Hepatitis C virus (HCV) continues to be a major disease burden on the world. In 1999, the WHO estimated a worldwide prevalence of about 3% with the virus affecting 170 million people worldwide. The World Health Organization (WHO) developed conservative estimate that 2.3-4.7 million new HCV infections may result from unsafe injections annually1. Hepatitis is caused by at least five distinct viruses. Each belongs to an entirely different family of viruses, and they have very little in common except the target organ that they affect, the liver and the certain degree of shared epidemiology 2. Two of the viruses (HAV and HEV are spread principally by fecal oral means and three (HBV, HCV, and HDV) are spread principally by exposure to blood, although HBV is frequently spread by unprotected sex 2. A new virus HGV was recently discovered and its prevalence and pathogenic role in various liver diseases (hepatitis) have been explained 3. The existence of the third type hepatitis HCV was not appreciated until 1975, when the application of recently developed diagnosis tests for hepatitis A and hepatitis B to store samples of prospectively studied cases of transfusion associated hepatitis revealed that the most of the cases were neither hepatitis A nor B 2 The average incubation period of ~7-10 weeks which is immediate between those of hepatitis A and hepatitis B and similar to mean incubation period of all cases of transfusion associated hepatitis in the 1950's and 1960's, suggesting that NANB 4 Although NANB hepatitis was transmitted to chimpanzee in 1978, thus establishing its infectious nature, it was not until 1989 that the virus was identified 2
The HCV GENOME:
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The Hepatitis C Virus is a member of Flaviviridae Family of Viruses. HCV is positive-strand RNA molecule ranging in size from 9.6 to 12.3 thousand nucleotides, with an open reading frame (ORF) encoding a polyprotein of 3000 amino acids or more. The structural proteins are encoded by the N-terminal part of the ORF, whereas the remaining portion of the ORF codes for the nonstructural proteins 5 The 5'UTR contains four highly structured domains, numbered I to IV, containing numerous stem-loops and a pseudo not 6 7.The 3'UTR IS organized in three regions including, a variable region of approximately 30-40 nucleotide t, a long poly(U)-poly(U/ UC) tract, and a highly conserved 3'-terminal stretch of 98 nt that includes three stem-loop structures SL1, SL2 and SL3 33 34 35 . The HCV ORF contains 9024 to 9111 nt depending on the genotype. The ORF encodes at least 11 proteins, including 3 structural proteins (C or core, E1 and E2), a small protein, p7, whose function has not yet been definitively defined, nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) and the protein which results from a frame shift in the core coding region
HCV Life Cycle:
In the blood of infected patients, HCV is physically associated with VLDL, LDL, and HDL. Entry into hepatocytes requires the tetraspanin CD81 36 the scavenger receptor class B type I (17), and the tight junction proteins claudin 37 38 which confer species specificity 39 HCV also binds to other molecules, such as glycosaminoglycans, entry process is controlled by viral surface glycoproteins that trigger the changes required for mediating fusion. At least two different classes of fusion proteins (I and II) can be distinguished 40 Enveloped, the LDL receptor, and the lectins DC-SIGN and L-SIGN, but these are not essential entry factors and do not confer tissue specificity. HCV polyprotein cleavage products, in particular NS3Â±5B, form a replicase complex associated with intracellular membranes that most likely contains cellular proteins. 41 42 It allows the production of viral proteins and RNA in a distinct compartment. Translation is initiated through an internal ribosomal entry site in the 5â€² untranslated region (UTR) and generates a single polyprotein of approximately 3,000 amino acids that is cleaved by cellular and viral proteases into 10 structural and non structural proteins. viral RNA than form membrane associated replication complexes and catalyze the transcription of negative-strand RNA intermediates from which, positive-strand RNA molecules are generated 43 Capsid proteins and genomic RNA assemble to form a nucleocapsid, which passes through intracellular membranes into cytoplasmic vesicles mature virions leave the cell via the secretory pathway.
Early clearance of HCV, to prevent the risk of developing cirrhosis and HCC and to decrease mortality has been the aim of antiviral treatment in patients with HCV infection. Treatment has traditionally been with Interferon alpha, although only small numbers of patients have shown clearance of virus by this method. In past, several studies have reported that using 3 million units (MU) of INF 3 times a week for 6 to 24 weeks or similar dose of IFN intravenously for 4 to 7 weeks are beneficial in clearing virus and normalizing ALT levels. Use of IFN (3 MU, 3 times in a week) intramuscularly for 4 weeks has shown similar results. Special precautions should be taken before giving interferon to patients with decompensate cirrhosis, severe neutropenia, uncontrolled thyroid functions, thrombocytopenia, drug or alcohol abuse and past or current psychiatric illness 44. The HCV genotype 3, younger age and female gender are significantly associated with high rate of spontaneous clearance of infection 45 Since the beginning of 2001, recombinant interferon has been replaced by newly developed pegylated IFN 2a and IFN 2b.The current standard therapy for hepatitis C treatment consists of combination of pegylated interferon-a (Peg-IFN-a) with Ribavirin (RBV). Modified forms of IFN, such as Pegylated IFN, etc. are available and have been shown to achieve a more sustained virologic response in chronic hepatitis C patients, particularly in those infected with genotype 1 (Manns et al., 2001). On the other hand, in the context of liver transplant patients with recurrent HCV infection, there is a strong need for new therapeutic options that halt the accelerated course of the disease which frequently leads to graft loss. Recently, Peg-IFN+RBV therapy has been explored in these patients, 45 but the efficacy is limited to approximately 30% of those treated, and can be associated with severe side effects and graft loss 46 This combination regimen is successful in patients with genotypes 2 and 3 infection suppresses the HCV rate to 75-90%. Nevertheless, this combination is much less successful in patients processing genotype 1 and 4 infections suppresses the HCV rate between 45% and 52% 47 48 The combination treatment can be administered to the relapse cases and people who do not respond to monotherapy. Therefore, the unavailability of an effective therapy, and the increase in the disease burden expected for the years to come, have fueled pharmaceutical companies to develop specific compounds directed against critical steps of the HCV life cycle.49
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HCV NS3 Protease
NS3 protease is a relatively small protein (180 residues) and belongs to the sub-class of small chymotrypsin-like proteases 1989, 50 two groups presented seminal comparative sequence studies suggesting the presence of a trypsin/chymotrypsin-like serine protease in the N-terminal one-third of the NS3 protein of flaviviruses and pestiviruses 50 51 it makes an economical use of loops, since it lacks a series of connecting elongations that are a common feature of the longer cellular proteases. The solution structure of the complex is similar to the solution structure of the free enzyme 51
HCV might encode a homologous trypsin/chymotrypsin-like serine protease as well. A catalytic triad of His1083, Asp1107, and Ser1165 based on the polyprotein number of HCV-H strainwas identified by sequence alignment of the HCV NS3 protein with many known viral and cellular serine proteases, which belong to the trypsin/chymotrypsin superfamily of serine proteases (52-55 HCV encodes a polymerase that specifically synthesizes The mature NS3 protein comprises 5 domains: the N-terminal 2 domains form the serine protease along with the NS4A cofactor, and the C-terminal 3 domains form the helicase new viral RNA (NS5B), and two proteases that cleave the polyprotein, the NS2/NS3 autocatalytic protease and the NS3-NS4A serine protease. Several compounds that influence the activity of the NS3 protease and the NS5B RNA-dependent RNA polymerase are currently in clinical trials, and will likely become the next generation of anti- HCV drugs. 50 The portion of HCV acts as helicase has attracted the attention not only of researchers interested in developing novel antiviral drugs, but also those studying how proteins interact with nucleic acids. HCV helicase has attracted the attention not only of researchers interested in developing novel antiviral drugs, but also those studying how proteins interact with nucleic acids.
HCV NS3 Protease Inhibitors
The NS3 protease domain has since been delineated to approximately 181 residues at the N-terminal third of NS3 (around residues 1027Â±1207 of the HCV polyprotein), within which are found the conserved residues His-1083, Asp-1107 and Ser-1165, characteristic of the catalytic triad found in trypsin-like serine proteases 56 The remaining C-terminal portion of NS3 contains motifs characteristic of a helicase} nucleoside-triphosphate-binding domain 57 Nonstructural protein 3 (NS3) of HCV contains a zinc-binding serine protease domain that has been shown to be responsible for processing of the downstream nonstructural proteins 58 59 are conserved in all HCV genotypes, 60 characterization of enzymatic activity of the purified GBV-B NS3 protein in vitro on HCV substrate sequences shows that this enzyme is a serine protease, that it is able to cleave all of the substrates cleaved in trans by the HCV NS3 protease, and that cleavage indeed occurs after a cysteine residue. The NS3-4A serine protease is responsible for the proteolytic cleavage at four junctions of the HCV polyprotein precursor: NS3/NS4A (self cleavage), NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B 60-63 In addition, NS4A forms a non-covalent complex with the NS3 serine protease, which was stable in the presence of non-ionic detergent 60 64 The nonstructural (NS) proteins include enzymes necessary for protein maturation (NS2/3 and NS3 proteases) and viral replication (NS3 helicase/nucleoside triphosphatase and NS5B RNA polymerase.
The first HCV protein crystallized was the portion of NS3 that acts as a helicase, an enzyme that tracks along a nucleic acid strands displacing annealed strands or RNA-binding proteins. 65 However, NS3-NS4A protease and NS5B have been so far the most successful HCV encoded drug targets 50 The NS3 protease is a serine protease with a shallow hydrophobic substrate binding region which thus represents a significant challenge for rational drug design. Nevertheless, numbers of of NS3 protease inhibitors have entered clinical development.
Development of HCV NS3 Protease Inhibitors
The NS3 protein contains a trypsin-like serine protease domain at its N-terminus, while its C-terminal domain has helicase activity. It has been clearly demonstrated that the NS3 protease requires the cofactor NS4A to efficiently cleave the rest of the non-structural proteins (NS3, NS4A, NS4B, NS5A and NS5B). Since NS3 is necessary for subsequent viral replication, it is thought that development of a specific inhibitor of NS3 protease activity would be an attractive target for new anti-HCV drugs 66 Addition yet to be made from your SN paper.99 The high rate of viral production linked to the low fidelity of the RNA polymerases 67 leads to genetic heterogeneity of HCV in infected patients 68 Inhibitors of the HCV serine protease were designed through a substrate-based approach 69 70
NS3/4A Protease Inhibitors
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Deletion studies indicate that the N-terminal 22 residues of NS3 and the above-mentioned central region of NS4A is involved in interaction between two proteins 51-54. The target NS3 or NS3-4A complexes are localized almost exclusively in the cytoplasm. Substitutions that disrupted the interaction between NS3 and NS4A also resulted in reduction or loss of protease activity, suggesting that formation of an NS3-NS4A complex could be a pre-requisite for a functional serine protease 71 Thus, the HCV NS3/4A protease is one of the most attractive targets for antiviral chemotherapy. BILN-261 (ciluprevir), was developed by Boehringer Ingelheim scientists who established for the first time a proof-of-concept for these types of inhibitors of HCV replication Ciluprevir showed promising results in phase I studies, but further development was halted because cardiotoxicy was detected in animals There are several other HCV NS3 protease inhibitors in development, such as telaprevir (VX-950), boceprevir (SCH- 503034), SCH6 (SCH446211), ITMN-191, TMC435350, MK-7009 and ACH-806 (GS-9132) 80-92 Some of these compounds well as a number of other compounds have been forwarded to later stages of development and are now in advanced clinical trial
Table 1.1: HCV NS3/4A Protease Inhibitors at Preclinical or Advanced clinical Trial, chemical structure and Developmental Status
HCV NS3/4A Protease Inhibitors chemical structure Developmental Status
HCV NS3 Protease Inhibitors at Preclinical or Advanced clinical Trials and their chemical structure
Medivir / Tibotec
Merck & Co.
I will their stage of development in separate column also
Am I supposed to give references of these inhibitors here inside this table or at the end?
I will defiantly search those structures or draw them in better pixels and form
Table 1.2: Novel Patented HCV NS3/4A Protease Inhibitors
Novel Patented HCV NS3/4A Protease Inhibitors
Can I add some of your compounds or there is in ignorance about their mechanism?
One of the unique properties of HCV helicase is that, unlike other helicases, the protein binds RNA and DNA in a sequence specific manner. Even the first studies of the protein noted that HCV helicase has a distinctive nucleic acid stimulation profile 92 clearly; the biological role of HCV helicase needs to be investigated in more detail. It has long been a mystery why RNA viruses that replicate outside the nucleus encode a helicase. A protein that resolves duplex RNA and DNA structures and displaces proteins bound to nucleic acids, like HCV helicase, could be valuable to 93 although the NS3 helicase is a potentially attractive target for anti-HCV drugs, no helicase inhibitors have yet entered clinical trials. A number of groups, including workers at the Universities of Cardiff 94 95 have research aimed at identifying inhibitors of the NS3 helicase.
Biological Modes of NS3 Protease Inhibitors
Cell Culture System
From in vitro studies using Cell Culture System, MAVS cleavage by NS3 protease in HCV-infected Huh7 cells in culture is completely abrogated by BILN 2061 treatment, demonstrating a dual therapeutic potential of protease inhibitors to restore antiviral innate signaling 92 Up to now only two reports have been published describing the replication of a transfected HCV genome in the human hepatoma cell lines Huh-7 or HepG2 96 IRES element was included to allow translation of the HCV NS proteins. Since we did not know whether NS2 was required for replication, two variants were generated spanning the NS2Â±5B or the NS3Â±5B region97
Addition yet to be made from your SN paper.99 Surprisingly, these selected cells carried large amounts of HCV RNAs detectable by Northern blot, or after metabolic radio labeling with [H]uridine, providing formal proof that these RNAs were actively replicating in the cells.
HCV NS3 Protease In Vitro Assay
Examination of processing using in vitro-translated NS4A/4B, NS4B/5A, and NS5A/5B polyprotein fragments from genotype 1a revealed no measurable differences in protease activities for genotype-1 and -3 single-chain NS4Apeptide-NS3 proteases 100 but a reduction in activity for the genotype-2 NS3 protease- NS4Apeptide complex relative to that of genotypes 1 and 3 101 Later, in vitro translated polyprotein substrates were used for monitoring activity of purified NS3 protease, which was generated as recombinant proteins in E. coli, baculovirus, or yeast expression systems 102-104 However, these assays using in vitro translated polyproteins are much less quantitative than those using synthetic peptides as substrates, which are cleaved by the HCV serine protease and the products are analyzed on high performance liquid chromatography (HPLC) There are also limitations in interpreting the results of in vitro experiments. For example, the concentrations of drugs used in this study were selected according to their relative potency in the HCV replicon system and may not reflect clinically relevant doses. Also, the effects of the drugs in vivo will be affected by many other factors, such as the physical properties and pharmacokinetics of the drugs. In addition, agents such as IFN and ribavirin could exert indirect antiviral activities, such as immunomodulatory effects, that are absent in the in vitro system 95 It remains to be determined whether the synergy observed between HCV protease inhibitors and IFN in vitro can ultimately be translated into clinical efficacy.
As the only animal model for the study of HCV, the chimpanzee was used to provide early characteristics of HCV. Even before HCV had been identified, the chimpanzee was involved in the study of Non-A, Non-B hepatitis (NANBH) virus transmission, in establishment and duration of disease, and in the chronic nature of NANBH infection 105 The role of the NS2/3 protease in HCV replication remains to be fully understood. NS2/3 cleavage is required for viral replication in vivo, as demonstrated by an HCV clone devoid of NS2/3 activity that fails to cause a persistent infection in a chimpanzee 106 In addition, efficient NS2/3 cleavage is required for replication of the viral RNA genome in a NS2-3-â€²UTR replicon system (Welbourn et al., 2005) However, it is extremely limited in its availability, as well as by its expense. As a consequence, many results have been generated in experiments with low numbers of animals. A number of NS3-based HCV vaccines have been developed and tested in non transgenic-mouse models 107 However, no study has tested whether vaccine-primed T cells can actually enter the liver, which is a prerequisite for a functional therapeutic vaccine. A recently developed codon-optimized vaccine encompassing the complete HCV NS3/4A protease has been found to effectively prime HCV-specific T cells that inhibit the growth of HCV-expressing tumor cells in vivo . vaccine to test the ability of the vaccine-primed T cells to enter the liver and eliminate HCV antigen-expressing hepatocytes, using a newly developed transiently transgenic-mouse model. Furthermore, immune responses to HCV in an vivo system was studied in mice transgenic for HLA-DR and human CD4 to analyze the specificity of murine responses to the HCV NS3 antigen in an effort to determine whether the epitopes recognized correspond to those recognized by human T cells 108 These models are technically challenging, but once optimized they promise to be extremely useful both for the study of HCV and for drug development
Challenges and future Prespectives
Presently it appears that HCV protease and HCV polymerase inhibitors will be developed as the next generation of anti-HCV drugs. Inhibitors targeting the NS3 helicase domain could affect its protease activity given that the NS3 sub-domains mutually regulate each other109 .. HCV NS3 protein has serine proteins and helicase activity and is one of the most conserved protease of HCV 110 It contains an immunodominant CD4+ T-helper epitope and several CTL epitopes, which have been associated with control of HCV in patients with self-limiting infection 111. These characteristics also make NS3 an appropriate vaccine candidate for HCV.
In addition, the antiviral activity of these treatments will have to be evaluated in individuals infected with non-genotype 1 HCV. For instance, the study C209 revealed that telaprevir shows little or no antiviral activity against genotype 3 HCV while efficiently targeting both genotype 1 and 2 HCV. Another challenge resides in the identification of new classes of NS3/4A protease inhibitors. Achillion Pharmaceuticals (CT) is developing ACH-1095, an NS4A antagonist, which is essential for the NS3 serine protease activity. This compound is currently in late-stage preclinical assessment. A major challenge for a successful anti-HCV therapy is to delay the emergence of drug resistance and virological breakthrough that were reported in the first two weeks under DAA (Direct Acting Antiviral) monotherapy.112 hence, specific therapeutic strategies have to be elaborated in order to cure HCV infection. Target-based antiviral drug discovery that mainly relies on the use of in vitro assays, has led to the identification of several anti-HCV compounds awaiting clinical validation through.
It is hoped that the introduction of HCV protease inhibitors in the clinic, either alone or in combination with IFN, will provide better treatment regimens for hepatitis C patients.ccc The combination of specific HCV NS3/4A inhibitors with Peg-IFN or Peg-IFN+RBV treatment regimes may be synergistic, opening the door to future combination therapies. Nevertheless, it will be very exciting to see HCV NS3-4A serine protease inhibitors progress through clinical developments and, hopefully, provide hepatitis C patients with much needed, more effective therapies.