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Hepatitis C, a life threatening disease, is the frequent and most significant cause of liver failure. Approximately 170 million people worldwide suffer with chronic hepatitis C infection. The causative agent of the disease is the hepatitis C virus (HCV), a non-cytopathic hepatotrophic virus belonging to the Flaviviridae family. HCV is a spherical, enveloped virus 40- 60 nm in diameter that infects only humans and chimpanzees. The most common means of HCV transmission is through blood transfusions, hemodialysis, and organ transplants. Six major HCV genotypes and numerous subtypes have been identified based on sequence homology (Penin et al., 2004). Differences in genotypes are trivial and can be as much as 40% at the nucleotide level. Genotypes 1, 2, and 3 have a worldwide distribution, while genotypes 4, 5, and 6 are localized to more specific geographic regions. Besides being limited to varied geographical areas, each genotype has its own unique pattern of disease succession and response to therapy. Early infection leads to the founding of an acute phase that is asymptomatic and therefore difficult to detect. Approximately 15%-20% of acutely infected individuals clear the infection devoid of medication and the remaining, 80% of acute infections become persistent thereby reaching a point of chronic state that threatens liver failure. Roughly one-fifth of chronically infected individuals go on to develop cirrhosis and among those 1%-3% develop hepatocellular carcinoma (Qureshi., 2006). Pegylated interferonÎ± along with ribavirin is the current therapeutic treatment effective for HCV genotype 2 and 3, but not so efficient in clearing HCV in patients infected with genotype 1. As a result of this many new agents with different other targets identified as a consequence of understanding the HCV lifecycle have been developed in the hope that they will revolutionize the current therapeutic outcomes for HCV. The key focus of the development or discovery of such drugs is to target viral processes during the viral replication cycle.
MOLECULAR BIOLOGY OF HCV
HCV is a 9.7kb single stranded RNA virus with positive polarity. It encodes a single polyprotein of 3011 amino acids. The polyprotein flanked on either side by 5' and 3' UTR (UnTranslated Region) is then processed by both host as well as viral proteases into structural ( Core, E,E2 and p7) and non-structural genes (NS2, NS3,NS4A, NS4B, NS5A and NS5B) (Lindenbach et al., 2005). C:\Users\Sundus\Desktop\Chipkoo's modified\3V2.jpg
a, The structure of the viral genome, including the long open reading frame encoding structural and nonstructural genes, and 5' and 3' UTRs. The polyprotein processing scheme is shown below. Closed circles refer to signal peptidase cleavage sites; the open circle refers to the signal peptide peptidase cleavage site. All other terms are defined in the text.
Â b, The topology of HCV proteins with respect to a cellular membrane.(Lindenbachet al., 2005)
Various steps are involved in the attachment, infection, replication and then release of the HCV virus to infect other cells. It is crucial to understand and study each of these steps as each and every individual step serves as a potential therapeutic target for rendering the viral infection and thereby the replication and release inactive as well.
HCV Life Cycle
The genetic material of the HCV virus is surrounded by an icosahedral capsid (protein) which in turn, is surrounded by glycoproteins that are derived from the host cell membrane. Many surface proteins have been considered as acting as cell surface receptors for HCV, but out of all of them the CD-81 receptor has emerged to be the most likely candidate to which the envelope proteins E1 and E2 of the virus attach (Pileri et al., 1998).
CD-81 receptor is expressed on other non-hepatic cells as well, but those cells are somehow not vulnerable to HCV infection which therefore leads to the conclusion that other liver-specific proteins act as receptors or co-receptors for virus attachment and entry into the cell (Tan et al., 2003).
Once the virus is bound to the host cell, the host cell membrane folds inwards. Low pH, in the endocytic compartment results in the fusion of the host and viral membrane and hence the release of the single stranded RNA of virus into host cell cytoplasm via endocytic uncoating (Lindenbach and Rice, 2005).
After gaining entry into the host cytoplasm HCV RNA is translated. The ribosomal units present on the RER bind to the internal ribosomal entry site (IRES) present at the 5' end of the viral genome. The 40S ribosome forms a complex with IRES initially, and then the eukaryotic initiation factor eIF-3, followed by the Met-tRNA-eIF-2GTP which results in the formation of 48S rRNA complex at the initiation codon AUG (Kim et al., 2004).This complex is then converted to the translationally active 80S complex. This translation results in the formation of a long polyprotein comprising of 3011 amino acids. Processing of the structural genes is then carried out by the host peptidases, whereas the proteolytic processing of the non-structural genes is performed by HCV proteases (He et al., 2003).
FIGURE: Various steps of the HCV Life Cycle
Functions of HCV Encoded Proteins:
The core protein is a multifunctional basic protein. It not only serves the purpose of encapsulating the viral genome but also acts as a trans-modulating factor in the IRES mediated translation of the cap-less mRNA (Boni et al., 2004). It is found in high concentrations on the surface of ER in host cell cytoplasm as well as mitochondria where it is involved in elevating production of Reactive Oxygen Species (ROS). Core also negatively impacts the Electron Transport Chain (ETC). The core protein is also responsible for activation of various signaling pathways including the NF-ÎºB pathway (Yoshida et al., 2001). Apart from this the Core also influences expression of various genes by interacting with various proteins.
HCV encodes two glycoproteins E1 and E2 that are processed by host signal peptidases. When both E1and E2 are expressed together they are retained in the endoplasmic reticulum (ER) where they form heterodimers. The E2 contains two hyper variable regions (HVR) called HVR1 and HVR2. Variability in sequence arises due to mutations occurring in these HVR's. Due to hyper mutation epitopes confined in these areas evolve rapidly making it challenging for the immune system to catch up (Pohlmann et al.,2003).
p7 is a 63 amino acid protein that functions as an ion channel. It is believed that infectivity and replication of HCV is dependent on the p7 gene product (Sakai et al., 2003).
NS2 is a cysteine protease that is semi-matured by the action of host signal peptidases; its complete processing is dependent on N-terminal region of non-structural gene NS3 (Failla et al., 1994). The N-terminal of NS3 is responsible for activation of NS2 as a result of which the processing of NS2-NS3 takes place autoproteolytically by NS2.
NS3 serves as a bifunctional protein. N-terminal of NS3 contains serine protease activity which requiresNS4 to function optimally (Failla et al., 1994). Fusion of these two proteins results in the processing of the remaining downstream polyprotein. The C-terminus of the NS3 harbors the RNA helicase domain whose exact role in viral replication cycle remains elusive. However biochemical studies have shown that the helicase functions in 3' to 5' direction and is capable of unwinding DNA:DNA, RNA:RNA and RNA:DNA duplexes (Tomei et al., 1996).
The 54 amino acid protein NS4A functions in providing stability to non-structural gene NS3. Without the NS4A protein acting as a co-factor NS3 would have a very small half life. NS4A also functions in escorting NS3 to ER due to the presence of hydrophobic amino acids stretch on its N-terminus (Wolk et al., 2000).
NS4B is an integral membrane protein, which plays a key role in HCV replication and is confined to the ER (Gretton et al., 2005). N-terminus of this protein contains an amphipathic helix (AH) rich region. The sequence within this region is conserved in all HCV genotypes and any mutations in it negatively influenceHCV replication (Elazar et al., 2004).
Moreover, NS4B also binds GTP and is a GTPase. Mutations that abolish GTP binding or inhibit GTPase activity strikingly reduce HCV replication. Recent studies have shown that NS4B influence host cell signaling pathways and hence maybe important for HCV pathogenesis (Zheng et al., 2005).
The NS5A protein consists of three domains whose functions remain unknown. NS5A protein is phosphorylated by various protein kinases but the significance of this in host cell phenotype remains unknown (Coito et al., 2004).
FIGURE: Hepatitis C virus genomic organization,replication,translation and generation of functional proteins. Proteins encoded by the hepatitis C virus (HCV) genome. HCV is formed by an enveloped particle harboring a plus-strand RNA of about 9.6 kb. The genome carries a long open-reading frame (ORF) encoding a polyprotein precursor of 3010 amino acids. Translation of the HCV ORF is directedÂ viaÂ a 5' untranslated region (UTR) functioning as an internal ribosome entry site (IRES); it permits the direct binding of ribosomes in close proximity to the start codon of the ORF. The HCV polyprotein is cleaved co- and post-translationally by cellular and viral proteases into ten different products, with the structural proteins core (C), envelop 1 (E1) and envelop 2 (E2) located in the N-terminal, whereas, the nonstructural (NS2, NS3, NS4A, NS4B, NS5A, NS5B) replicative proteins are located in the remainder. Reputed functions of the cleavage products are shown. (from World J Exp Med.Â 2012;Â 2(2): 7-25)
This gene also harbors within it a stretch of 40 amino acids. This length of amino acids is referred to as the Interferon Sensitivity Determining Region (ISDR) which was once thought to serve as a predictor of how a patient might respond to IFN-Î± therapy. The studies carried out on the ISDR region have lead to the fruitful predictions of how a patient will respond to IFN therapy. (Enomoto et al., 1996)
NS5B is the RNA dependant RNA polymerase (RdRp) enzyme and functions by carrying out replication of Hepatitis C Virus. The NS5B is attached to cell membrane by the help of 21 amino acids on its C-terminus. These amino acids are preserved in all HCV isolates. The RNA dependent RNA polymerase like all other RNA polymerases is an error prone enzyme with a misincorporation rate of 10-3 per nucleotide per generation (Schmidt et al., 2001). As a consequence of its poor fidelity rate HCV evolves rapidly and constantly produces viral quasi-species which facilitate HCV to escape immune system.
Assembly of The RNA Replication Complex
The non-structural proteins ranging from NS3-NS5B facilitate HCV RNA replication, first by assembling the membrane associated replication complex (replicase) which is responsible for synthesizing RNA and then by recruiting the genomic RNA into the complex. All positive strand RNA virions replicate in association with altered cytoplasmic membranes. This most likely helps physically sequester the replicase from host defenses and also simultaneously offers mechanical support and concentrates viral products (Lindenbach and Rice, 2005). HCV is no exception. In cell culture, the HCV non-structural proteins, RNA Dependant RNA Polymerase (RdRp) , associate with ultrastructural vesicular structures, termed as the " membranous web" which seem to resemble the altered membranes seen in HCV infected hepatocytes .
The NS4B protein can induce these modifications in cellular membranes which results in formation of membranous webs that serve as scaffolds for protein assembly. C-terminus of non-structural protein NS3 encodes the RNA helicase that maybe involved in the initiation of RNA synthesis. This helicase incrementally separates small stretches of substrate double-stranded RNA, ultimately releasing the newly formed RNA from its template. The NS5A protein as mentioned earlier has three structurally distinct domains, one of which may be involved in RNA binding. The other two domains may influence the efficiency of RNA replication (Lindenbach and Rice, 2005).
Although all of the non-structural proteins are essential for replication, the NS5B protein encodes the RNA dependant RNA Polymerase (RdRp) which is responsible for RNA synthesis. The NS5B forms a right hand polymerase structure such that the finger and the thumb encircle the active site in the palm, thus forming a channel for single-stranded RNA template. Structures protruding from the polymerase thumb which is likely to assist in positioning the template correctly (Lindenbach and Rice, 2005).
Adaptive mutations that increase replication efficiency occur in NS3, NS4B, NS5A or NS5B. Mutations in NS3 tend to have weak effects unless combined with mutations in another protein, whereas mutations in other proteins strongly enhance replication but are incompatible when combined with any mutation other than those in NS3 (Lindenbach and Rice, 2005).
The composition of the membranous webs likely influences the rate of HCV replication. Replication is stimulated by increased saturated and monosaturated fatty acids. The composition of the cell membrane can be modified via the mevalonate pathway (Goldsteint et al., 1990). Controlling this pathway has proven beneficial in other clinical conditions, primarily hyperchlolesteromia. The mevalonate pathway produces cholesterol and nonsterol isoprenoid products, including franesyl and geranylgeranyl. These isoprenoids attach to the membrane proteins through protein prenylation (Nettles et al., 2008). Researchers have shown that inhibiting protein geranylgeranylation leads to disassembly of HCV replication complex and consequently to the disruption of HCV RNA replication. Statins, which are 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, deplete mevalonate, thereby decreasing farneysl and geranylgeranyl pyrophosphates, which are donors of prenyl groups (Goldsteint et al., 1990). This limits protein prenylation and thus HCV replication. In 2003, Ye et al demonstrated that lovastatin inhibits replication of HCV in cell culture. The effect was reversed by adding geranylgeranyl, a donor of prenyl groups (Ye et al., 2003). In other words changing the lipid composition of the membranous web greatly influences HCV replication. HMG-CoA reductase inhibitors deplete lipid products necessary for HCV replication. When these products are returned to the milieu, HCV replication resumes.
FIGURE: (David Evans. 2012)
HCV has a higher rate of replication, producing nearly one trillion particles per day in a chronically infected individual. Due to lack of proofreading by the HCV RNA polymerase, HCV also has an exceptionally high mutation rate, which may help the virus elude the host immune response (Ye et al., 2003).
RNA synthesis within the replication complex is poorly understood. On the basis of knowledge attained from observing other RNA viruses, it is assumed to be semi-conservative and asymmetric. A positive- strand genome serves as a template, producing a negative strand intermediate, which then acts as a template to produce multiple positive genome copies that are then used for either polyprotein translation, production of more negative-strand RNA, or are packed into new virus particles (Lindenbach and Rice, 2005).
Virus Assembly and Release
The last stages of the viral life cycle are poorly understood. The virus is signaled to switch from replication mode to assembly mode; this may be triggered by phophorylation of NS5A.Nucleocapsid assembly involves packaging genomic RNA in the icosahedral shell of protein (capsid) and then encapsulating this structure with a lipid envelope made from modified host cell membrane. Core protein associates with the endoplasmic reticulum membrane, and a region of the core protein mediated interaction with the genomic RNA. The core undergoes post translational glycosylation-an enzymatic process that links sugars to the protein-in the lumen of endoplasmic reticulum as well as Golgi apparatus (Lindenbach and Rice, 2005). Envelope proteins are similarly modified. Without extensive N-glycosylation, the envelope proteins are unable to undergo complex folding which is essential for virus function. The assembled virus then buds into an endoplasmic reticulum vesicle (cellular secretory pathway), and is released from the infected hepatocyte.
HOST RESPONSE TO HCV INFECTION
Intracellular and Extracellular Host Response
Once the HCV virus gains entry into the host cell (hepatocyte), the innate host cell response is initiated. This involves the activation of certain signaling pathways and expression of specific genes, which collectively work in order to produce an anti-viral state within the infected cell. The pathway activated extracellularly in infected liver cells is in response to the double stranded RNA (dsRNA). The presence of this dsRNA is sensed by certain specific receptors, which further trigger downstream proteins that end up activating protein kinases TBK1 and IKK-É› (Kawai et al., 2005).
HCV dsRNA is detected by retinoic acid inducible gene-1(RIG-1) intracellularly. Once the dsRNA binds to it, RIG-1 undergoes specific conformational alterations as a result of which it binds to a specific adaptor protein termed Cardif, which activates protein kinases TBK1 and IKK-É› (Yoneyama et al., 2004).
Activation of both these kinases results in phophorylation of certain transcription factors which in turn are responsible for inducing the expression of IFN-Î² as well as other cytokine (Hiscott et al., 1999). Phophorylation of certain other Interferon regulatory Factors (IRF) namely IRF-3 and IRF-7 leads to the expression of IFN- Î± (Gilmour et al., 1995).Both IFN-Î² and IFN- Î± mediate their effect in an autocrine and paracrine manner via the IFNÎ±/Î² receptor. The extent of the innate host response is analyzed by the quantity of IFN produced and how well expression of Interferon Stimulate Genes (ISG) functions. All this determines whether if the HCV infection will proceed from the acute to the chronic stage or not.
Elusion of the Host Innate and Adaptive Immune Response
The acute phase of the HCV infection is asymptomatic and hence difficult to detect. Although the infected liver cells continuously produce IFN and express ISG during this phase. Progression from the acute phase to the chronic phase requires the proteins encoded by the virus, as these proteins are the key components that are responsible for modifying the host signal transduction pathways and by doing so they weaken the host innate immune response, thereby creating an ideal milieu for the replication, assembly and release of HCV (Gale et al., 2005).
NS3-4A inhibits action of RIG-1 and TRIF, as a consequence of which expression of both IFN-Î² and IFN-Î± is disturbed (Li et al., 2005). Besides NS3-4A, the core protein also plays a crucial role in escaping the host immune system. It achieves this by triggering the expression of suppressor of cytokine-signaling-3 (SOCS-3) which is involved in inhibiting the JAK-STAT pathway. Latter is responsible for formation of ISGF-3, which is required for expression of ISG's (Bode et al., 2003).
The NS5A non-structure viral protein contributes its share by stimulating the P13 kinase signaling pathway and attenuating the RAS ERK Mitogen Activated Protein Kinase (MAPK). Stimulation of PI3 kinase pathway prevents apoptosis of HCV infected liver cells (Street et al., 2004). NS5A along with E2 glycoprotein also inhibit activity of PKR, by preventing the binding of the dsRNA to PKR.
As a whole acute hepatitis progresses or develops into chronic hepatitis as a result of constitutive expression of HCV genome within the hepatocytes and also by the suppression of IFN producing genes.
The current therapy available to patients with either acute or chronic hepatitis C is comprised of a combination of weekly injections of pegylated interferon alpha and twice a day oral administration of ribavirin. Since the original interferon alpha used had a short life in systemic circulation, the new generation of poly ethylene glycol (PEG) conjugated with interferon-Î± was introduced as they are longer lasting and produce significantly more effective anti viral response (Silva et al., 2006). The mechanism by which ribavirin acts remains unclear.
Potential Targets for Therapeutic Intervention
No vaccine against HCV has been developed; this is primarily due to rapid viral evolution (Yu et al., 2004). Another area under investigation is attacking HCV during its travel through the cytoplasm which could result in the destruction of HCV RNA and thus prevent or decrease HCV polyprotein production. Short RNA segments (oligonucleotides and short-interfering RNAs, or siRNAs) have been produced that complement segments of the HCV RNA. When the complementary RNA binds, it marks the genomic RNA for destruction. Sequences that bind the IRES prevent ribosomal binding and hence replication as well (McHutchison et al., 2006). Alternatively, attachment of siRNAs results in sections of double-stranded RNA which is naturally earmarked for degradation via endogenous RNases (Stevenson., 2003). Although this is exciting technology, these targeted agents remain in early development.
Another potential target is the inhibition of the NS3 serine protease as a result of which non-structural protein processing, RNA replication, and viral assembly is compromised severely. This enzyme which serves as a serine protease is the target of the highly anticipated protease inhibitors currently in late stages of development. Although designing an inhibitor was difficult due to the shallow substrate binding groove on NS3, two protease inhibitor compounds (telaprevir and boceprevir),and a number of other such agents are likely to be introduced in the next two-three years..
The specifically targeted antiviral therapies for HCV or the STAT-C class of agents are enzyme inhibitors that target viral enzymes required for HCV replication. The STAT-C agents include protease inhibitors and polymerase inhibitors.
Telaprevir (VX-950) is a reversible inhibitor of the HCV NS3-4A serine protease. In the in vitro replicon system, telaprevir demonstrated potent antiviral activity against HCV with more than a 4-log reduction in HCV RNA after two week incubation (Lin et al., 2006). Telaprevir continued to demonstrate potent suppression in clinical trials. In North American multicenter trial PROVE 1 trial, 81% of genotype 1 treatment-naive patients treated with triple therapy (telaprevir, Peg-IFN, and RBV) achieved a Rapid Virologic Response (RVR; HCV PCR-negative at week four of therapy). In addition, 61% of patients receiving a twenty-four week treatment course (twelve weeks of triple therapy followed by twelve weeks of standard of care [Peg-IFN/RBV]) achieved a Sustained Virologic Response (SVR) (McHutchison et al., 2009). The multicenter European study, PROVE 2, demonstrated similar efficiency and confirmed the continued need for RBV in combination with telaprevir and Peg-IFN; 69%-80% of patients receiving triple therapy achieved an RVR compared with 50% of patients who received only Peg-IFN and telaprevir. This difference was even more prominent when comparing SVR: 60% of triple-therapy recipients compared with 36% of Peg-IFN/telaprevir patients effectively cleared virus after twelve weeks of therapy (Hezode et al., 2009). PROVE 3 is a phase 2b trial investigating the effects of telaprevir in patients with prior suboptimal response to Peg-IFN/RBV. Approximately 50% of patients on triple therapy achieved an SVR; 69%-76% of patients who had relapsed (defined as HCV-negative on therapy with return of viremia upon treatment discontinuation) attained an SVR (Manns et al., 2009).
Boceprevir (SCH 503034) also reversibly binds to the NS3 active site. It also dramatically suppresses HCV in an HCV replicon system, decreasing RNA levels 1.5-2 log at seventy-two hours and 3.5-4 log after fifteen days (Malcolm et al., 2006). This agent continued to exhibit compelling inhibition of HCV replication in clinical trials, especially when combined with Peg-IFN and RBV. Data from the phase 2 SPRINT-1 (HCV Serine Protease Inhibitor Therapy-1) trial showed that boceprevir in combination with Peg-IFN and RBV given for twenty-four or forty-eight weeks achieved an SVR in 74%-94% of HCV genotype 1 treatment-naive patients who attained an RVR (Kwo et al., 2009). Inhibition of NS3 may also improve interferon responsiveness by restoring virally suppressed interferon pathways. Replicating HCV prevents phosphorylation of interferon regulatory factor 3(IRF-3) (Foy et al., 2003).This may explain why when combined with interferon, protease inhibitors synergistically enhance therapeutic response.
As with many medications, these agents are hampered by side effects. Rash, gastrointestinal side effects and anemia have been problematic for telaprevir, although they rarely result in treatment discontinuation. (McHutchison et al., 2009)Similarly, boceprevir has been associated with anemia and gastrointestinal problems (Kwo et al., 2009). Both agents have also been associated with viral resistance, especially when used as monotherapy. These mutations may present resistance to other protease inhibitors.
Blockage of NS4A binding also decrease NS3 activity by preventing it from assuming a more active configuration ACH-806 (also known as GS-9132) binds to NS4A in genotype 1 virus. This agent was shown to decrease virus by approximately 1 log after five days (Pottage et al., 2007). Unfortunately, development was stopped due to nephrotoxicity.
The STAT-C agents also include anti-HCV drugs that target the NS5B RNA-dependent RNA-polymerase. Several polymerase inhibitors have been developed. However, despite efficacy, tolerability/adverse events have halted further development of many compounds. Currently, one nucleoside inhibitor, R7128, and several non-nucleoside inhibitors (PF-00868554, GS 9190, VCH-759) are in phase 2 trial (Rodriguez et al., 2008). In a phase 2a trial involving genotype 1 treatment-naive subjects with chronic hepatitis C, R7128 given twice a day in combination with Peg-IFN/RBV achieved an RVR (again, defined as HCV RNA PCR-negative at week 4 of treatment) in 85%-88% of subjects compared with 20% in patients who received Peg-IFN/RBV alone. This drug appeared to have clinically significant antiviral potency regardless of race/ethnicity, and side effects were minimal (Rodriguez et al., 2008). Interim data also suggest that this agent will be efficacious in non-genotype 1 patients as well (Gane et al., 2008). All 3 non-nucleoside inhibitors (PF-00868554, GS 9190, VCH-759) have demonstrated excellent short-term antiviral activity. (Hammond et al., 2008)Studies are under way to evaluate the efficacy of these agents for longer-term treatment when given in combination with the standard of care, Peg-IFN/RBV.
Therapeutic Strategies Targeting Host-Directed Glycosylation
Disrupting the glycosylation process should promote improper envelope folding and consequently interruption of vital functions that are dependent on HCV.
Celgosivir (MX-3253) is an inhibitor of the host enzyme alpha-glucosidase I which plays a role in glycosylation of viral proteins (Kaita et al., 2007). Administration of this oral drug prevents proper envelope folding and thus prevents viral assembly. Data confirm that celgosivir offers additional viral suppression when combined with Peg-IFN + RBV.When given as monotherapy, however, it had minimal antiviral effects.
Further Therapeutic Targets
Although the function of NS5A remains vague, it is vital to replication, and therefore, agents that target this protein are likely to inhibit HCV replication. A dose-finding study involving the NS5A inhibitor BMS-790052 demonstrated significant long-lasting inhibition after a single dose. (Nettles et al., 2008)
Since RNA-dependent RNA polymerase is responsible for synthesis of both negative-strand RNA intermediate template and positive-strand genomic RNA, it has also emerged as a sound therapeutic target. Several HCV polymerase inhibitors are in early stages of development. These antiviral drugs can either be nucleoside or non-nucleoside inhibitors. Nucleoside inhibitors are analogs of their natural substrates. The polymerase attempts to incorporate the analog into the growing RNA chain, resulting in chain termination and binding of the enzyme's active site. It is thought that resistance will be uncommon with these agents because they incapacitate the active site (Manns et al., 2007). Non-nucleoside inhibitors achieve the same effect by binding directly to the enzyme, causing conformational changes in the polymerase. Drug resistance is more common with non-nucleoside inhibitors because they bind areas distant to the active site. A compensatory mutation that enables drug to escape may not impair the activity of the enzyme.