1.61 log10 viral load reduction. incorporation of ribonucleoside triphosphate to Combination of boceprevir with PEG-IFN-α2b homopolymer or heteropolymer RNA template. In resulted in a 2.88 log10 viral load reduction compared contrast to NS3 protease, high-throughput screening [129] to only 1.3 log10 with PEG-IFN-α2b alone . In a of small molecule libraries successfully led to the Phase II trial with treatment naive patients, triple identification of multiple classes of NS5B inhibitors. therapy including 800 mg q8h boceprevir resulted in The structure of NS5B was solved in 1999 [1,13,76], 67%-75% SVR compared to 38% with PE-IFN-α2b/ which helped characterizing binding of these ribavirin. However, treatment with boceprevir caused inhibitors to NS5B. The structure of NS5B, like many higher incidence of anemia in addition to fatigue, other viral polymerases, resembles the shape of a right nausea and headache that were typically associated hand consisting of finger, thumb and palm domains. with SoC [67]. Phase III trials with boceprevir in There are two major classes of polymerase inhibitors, treatment naive and failure patients are also on-going. nucleosides and non-nucleosides. As shown in Table 1, a number of other NS3 Nucleoside inhibitors (NIs) bind competitively with protease inhibitors are in early phases of clinical natural nucleoside triphosphate substrates to the active development. Many of these compounds have shown site of polymerase and, once incorporated, serve as much improved potency (replicon EC50<10 nmol/L) chain terminator to block further extension of viral and longer half-life which enables more convenient RNA nascent strand. The first nucleoside analog dosing (once or twice daily). The efficacy and safety demonstrating clinic efficacy against HCV is of these potentially better second generation protease valopicitabine (NM283) [111]. At 800 mg/day it reduced

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viral load by 1.2 log10 after 15 days of treatment. other classes of HCV inhibitors. However, it appears However, further development of the compound was to be more difficult to develop resistance against terminated due to dose-limiting GI toxicity and nucleoside inhibitors vs. non-nucleoside polymerase insufficient efficacy/safety benefits. The second HCV inhibitor or protease inhibitor[96]. This is mainly due to NI, R1626, achieved a more profound viral load the fact that nucleoside inhibitors bind to the highly reduction of 3.5 log10 after 14 days at the highest dose conserved active site of NS5B. Any mutation at the tested, 4,500 mg twice per day (bid). Combination of active site conferring resistance also leads to a 1500 mg bid R1626 with PEG-IFN-α2a/ribavirin resulted significant cost to the fitness viral polymerase and [114,122] in a 5.2 log10 viral load reduction after 4 weeks . replication. However, further development of R1626 was also Non-nucleoside inhibitors (NNI) of NS5B are discontinued due to higher incidence of neutropenia. non-competitive with regard to nucleoside substrate Currently, the most advanced HCV NI is R7148. In and bind to the surface of the protein. Interestingly, the Phase I monotherapy trial, 1,500 mg bid of R7148 there have been at least four allosteric binding pockets resulted in a 2.7 log10 viral load reduction after 14 identified for HCV NNIs. days. Combination of 1000 mg or 1500 mg bid of NNI site 1 is also referred as thumb pocket 1. R7148 with PEG-IFN-α2a/ribavirin resulted in 85% Inhibitors of this site are hypothesized to displace the rapid virological response (RVR) compared to 19% Λ finger loop from the upper thumb domain of NS5B with SoC [124]. Two liver-targeting prodrugs of and interfere with a conformational change required nucleoside analogs, IDX184 and PSI-7851, recently during RNA synthesis. BILB1941 was the first site 1 entered clinical development. These compounds are NNI that reported proof-of-concept (PoC) clinical [29] designed to achieve higher concenrations of the active efficacy . It has a replicon EC50 of 153 nmol/L and metabolites in the liver while reducing systemic 84 nmol/L against genotype 1a and 1b, respectively. A exposure thereby limiting potential side effects. greater than 1 log10 viral load reduction was achieved IDX184, a nucleotide prodrug of 2'-methyl guanosine, when patients received 450 mg q8h of the compound resulted in a 0.47 log10 viral load reduction after for 5 days. Unfortunately further development of the dosing 25 mg once daily (qd) for three days and 0.74 compound was terminated due to GI intolerance. log10 at 100 mg qd. Combination of 50 mg qd Recently it was reported that a follow-up compound, IDX-184 and PEG-IFN-α/ribavirin resulted in a 3.66 BI-207127 achieved a dose-dependent viral load [71] log10 reduction after 14 days vs. only 1.7 log10 with reduction in the range of 0.6-3.1 log10 after 5 days . PEG-IFN-α/ribavirin alone [68]. PSI-7851 is a phos- Another site 1 compound, MK-3281, an indole-based phoramidate prodrug of β-D-2’-deoxy-2’-fluoro-2’-C- inhibitor of 40 nmol/L replicon EC50, also reported methyluridine-5’-monophosphate. When administered clinical efficacy, with much greater viral load as a monotherapy, 400 mg qd of the compound reduction in genotype 1b patients (3.75 log10) than [125] suppressed HCV RNA by 1.95 log10 after 3 days . genotype 1a patients (1.28 log10) after dosing 800 mg Nucleoside inhibitors are generally less potent than bid for 7 days [12].

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NNI site 2, also known as thumb pocket II, is a EC50=29.2 nmol/L). In a three-day Phase I study, a hydrophobic pocket located at the base of the thumb median viral load reduction of 2.3-2.9 log10 was domain. A series of thiophene carboxylic acid based achieved with 200-800 mg bid of the compound. The inhibitors were discovered to bind to this pocket. The antiviral effect was more pronounced in genotype 1b first compound of this series demonstrating clinical patients than in 1a patients, consistent with in vitro [74] efficacy was VCH-759, which had an EC50 of 0.34 potency of the compound . Combination of 200 mg and 0.27 µmol/L against genotype 1a and 1b replicon, bid ANA598 and SoC for 12 weeks reduced viral load respectively. Ten days of monotherapy with VCH-759 to undetectable level in 73% of patients. However, a at 400 and 800 mg tid (three times daily) resulted in higher incidence of skin rash was observed with

1.9 and 2.5 log10 viral load reduction in HCV patients ANA598 treatment. Further evaluation of the com- [25]. A second, slightly more potent compound, VCH- pound is on-going.

916 (1a/1b replicon EC50=79/110 nmol/L), produced NNI site 4, also known as palm site II, partially [73] a 1.5 log10 viral load reduction after 3 days . overlaps with site 3 but is closer to the active site and Currently the most advanced inhibitor of the series is the junction between the palm and thumb domains. A

VCH-222 (VX-222), which has an EC50 of 65 and 41 series of benzofuran based inhibitors were identified nmol/L against genotype 1a and 1b replicon, to bind to site 4. The most notable compound is respectively. In genotype 1 HCV patients, 250-750 mg HCV-796, a very potent inhibitor of both genotype 1a [57] bid or 1500 mg qd of VX-222 resulted in a >3 log10 and 1b HCV (replicon EC50=10 nmol/L) . In a viral load reduction after 3 days [126]. Another class of Phase 1b trial patients receiving 500-1000 mg bid site 2 inhibitors is represented by filibuvir (PF- HCV-796 monotherapy had a peak viral load

868554), which is partially cross-resistant with reduction of 1.4 log10. However, the viral load started thiophene carboxylic acids as their binding pockets rebounding in most patients at day 4, which was overlap. The compound had a replicon EC50 of 59 associated with the emergence of resistance mutations. nmol/L in vitro [135]. Monotherapy with the compound In a Phase II trial, combination with PEG-IFN-α2b at 450 mg bid or 300 mg tid resulted in a maximum resulted in 3.3-3.5 log10 HCV RNA reduction at day viral load reduction of 2.1 log10. These site 2 14 vs. only 1.6 log10 with PEG-IFN-α2b alone. inhibitors all lost activity against other HCV However, the compound was discontinued due to genotypes, presumably due to sequence variation significant hepatotoxicity findings in two patients (polymorphism) around the binding pocket. receiving the compound in combination with NNI site 3 is located at the palm domain of NS5B. PEG-IFN-α2b/ribavirin for 12 weeks [31]. Benzothiadiazine inhibitors targeting site 3 were first discovered in 2001 and subsequently followed up by INHIBITORS OF NS5A AND OTHER several groups [18,51,105,117,133]. ANA598 belongs to this VIRAL TARGETS series and has great in vitro potency, particularly Apart from NS3 and NS5B, the other viral proteins against genotype 1b (replicon 1b EC50=2.8 nmol/L, 1a have also been pursued as potential antiviral targets.

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The most promising one is NS5A. NS5A is a to date no data has been published demonstrating a multifunctional protein. NS5A interacts with NS5B direct binding of the inhibitors to NS5A protein. and is part of the replication complex, thus it is Besides the cell-based replicon assay, there is no required for viral replication. More recently it was defined function of NS5A that can be used to measure demonstrated that NS5A is also involved in viral and optimize the activity of the inhibitors. assembly. NS5A exists in both basally and NS4A is the co-factor of the NS3 protease. A series hyperphsophorylated forms, the function of which of acyl thiourea inhibitors were identified through may be regulated by cellular kinases. NS5A protein is replicon screening. It was proposed that these consisted of three domains. The structure of domain 1 compounds bind to NS4A and interfere with the was solved recently by two independent groups [85,141], interaction between NS3 and NS4A. The lead interestingly revealing two different conformations, compound ACH 806 (GS-9132) reduced viral load by which were hypothesized to be associated with 0.91 log10 at 300 mg bid for 5 days in HCV patients different roles that NS5A may play at different steps but was terminated due to nephrotoxicity [85,141,155]. of viral life cycle. NS4B is responsible for anchoring the replication Besides the intriguing biology associated with complex to the ER membrane and is required for viral NS5A, the discovery of a NS5A inhibitor BMS- replication. Recently it was reported that NS4B also 790052 has greatly increased the interest on this contributes to virus assembly and release. There have protein as a drug target. It is the most potent HCV been several reports of potential NS4B inhibitors [19,27,121,134] inhibitor reported to date, with a replicon EC50 of 9 , mainly based on the observation that these pmol/L against genotype 1b and 50 pmol/L against inhibitors select for specific resistance mutations in genotype 1a. The exceptional potency of the NS4B. Because of the lack of a functional assay for compound also translated to clinical efficacy: a single NS4B and the absence of direct-binding data, the dose of BMS-790052 resulted in a 3.6 log viral load mechanism of action of these inhibitors remains to be reduction in HCV patients after 48 h which was elucidated. maintained for 6 days [41]. The compound is currently The p7 protein of HCV was shown to be required being evaluated in Phase II trials in combination with for viral replication in chimpanzees [128]. The function PEG-IFNα/ribavirin. Several other NS5A inhibitors of p7 has been unknown until recently several groups are also reportedly in early clinical or preclinical showed that p7 has cation channel activity in vitro development (Table 1). [47,109] and appears to play an important role during It should be noted that the mechanism of action of virion secretion in culture [60,138]. The structure of p7 these compounds has not been completely elucidated. was solved recently [87], displaying some similarity to They were claimed to be NS5A inhibitors mainly M2 ion channel of virus, both of which can because they select for specific resistant mutations in be blocked by the inhibitor amantidine [47]. P7 can also NS5A. Some of the compounds were shown to be inhibited by iminosugars [109] and hexamethylene modulate the phosphoylation of NS5A [75]. However, amiloride [115]. Compounds that block its activity in

256 Virol. Sin. (2010) 25: 246-266 vitro also inhibit viral particle production in cell cis-trans isomerases (PPIase), which are involved in culture [46,139]. BIT225, a compound with known many cellular processes such as protein folding and activity against HIV-1 Vpu ion channel [63], blocks p7 trafficking. It has been shown that cyclophilins and is currently in early clinical trials for HCV [88]. particularly cyclophilin A (Cyp A) is required for The C-terminal two-thirds of NS3 is a helicase/ HCV replication. Knock-down of Cyp A with specific NTPase, which has been well characterized. The siRNAs blocked HCV replication [40,99,100,147]. The structure of the NS3 helicase is also available. HCV inhibitory activity of cyclosporin analogs However, helicase is traditionally a difficult target. correlated with their cyclophilin-binding affinity, but Despite a number of screening and early drug not immunosuppressive or P-gp inhibitory activity [89]. discovery efforts [10,11,44,136,144], no potent and selective Although the functions of cyclophilins in HCV remain NS3 helicase inhibitors have been identified that are to be fully elucidated, increasing evidence suggests suitable for further development. that cyclophilins (mainly A and B) are involved in Cleavage of HCV polyprotein between the NS2 and HCV replication by (1) interacting directly with viral NS3 is mediated by an autoproteolytic activity that proteins (NS5A and NS5B) as part of the replication requires both the C-terminal portion of NS2 and the complex and/or (2) mediating the correct folding and N-terminal of NS3. Assays have been established to trafficking of viral proteins to the site of replication measure the protease activity, which could enable (cytosolic side of ER membrane) through their PPIase screen for inhibitors [26,142,150]. However, NS2 remains activity [15-17,21,32,40,43,49,50,62,81,123,148,154]. Cyclophilin to be a difficult target because of the hydrophobic inhibitors block the interaction of cyclophilins with nature of the protein and the challenge to inhibit an HCV proteins and hence the formation of a functional autoproteolytic reaction. It was recently discovered viral replication complex. that NS2 is also involved in viral assembly and Three cyclophilin inhibitors have entered the clinic production, however the autoprotease activity of NS2 and shown efficacy in HCV patients. NIM811, a does not appear to be required [59]. non-immunosuppressive cyclosporin analog, is a potent HCV inhibitor in vitro [89]. The combination of CYCLOPHILIN INHIBITORS AND NIM811 with IFN-α and NS3 protease or polymerase HOST TARGETS inhibitors not only enhanced anti-HCV activity but There are only ten viral proteins of HCV and not all also helped to suppress the emergence of resistance of them are druggable targets, but there many more [95]. In a Phase 1b trial in genotype 1 HCV patients host proteins involved in viral replication, which who had relapsed in prior interferon therapy, patients greatly expand the list of potential antiviral targets. receiving 600 mg bid NIM811 plus PEG-IFN-α2a had

Targeting host factors also has the advantage of an HCV viral load reduction of 2.78 log10 compared to [72] presenting a higher genetic barrier to resistance. One only 0.58 log10 with PEG-IFNα-2a alone . of such targets is cyclophilin. Cyclophilins are a Alisporivir (Debio-025) is a more potent cyclosporin family of highly conserved cellular peptidyl-prolyl analog [106]. In a Phase I trial in HIV-HCV co-infected

Virol. Sin. (2010) 25: 246-266 257 patients, 1200 mg bid of alisporivir monotherapy Several new forms of long-acting IFNs are in clinical resulted in a 3.4 log10 reduction of HCV RNA after 14 development. The most advanced is albinterferon-α2b days [37]. In a Phase II combination study, 600 mg qd (albIFN), a fusion protein of human albumin and alisporivir plus PEG-IFN-α2a led to 4.6 log10 viral IFN-α2b. It has an extended half-life of 144 h, which load reduction after 28 days in genotypes 1 and 4 is even longer than those of pegylated IFNs and allows [35] patients and 5.9 log10 in genotype 3 patients . The dosing every two or four weeks while maintaining a third cyclosporin analog that has shown clinical comparable efficacy and safety profile [3,101,158]. efficacy is SCY-635, which had a 2.2 log10 viral load Toll-like receptor TLR7 recognizes single-stranded reduction in a 15-day monotherapy trial in genotype 1 RNA virus and activates type 1 IFNs as part of innate HCV patients [55]. immune response. Small molecule agonists of TLRs Alpha-glucosidase I is another host target being such as isatoribine could exert antiviral effect through pursed, which is involved in glycoprotein processing activation of TLR and IFN pathway. Isatoribine and and is important for viral maturation and release. an oral prodrug of isatoribine, ANA975, showed Inhibition of alpha glucosidase leads to misfolding of clinical efficacy in HCV patients [56,152]. However, HCV envelop protein thus blocks viral assembly and further development of these drugs was discontinued release [151]. Monotherapy with an alpha-glucosidase I due to significant side effects and insufficient inhibitor, celgosivir, resulted in only a modest therapeutic window. In 2009, it was reported that a antiviral effect. In a 12-week Phase II trial, only 2 out novel oral TLR7 agonist ANA773 demonstrated of 35 patients had greater than 1 log10 viral load significant antiviral response in hepatitis C patients. reduction [157]. Combination of 400 mg celgosivir with Patients receiving 2000 mg of ANA773 every other

PEG-IFNα/ribavirin resulted in greater than 2 log10 day for 10 days has a mean maximal viral load decline viral load reduction in 45% patients vs. 10% with PEG- of 1.3 log10, compared to 0.3 log10 with placebo. No [61] IFN-α/ribavirin only . Further combination trials serious adverse events were reported. Further clinical with different dosing regime are currently on-going. trials of ANA773 in combination with other HCV Nitazoxanide is a drug previously approved for agents are expected. parasitic infestations. Recently, it was shown to inhibit There have also been limited efforts in developing HCV through inducing phosphorylation of eukaryotic therapeutic vaccines for HCV. GI-5005 is a heat- initiation factor 2α, a known mediator of host antiviral inactivated recombinant Saccharomyces cerevisiae defense [28,66]. In a Phase II study in genotype 4 that has been genetically modified to express HCV patients in Egypt, the combination of nitazoxanide NS3 and core proteins [48]. In a Phase I trial, GI-5005 with PEG-IFN-α2a and ribavirin resulted in 79% SVR showed a modes antiviral effect reducing viral load by [127] vs. 50% with PEG-IFN-α2a and ribavirin alone . 1.4 log10. It is currently being evaluated in triple combination therapy with SoC. A number of other NOVEL , IMMUNOMODULATORS vaccine strategies such as DNA and T cell based AND IMMUNOTHERAPY vaccines have also been pursued. IC41, a peptide-

258 Virol. Sin. (2010) 25: 246-266 based vaccine is currently in early clinical deve- Despite these progresses, it remains to be determined lopment [33,34,65,149]. whether an IFN-free regimen is capable of completely clear the virus, i.e. cure HCV infection. The main FUTURE PERSPECTIVES issue for DAAs is the development of resistance. It The first generation of orally active direct acting has been demonstrated both in vitro and in patients antiviral agents (DAA), specifically NS3 protease that drug-resistant mutants can emerge quickly, even inhibitors, are expected to be approved within two with the most potent inhibitors of viral protease or years to be used in combination with current SoC polymerase [130]. This has been attributed to several (PEG-IFN-α and ribavirin). These new triple factors: (i) HCV replicates at a high rate in patients, combination therapies will significantly improve producing an estimate of 10[10-12] virions per day [103]; treatment response over current SoC. However, the (ii) the RNA-dependent RNA polymerase of the virus side effects associated with these new oral agents in lacks proof-reading function and has an error rate of addition to the significant side effects already caused about 10-4 mutations per genome per replication cycle. by IFN and ribavirin suggest that even less patients As a result there is an extremely high degree of can tolerate the new therapies. Moreover, the first heterogeneity of viral population (quasispecies) in generation DAA has short half-life and requires each patient; (iii) since viral targeted inhibitors frequent dosing (q8h). Patient compliance could typically bind to a defined pocket of a viral protein, become a real issue which may significantly typically a single mutation in viral genome is compromise the efficacy and utility of the drugs. sufficient to disrupt the binding of inhibitor and lead With multiple classes of small molecule DAAs in to resistance. Theoretically all the possible single, development, the future objective of HCV therapy is double or even triple mutations are already pre- an IFN-free, oral cocktails of DAAs, similar to the existing in HCV patients, therefore it was estimated at highly active antiretroviral therapy (HARRT) for least three DAAs are required to completely suppress HIV-1 infection. The first of such combinations has the emergence of resistance. Compounds with yielded promising results in the clinical trial. The relatively high resistance barrier such as cyclophilin combination of an NS3 protease inhibitor RG7227/ inhibitors could provide the key advantage in an IFN- ITMN-191 and an NS5B polymerase inhibitor free regimen. Thus, the combination of host and viral RG7128 administered without PEG-IFN-α or ribavirin targeted inhibitors could be an attractive strategy in for 13 days reduced HCV RNA to an undetectable maximizing antiviral efficacy and suppressing the level in 88% of treatment-naive patients and 50% of emergence of resistance. null responders to prior IFN therapy [42]. Several other clinical trials exploring different oral combinations References have already been initiated, including telaprevir plus 1. Ago H, Adachi T, Yoshida A, et al. 1999. Crystal structure of the RNA-dependent RNA polymerase of VX-222, BMS-790052 plus BMS-650032, and GS- hepatitis C virus. Structure, 7: 1417-1426. [22] 9256 plus GS-9190 . 2. Arasappan A, Bennett F, Bogen S L, et al. 2010.

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