2.1 General Reviews
Virus binding, internalization, cytoplasmic releasing and uncoating, IRES-mediated translation, polyprotein processing, virion packing, assembly, maturation, and release of infectious particles protease are the important steps for in the viralreplication life cycle.
All steps of virus life cycle are the targets for anti-viral drug discovery. The complete HCV life cycle is difficult to investigate due to the lack of a suitable in vitro and in vivo model system for HCV infection. Inadequate virion productions for study are the main problems of the model system.
IRES at the 5’ region of HCV RNA genome is also a target for anti-HCV drug discovery (183, 195). Ribozyme and antisense oligonucleotides for viral RNA are currently being evaluated in clinical trials (183). RNA interference technologies are used for specific purposes against HCV viral genome recently (66, 100, 155, 199, 208).
HCV E2 glycoprotein is very important for viral attachment to host cell, which is another potential anti-viral target. Monoclonal antibody against HCV E2 protein has been assessed in clinical phase Ib (183). Until the subgenomic replicon system has been developed (19, 117), partial HCV life cycle could not be studied in vitro. Although the subgenomic replicon system could help us to observe highly viral RNA replication, no virion could be obtained.
NS3 viral protease and NS5B RdRp are viral specific proteins and has been conserded as good candidates as anti-viral targets. Several biochemical and cell-based
assay systems have been established to identify selective inhibitors, for NS3/4A protease and NS5B polymerase. The success of protease and polymerase inhibitors in controlling human immunodeficiency virus (HIV) infection justfies the conviction that the HCV NS3 and NS5B be the choice of drug targets. Moreover, the combination therapy of protease and reverse transcriptase inhibitors has been observed the most effective treatment against HIV infections. Structure-activity relationship (SAR) studies and in-depth investigations on biochemical property of target proteins provided the necessary information for designing inhibitors and for optimization of their activity. Indeed, a NS3 protease inhibitor and a NS5B inhibitor have reached phase II clinical studies (183).
The antiviral targets of the HCV have been reviewed recently (40, 176, 201). We’ll majorly address on replicon system, NS3/4A viral protease and NS5B polymerase.
2.2 Antiviral drug Screening Using HCV Subgenomic Replicon System
The HCV subgenomic replicon system facilitates a robust anti-HCV drug screening plateform. Numerous studies report the discovery of novell anti-HCV compounds/drugs based on the HCV subgenomic replicon system (109). The HCV replicon system enables high-level replication of viral subgenomic RNA. Although the life cycle of the HCV could not be fully evaluated by this system, informations on HCV replication and host factors involved in HCV replication could be obtained. The disadvantage of screening drugs based on the replicon system is that compounds identified need to be further investigated for their modes of actions.
2.3 NS3/4A as an antiviral target
NS3 viral protein is a multifunctional enzyme, which contains a protease and a helicase/NTPase domain. The N-terminal one-third part, adjacent to the C-terminus of NS2, is the protease domain and the other two-third is the helicase/NTPase domain.
Both the protease activity and helicase activity are commonly considered as targets for anti-viral drug discovery. Many assay systems of both enzyme activities have been well established for screening of inhibitors. Inhibitors could be identified through molecular modeling based on the NMR and X-ray crystallographic studies (144, 149). Three strategies for developing inhibitors of the HCV NS3/4A protease have been attempted:
abolishing the NS3/NS4A interaction, interfering with zinc binding and preventing substrate binding in the active site. Typically, the serine protease inhibitors derived from its own substrates by substitution of the scissile amide bond with an electrophilic moiety is able to form a covalent adduct with the catalytic serine residues (47). The penta- or hexapeptides NS3 protease inhibitors derived from the N-terminal NS3 cleavage products were named peptidomimetic inhibitors (83, 140, 212). The biopharmaceutical classes have been reported that structurally diverse NS3-protease substrate-based inhibitors including α-ketoamide (73, 74), Pyrrolidine-5,5-trans-lactams (4, 174, 175), azapeptides (6, 212), α-ketoacids (33, 132, 137), and boronic acids (153).
VX-950 (LY-570310) is a lead NS3-protease inhibitor compound and is in preclinical stage (183) that is identified by using structure-based computational and combinatorial-chemistry techniques. Using structure-activity relationship study another important type of peptidomimetic inhibitors derivative was found from the N-terminal cleavage products by NS3-protease (114). BILN 2061 is a macrocyclic peptidyl
carboxylic acid (53, 115) that is a very potent competitive inhibitor of the NS3/4A protease. BILN 2061 has good bio-availablity and inhibits HCV replication in cell-based assay (103, 162). BILN 2061 was at phase I clinical trials without serious adverse events and did not damage liver enzymes, only minor gastrointestinal disturbances were observed. These results indicate that BILN 2061 may be safe and effective in the treatment of hepatitis C infection. However, the BILN 2061 resistant mutant of the HCV has been found (141). Only one amino acid change in the NS3 protease region would result in the development of resistant strain..
2.4 RNA-dependent RNA polymerase (RdRp ; NS5B ) as an antiviral target
Viral polymerases are attractive targets for antiviral therapy as demonstrated by the clinical success of nucleoside and non-nucleoside inhibitors of HBV and HIV replication.
According to the chemical structure and mechanism of action, three categories of the viral polymerase inhibitors could be classified (38): (i) nucleoside analogues (49), (ii) non-nucleoside inhibitors (15, 16, 79, 166), and (iii) pyrophosphate mimics (180).
Nucleoside analogues are substrate analogues that need to be phosphorylated to their corresponding nucleotide in cytoplasm of infected cells. Then, the prodrug becomes active against the viral polymerases. The nucleotide could be incorporated by the polymerase during progressive gene synthesis, leading to early termination of the elongation reaction and thus inhibition of the viral replication. HCV NS5B polymerase is also an anti-HCV target (201) since the NS5B activity is essential for HCV viral replication. The biochemical properties (110, 119, 214) and crystal structures (24, 108) of
NS5B have been characterized. HCV NS5B uses di- or tri- nucleotides to initiate RNA replication and forms replication complex at 3’-end of HCV RNA genome. The replication complex contains NS5B polymerase, template, primer, nucleotides, and other associated factors. NS5B polymerase forms complex with nucleotides is needed for de novo initiation (23, 156). De novo initiation must then be followed by RNA elongation, termination of polymerization and release of nascent strand. In principle, each of these steps could be seen as a target for anti-viral therapy. SAR studies have led to the identification of both catalytic and regulatory nucleotide binding site in the HCV RdRp (160) such information is essential for the design of novel nucleotide analogues inhibitors.
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Benzimidazole and bezothiadiazine derives are the two major classes of the non-nucleoside type inhibitors of NS5B polymerase. These inhibitors comprise structurally heterogenous compounds, which usually bind to a site on the enzyme surface away from active site, such as an allosteric site (191). These two structural classes of inhibitors abolish the NS5B activity by binding to different allosteric interaction sites (192). A series of diketo acids were reported to selectively and potently inhibit the HCV NS5B polymerase elongation activity in vitro (181). The mechanism of action of these related compounds was found to be noncompetitive with respect to both the RNA template and to the nucleotides.