Development of Novel Antiviral Therapies for Hepatitis C Virus Kai Lin** (Novartis Institutes for Biomedical Research, Inc
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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 Hepatitis C 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 interferon-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 infections 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 infection is pegylated interferon alpha (PEG- patients will die from the consequences of HCV IFN-α) in combination with ribavirin 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. Virol. Sin. (2010) 25: 246-266 249 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].