NS5A Review - Small molecule inhibitors of the hepatitis C virus-encoded NS5A protein
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Virus Research Dec 2012
Received 14 August 2012
Received in revised form
13 September 2012
Accepted 13 September 2012
Available online 23 September 2012
Oscar Belda, Paul Targett-Adams*
Medivir AB, PO Box 1086, Lunastigen 7, Huddinge, SE-141 22, Sweden
"NS5A-targeting molecules are probably the most potent antiviral molecules ever discovered"
"At least 4 NS5A-targeting molecules are being evaluated in combination with pegIFN/RBV: BMS-790052, GS-5885, ABT-267, and GSK23368005 (Table 1 and Table 2)."
"BMS-790052 serves as the prototype molecule for this class of HCV inhibitor; defined by exquisite low picomolar EC50 values for HCV genotype 1 (Table 1.). These molecules are over a 1000-fold more potent than the current licensed HCV protease inhibitors in cell culture-based HCV replicon assays ( Flint et al., 2009, Gao et al., 2010 and Lin et al., 2006); they are probably the most potent antiviral molecules ever discovered."
"Both Achillion's and Merck's NS5A inhibitors (ACH-3102 and MK-8742, respectively) retain substantial levels of in vitro potency against NS5A resistance polymorphisms that plague early NS5A inhibitors, such as BMS-790052 and GS-5885 ( Liu et al., 2012 and Yang et al., 2012). Accordingly, they can be viewed as 'second generation' NS5A inhibitors and time will tell whether these favorable preclinical profiles will also translate to superior clinical efficacy."
"NS5A inhibitors are remarkable molecules but they have an Achilles heel. Specifically, current NS5A inhibitors are susceptible to certain drug-resistant virus variants, particularly in patients infected with HCV genotype 1a.......(from jules: full suppression by an adequately potent multi-drug regimen can prevent resistance).....typically substitution at NS5A residues 28, 30, 31, and 93 for genotype 1a and residues 31 and 93 for genotype 1b, thus confirming the utility of the replicon system to predict in vivo virologic behavior
all-oral combination regimens composed of DAA components representing multiple mechanistic classes will have the capability to combat HCV drug resistance and the true benefit of NS5A inhibitors will likely be as components of tailored HCV DAA drug cocktails. Here, they can utilize their exquisite potency to rapidly drive down virus replication within the protection provided by the presence of other HCV DAAs, so that viral resistance to any one DAA component can be suppressed by others present in the same combination. The future is looking promising for the millions of people currently afflicted with HCV; hepatitis C is a disease that we can ultimately cure, and NS5A inhibitors could play a pivotal role."
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BMS052, ABT267 & GS5885 are in the final stages of development. GSK8005 is being studied in an initial combination study with ALS2200, the Vertex nucleotide, and Vertex is also studying ALS2200 in combination with protease TMC435. MK8742 is in a combination study now with the Merck 2nd generation protease MK5172. IDX719 is starting a study in combination with protease TMC435+TMC647055 (non nuc polymerase inhibitor). ACH3102 is in a planned study IFN-free in combination with Achillion's 1st gen protease sovaprevir.
AASLD: High Rate of Sustained Virologic Response With the All-Oral Combination of Daclatasvir (NS5A Inhibitor) Plus Sofosbuvir (Nucleotide NS5B Inhibitor), With or Without Ribavirin, in Treatment-Naive Patients Chronically Infected With HCV GT 1, 2, or 3- (11/13/12) with 12 or 24 weeks 2-drug therapy 100% cure rates
AASLD: An Interferon-Free, Ribavirin-Free 12-Week Regimen of Daclatasvir (DCV), Asunaprevir (ASV), and BMS-791325 Yielded SVR4 of 94% in Treatment-Naïve Patients with Genotype (GT) 1 Chronic Hepatitis C Virus (HCV) Infection - (11/13/12)
AASLD: A 12-week Interferon-free Treatment Regimen With ABT-450/r, ABT 267, ABT-333, and Ribavirin Achieves SVR12 Rates (Observed Data) of 99% in Treatment-naïve Patients and 93% in Prior Null Responders With HCV Genotype 1 Infection- (11/13/12)
AASLD: Once Daily Sofosbuvir (GS-7977) Regimens in HCV Genotype 1-3: The ELECTRON Trial - (11/14/12)
AASLD: GILEAD ANNOUNCES 100 PERCENT SUSTAINED VIROLOGIC RESPONSE RATE (SVR4) FOR AN INTERFERON-FREE REGIMEN OF SOFOSBUVIR (GS-7977), GS-5885 AND RIBAVIRIN IN TREATMENT-NAïVE GENOTYPE 1 HEPATITIS C INFECTED PATIENTS - (11/14/12)
BMS-790052 is a First-in-class Potent Hepatitis C Virus (HCV) NS5A Inhibitor for Patients with Chronic HCV Infection: Results from a Proof-of-concept Study
A phase 1, randomized, placebo-controlled, 3-day, dose-ranging study of GS-5885, an NS5A inhibitor, in patients with genotype 1 hepatitis C
Safety and Antiviral Activity of ABT-267, a Novel NS5A Inhibitor, During 3-Day Monotherapy: First Study in HCV Genotype-1 (GT1)-Infected Treatment-Naïve Subjects
In Vitro Resistance Analysis of Merck's HCV NS5a Inhibitor MK-8742 Demonstrates Increased Potency AgainstClinical Resistance Variants and Improved Resistance Profile
GSK2336805 HCV NS5A Inhibitor Demonstrates Potent Antiviral Activity in Chronic Hepatitis C (CHC) Genotype 1 Infection: Results from a First Time in Human (FTIH) Single and Repeat Dose Study
In vitro profiling of GSK2336805, A Potent and Selective Inhibitor of HCV NS5A
PRECLINICAL CHARACTERISTICS OF ACH-3102: A NOVEL HCV NS5A INHIBITOR WITH IMPROVED POTENCY AGAINST GENOTYPE-1A VIRUS AND VARIANTS RESISTANT TO 1ST GENERATION NS5A INHIBITORS
IDX719, HCV NS5A Inhibitor, Demonstrates Pan-Genotypic Activity after Three Days of Monotherapy in Genotype 1, 2, 3 or 4 HCV-Infected Subjects
PPI-668, A Potent New Pan-Genotypic HCV NS5A Inhibitor: Phase 1 Efficacy and Safety
Hepatitis C virus (HCV) is a modern-day pandemic; 2-3% of the world's population are thought to be infected with the virus and are subsequently at risk of developing end-stage liver diseases. The traditional standard of care (SOC) for HCV-infected patients has been limited to a regimen of pegylated-interferon alpha (pegIFN) and ribavirin; displaying low cure rates in a majority of patients and severe side effects. However, in 2011 the first direct-acting antivirals (DAA) were licensed to treat HCV-infected patients in combination with SOC, which served to elevate treatment response rates. The HCV drug development pipeline is currently populated with many additional and improved DAAs; primarily molecules that target the virus-encoded protease or polymerase enzymes. These molecules are being evaluated both in combination with the traditional SOC and together with other DAAs as all-oral pegIFN-free regimens with the ultimate goal of developing multiple DAA-containing HCV therapies that do not rely on an pegIFN backbone. A recent addition to the arsenal of HCV inhibitors in development is represented by an entirely new DAA class; molecules that target the HCV-encoded non-enzymatic NS5A protein. NS5A is essential for HCV propagation and, although its actual functions are largely unknown, it is likely a key regulator of viral genome replication and virion assembly. The protein is exquisitely sensitive to small molecule-mediated inhibition; NS5A-targeting molecules are probably the most potent antiviral molecules ever discovered and exhibit a number of other attractive drug-like properties, including activity against many HCV genotypes/subtypes and once-daily dosing potential. Although their mechanism of action is unclear, NS5A-targeting molecules are already proving their utility in clinical evaluation; particularly as components of pegIFN-sparring DAA combination regimens. This review will aim to amalgamate our current understanding and knowledge of NS5A-targeting molecules; their discovery, properties, applications, and insight into their future impact as components of all-oral pegIFN-free DAA combination therapies to combat HCV infection.
⇒ NS5A inhibitors target the hepatitis C virus-encoded NS5A protein. ⇒ The molecules exhibit remarkable capacity to inhibit HCV RNA replication. ⇒ Their mechanism of action is a subject of intense research focus. ⇒ NS5A inhibitors demonstrate impressive clinical efficacy. ⇒ They are destined to become components of interferon-free combination therapies.
Hepatitis C virus (HCV) is a global health burden; approximately 130-170 million people are thought to be chronically infected worldwide and HCV has superseded HIV as the leading cause of mortality due to an infectious agent in the US (Lavanchy, 2009, Ly et al., 2012 and TGBH, 2004). The virus is transmitted parentally by contaminated blood and the vast majority of individuals will develop a life-long chronic infection, which predisposes patients to a range of life-threatening clinical manifestations including cirrhosis and hepatocellular carcinoma (Alberti et al., 1999). HCV-associated liver diseases often manifest following decades of undiagnosed infection during which virus-infected individuals may unknowingly transmit the virus to others. The burden of HCV-associated disease is predicted to rise for the next 20 years and there is no HCV vaccine available (Deuffic-Burban et al., 2007). Prior to 2011, the standard of care (SOC) for HCV-infected patients was a combination of injected pegylated-interferon alpha (pegIFN) and oral ribavirin (RBV) administered for 48 weeks and sometimes beyond (Ghany et al., 2009 and Tan et al., 2002). This regimen is capable of achieving sustained virological response (SVR) rates above 80% for some non-genotype 1 infections (Ghany et al., 2009). However, the effectiveness of pegIFN/RBV is dramatically reduced for patients infected with genotype 1, which accounts for approximately 60% of global infections, and only 40-50% of these patients typically achieve SVR (Ghany et al., 2009 and Magiorkinis et al., 2009). Moreover, pegIFN/RBV treatment is associated with serious, and sometimes life-threatening, side effects (Ghany et al., 2009). Intensive research efforts driven by dissatisfaction with the traditional SOC combined with a growing knowledge of HCV molecular biology have culminated in the discovery, development, and availability of the first direct-acting antiviral molecules (DAAs) to combat HCV infection. Specifically, small molecule inhibitors of the HCV-encoded protease NS3 were granted FDA approval in 2011 for use in combination with SOC for patients infected with HCV genotype 1 (Ghany et al., 2011). In Phase III trials, these drugs, termed telaprevir and boceprevir (marketed as Incivek/Incivo and Victrelis, respectively) achieved SVR rates of up to 75% in treatment naïve HCV genotype 1-infected patients (Jacobson et al., 2011 and Manns et al., 2012). However, these DAA-containing regimens still exhibit several major limitations: (i) they contribute additional side effects to pegIFN/RBV therapy; (ii) they are associated with a high pill-burden and strict dosing regimen; (iii) their use is contraindicated with several other medicines; (iv) they are less effective when administered to patients with HCV-related liver diseases or patients who have previously responded poorly to pegIFN/RBV; (v) although treatment duration can be reduced to 24 weeks many patients still require medication for up to 48 weeks; (vi) the virus develops resistance against the DAAs (Pearlman, 2012); (vii) they are limited to genotype 1 infections (Ferenci and Reddy, 2011). Advances in rational-based drug design and the availability of systems to screen large compound libraries for inhibitors of HCV replication in tissue culture have facilitated an explosion in the identification of new DAAs, and the HCV pipeline is now populated with over 50 DAAs in clinical development (Pawlotsky, 2012). However, optimism must be guarded; attrition rates are high during drug development and up to 60% of anti-infectives fail in Phase II evaluations (Kola and Landis, 2004). Therefore, continued development of additional treatments for HCV must remain a priority particularly given the universal desire to develop pegIFN-sparing regimens and the widely held belief that, to limit emergence of drug-resistant viral variants, effective therapeutic strategies will consist of DAA combinations containing multiple drug classes (Rong et al., 2010).
1.1. HCV molecular virology
An extensive summary of the full molecular characteristics of HCV is beyond the scope of this review and several excellent texts already exist (Bartenschlager et al., 2010, Bartenschlager et al., 2011 and Moradpour et al., 2007). Therefore, this section will serve more as an introduction to HCV RNA replication. HCV is classified within the Hepacivirus genus of the Flaviviridae family, and is subdivided into seven genotypes ( Nakano et al., 2012, Simmonds et al., 1993 and Simmonds et al., 2005). HCV has a positive-sense, single-stranded RNA genome of approximately 9.6 kilobases, flanked by untranslated regions containing elements that regulate HCV RNA translation and replication, and encoding a polyprotein of about 3000 amino acids ( Moradpour et al., 2007 and Penin et al., 2004). The N-terminal segment of the polyprotein encodes the structural components of the virion, which consist of core protein and two glycoproteins termed E1 and E2. Core forms the capsid shell that houses the virus genome while the glycoproteins are located to a lipid envelope surrounding the capsid. A viroporin called p7 (Griffin et al., 2003) is located immediately downstream from E2 but it is unclear whether this small protein is also a virion component. The C-terminal portion of the polyprotein contains non-structural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) required for synthesis of viral RNA ( Appel et al., 2006, Blight et al., 2000 and Lohmann et al., 1999).
1.1.1. HCV RNA replication
Following HCV entry into hepatocytes the viral genome is released into the cytoplasm and is translated to the polyprotein by the host cell. Mature HCV proteins are generated following co- and post-translational proteolytic cleavage of the polyprotein by the virus-encoded NS2 and NS3 proteases and cellular enzymes. HCV RNA replication is believed to occur in membrane-bound vesicles, probably derived from the endoplasmic reticulum (ER) (Egger et al., 2002, Gosert et al., 2003, Quinkert et al., 2005 and Stone et al., 2007), and membrane-targeting sequences have been identified in each of the NS proteins with the exception of NS3, which localizes to the ER membrane through interaction with NS4A (Brass et al., 2002, Hugle et al., 2001, Moradpour et al., 2004 and Wolk et al., 2000). It is thought that the NS proteins, viral genomes and specific host-encoded factors interact to form multi-protein assemblies termed 'replication complexes' (RCs) that accumulate on rearranged ER membranes and are believed to be the locations of viral RNA synthesis catalyzed by NS5B; the virus-encoded RNA-dependent RNA polymerase (Gosert et al., 2003, Hamamoto et al., 2005, Okamoto et al., 2006 and Wang et al., 2005). Evidence suggests that oligomerization of NS4B may be responsible for rearranging ER membranes to generate intracellular docking sites for viral RNA replication, termed the 'membranous web' (Egger et al., 2002, Hugle et al., 2001, Lundin et al., 2003 and Yu et al., 2006). Both NS proteins and HCV RNA have been observed in close association with this structure (Egger et al., 2002 and Gosert et al., 2003). In HCV-infected cells, RCs are juxtaposed to intracellular lipid storage organelles termed lipid droplets (LDs), which are coated with the HCV core protein, and probably serve as platforms to accept replicated genomes from RCs to initiate capsid assembly and envelopment at the LD/ER interface (Jones and McLauchlan, 2010, Miyanari et al., 2007 and Targett-Adams et al., 2008).
1.1.2. The HCV-encoded NS5A protein
NS5A is a zinc-binding phosphoprotein essential for HCV RNA replication (Blight et al., 2000, Lohmann et al., 1999, Lohmann et al., 2001 and Tellinghuisen et al., 2008b). The protein is also required for virion morphogenesis (Appel et al., 2008, Hughes et al., 2009, Miyanari et al., 2007 and Tellinghuisen et al., 2008a), although its precise roles in the HCV life cycle are undefined. NS5A does not appear to display any enzymatic activity but the protein does exhibit a number of biological properties that probably enable it to perform a variety of roles within the virus replicative cycle. These include the capacity to exist as differentially phosphorylated species (Evans et al., 2004, Huang et al., 2007, Lindenbach et al., 2007 and Tellinghuisen et al., 2008a), the ability to bind HCV RNA (Foster et al., 2010, Huang et al., 2005 and Hwang et al., 2010), the facility to interact with other HCV-encoded proteins (Dimitrova et al., 2003, Masaki et al., 2008 and Shimakami et al., 2004), and the propensity to bind certain host factors (Hamamoto et al., 2005, Okamoto et al., 2006, Waller et al., 2010 and Wang et al., 2005). NS5A is peripherally anchored to ER membranes by an amino-terminal amphipathic helix (Brass et al., 2002 and Elazar et al., 2003), and structurally, the protein is divided into three domains. Domains II and III appear unfolded when examined in isolation (Hanoulle et al., 2009, Hanoulle et al., 2010 and Liang et al., 2007), whereas domain I of NS5A has been crystallized in alternative dimeric structures (Love et al., 2009 and Tellinghuisen et al., 2005) (Fig. 1). Thus, NS5A may exhibit a degree of conformational flexibility facilitating involvement at multiple stages within the HCV life cycle. Despite our current lack of knowledge concerning the full details of the HCV life cycle, it is likely NS5A functions as a key modulator of this process.
2. Discovery and properties of NS5A-targeting DAAs
2.1. Chemical genetics
Identification of chemical inhibitors of the HCV-encoded enzymatic gene products can be accomplished by screening compound libraries against the purified proteins of interest whose enzymatic activity can be recapitulated and assayed in vitro. Accordingly, inhibitors of a defined mechanism-of-action (MOA) are revealed and medicinal chemistry is used to define a structure-activity relationship (SAR) to build drug-like qualities into lead molecules, e.g. increase potency, minimize off-target effects, and refine pharmacokinetic properties. Discovery of novel HCV DAAs that target virus RNA replication beyond NS3 and NS5B inhibitors requires a different approach; termed chemical genetics (Bell, 2010). This involves a mechanistically unbiased screen of tissue culture cells that support HCV replication using large libraries of small organic molecules. Compounds that abrogate HCV replication are selected and the corresponding protein targets are subsequently identified. However, in practice, this is not straightforward since such strategies can uncover vast numbers of compounds with anti-HCV activity whose molecular targets can be difficult to identify. Nevertheless, a notable success was reported in 2010 by Bristol-Myers Squibb (BMS) who employed a chemical genetics strategy to ultimately discover BMS-790052; an extraordinary HCV inhibitor that targets the virus-encoded NS5A protein ( Gao et al., 2010, Lemm et al., 2010 and Lemm et al., 2011).
2.2. Discovery of BMS-790052
Following a high-throughput screen of over 1 million compounds, a team from BMS discovered a novel series of molecules that specifically inhibited HCV RNA replication (Gao et al., 2010 and Lemm et al., 2010). The screening protocol simultaneously measured inhibition of HCV replication, selectivity relative to a related virus (bovine viral diarrhea virus), and cellular toxicity in a 96-well format (O'Boyle et al., 2005). The HCV replication assay utilized human liver cells (Huh-7) transfected with an HCV 'replicon'; an autonomously replicating HCV subgenomic RNA molecule encoding HCV NS3 through NS5B (Lohmann et al., 1999). This assay permitted the simultaneous evaluation of 3 key parameters required for the identification of antiviral molecules worthy of follow-up studies; specificity, selectivity, and cytotoxicity. Hit compounds were subjected to a second screen to discard inhibitors of unrelated viruses and of conventional HCV targets, such as NS3 and NS5B (Gao et al., 2010 and Lemm et al., 2010). This campaign culminated in the identification of a novel class of HCV RNA replication inhibitors containing a thiazolidinone core, exemplified by BMS-824 (Lemm et al., 2010 and Romine et al., 2007) (Fig. 2). This compound exhibited EC50 values of ~5 nM and >10 μM for HCV genotype 1b and 1a replicons, respectively (Lemm et al., 2010).
Compound activity was specific to HCV and also yielded a broad therapeutic window; CC50 values were >50 μM (Lemm et al., 2010). BMS researchers subsequently discovered that BMS-824 underwent transformation to other inhibitory species after incubation in cell culture medium (Lemm et al., 2011 and Romine et al., 2011). In fact, mass spectral and nuclear magnetic resonance data determined the active compound species was actually a dimer of BMS-824 derived from an intermolecular radical-mediated reaction of the parent compound. Based upon an analysis of the structural elements of the dimer required for anti-HCV activity, the stilbene derivative BMS-665 was synthesized (Lemm et al., 2010) (Fig. 2). While the potency of BMS-665 was similar to that of BMS-824 for HCV genotype 1b, the molecule now possessed activity against genotype 1a with an EC50 of 393 nM (Lemm et al., 2010). Further refinement of the stilbene dimer yielded BMS-346 (Fig. 2), which improved activity against the HCV genotype 1b replicon 70-fold and shifted potency into the picomolar range with an impressive EC50 of 86 pM (Lemm et al., 2011 and Romine et al., 2011).
Identification of the stable, active pharmacophore associated with these compounds provided the foundation for the design of more potent inhibitors culminating in the identification of the symmetrical homodimeric biphenyl-based molecule BMS-790052 (Gao et al., 2010) (Fig. 2). BMS-790052 (later renamed daclatasvir) exhibited unprecedented levels of antiviral activity with astonishing mean EC50 values of 9 pM and 50 pM against HCV genotype 1b and 1a replicons, respectively (Gao et al., 2010). Importantly, BMS-790052 displayed a therapeutic index (CC50/EC50) of at least 100,000 in vitro and inactivity against a panel of 10 RNA and DNA viruses confirmed specificity for HCV (Gao et al., 2010). Subsequently, other drug discovery companies have developed their own versions of BMS-790052 (Table 1). Although the molecular structures of these other compounds are not known for the majority, patent literature suggests they are often BMS-790052-like. Thus, BMS-790052 serves as the prototype molecule for this class of HCV inhibitor; defined by exquisite low picomolar EC50 values for HCV genotype 1 (Table 1.). These molecules are over a 1000-fold more potent than the current licensed HCV protease inhibitors in cell culture-based HCV replicon assays ( Flint et al., 2009, Gao et al., 2010 and Lin et al., 2006); they are probably the most potent antiviral molecules ever discovered.
2.3. Linking compound MOA to NS5A inhibition
It is widely believed that BMS-790052-like compounds target the HCV-encoded NS5A protein (Gao et al., 2010, Lemm et al., 2010 and Targett-Adams et al., 2011). However, assigning NS5A as the molecular target of a small molecule is not straightforward since no direct screening assays for definitive NS5A functions exist, and no specific binding of compound to purified NS5A in vitro has been reported. Therefore, attributing NS5A as the target of specific small molecules requires a weight of indirect evidence rather than a direct indication. The biological properties of this class of molecule, detailed in this section, provide evidence to support NS5A as the molecular target for BMS-790052-like inhibitors and helps formulate a hypothesis regarding their possible MOA.
2.3.1. Resistance mutations map to the N-terminal domain I of NS5A
A relatively straightforward procedure to help determine whether an anti-HCV compound of unknown MOA directly targets a virus-encoded protein is to expose HCV replicon-containing cells to sub-maximal doses of the molecule for extended periods of time to force selection of adapted replicons capable of efficient growth in the presence of the inhibitor. Replicon RNA is subsequently isolated and the nucleotide sequence is determined to identify whether loss of compound activity correlates to specific HCV genetic loci. Using this strategy, mutations have been identified in the N-terminal domain I of NS5A, which confer varying degrees of resistance to the antiviral activity of BMS-790052-like molecules (Fridell et al., 2010, Gao et al., 2010, Lemm et al., 2010, Lemm et al., 2011 and Targett-Adams et al., 2011). For HCV genotype 1b, these resistance polymorphisms typically encode amino acid changes at NS5A residues 31 (from L to F/V) or 93 (from Y to H/N) (Fridell et al., 2010 and Targett-Adams et al., 2011). For genotype 1a, changes at NS5A residues 28 (from M to T), 30 (from Q to E/H/R), 31 (from L to M/V), and 93 (from Y to C/H/N) are primarily involved (Fridell et al., 2010). Thus, these findings provide genetic evidence to link the antiviral activity of molecules such as BMS-790052 to the N-terminal domain I of NS5A. The level of resistance conferred by these various amino acid substitutions varies considerably depending on the particular amino acid residue and the nature of the substitution. The level of protection such resistance substitutions provide genotype 1a is also considerably greater than for the corresponding mutations in genotype 1b (Fridell et al., 2010). For example, the Y93N mutation in genotype 1b results in an EC50 increase of 28-fold for BMS-790052, but for genotype 1a the corresponding EC50 increase is greater than 47,000-fold (Fridell et al., 2010). HCV replicons encoding multiple resistance substitutions in NS5A are further protected; a combination of L31V + Y93H in genotype 1b results in an EC50 increase of 8336-fold for BMS-790052, but for genotype 1a the corresponding EC50 increase is greater than 166,000-fold (Fridell et al., 2010). The liability of BMS-790052 for resistance mutations in NS5A of genotype 1a has profound clinical implications for all-oral DAA combinations containing this molecule (Lok et al., 2012b) (discussed in Section 3).
The prevailing theory regarding the MOA of NS5A-targeting molecules is that they perturb domain I-associated functions of NS5A (Lemm et al., 2010 and Targett-Adams et al., 2011). Domain I of NS5A is conserved amongst HCV genotypes and contains the amphipathic membrane-anchoring helix (Brass et al., 2002), a zinc-binding motif (Tellinghuisen et al., 2004), and exhibits affinity for HCV RNA (Foster et al., 2010, Huang et al., 2005 and Hwang et al., 2010). Exactly how NS5A-targeting molecules inhibit domain I-associated functions remain unknown but they do not appear to affect the capacity of NS5A to bind HCV RNA in vitro (Targett-Adams et al., 2011). Substitution at NS5A residue Y93 in domain I can be regarded a class-defining resistance polymorphism for NS5A-targeting molecules since it is a frequently observed resistance mutation ( Fridell et al., 2010, Lawitz et al., 2012b, Lin et al., 2012 and Targett-Adams et al., 2011). The crystal structures of NS5A domain I from genotype 1b indicate a dimeric molecule with each domain I monomer contributing a Y93 residue to opposing surfaces of the dimer interface (Fig. 1) ( Love et al., 2009 and Tellinghuisen et al., 2005). The propensity of resistance substitution at Y93 suggests subtle modifications to the nature of the monomer contacts are capable of decreasing sensitivity of the protein to NS5A-targeting compounds. However, inhibition of NS5A dimerization per se is not thought to be a mechanism through which NS5A inhibitors operate ( Lee et al., 2011 and Lim et al., 2012). Although it is believed the domain I dimer interface constitutes the compound interaction site on the NS5A protein, especially since the dimeric nature of NS5A-targeting molecules complements the crystal structures of NS5A, this has not been confirmed experimentally.
2.3.2. Indirect association of biotin-tagged tool molecules with NS5A
Experimental determination of a specific interaction between an agonist/antagonist and a target moiety provides strong evidence to link compound to a MOA. Conclusive demonstration of an HCV inhibitor directly bound to NS5A protein in vitro has not been reported; likely due to difficulty recapitulating the exact intracellular conformation of NS5A required for compound recognition. However, to reinforce the hypothesis that BMS-790052-like molecules do operate through a NS5A-related MOA, a biotin-tagged derivative of BMS-346 (Fig. 2) has been shown to associate with NS5A-containing protein complexes isolated from HCV replicon-containing cells by affinity capture ( Gao et al., 2010 and Targett-Adams et al., 2011). Stereoisomers that exhibited reduced anti-HCV activity were less capable of isolating NS5A-containing protein complexes under identical conditions, indicating specificity of the interactions ( Gao et al., 2010 and Targett-Adams et al., 2011). Notably, preincubation of replicon-containing cells with biotin-tagged compounds was an absolute requirement for affinity isolation of NS5A-containing complexes; no binding to compound was detected using replicon cell lysates or purified NS5A protein (Targett-Adams et al., 2011). Furthermore, minimal binding was detected upon intracellular expression of NS5A only or following expression of a NS3-NS5B polyprotein (Targett-Adams et al., 2011). Therefore, interaction of compound with NS5A presumably requires the presence of functional HCV RNA replication machinery and current evidence suggests NS5A-targeting molecules inhibit the formation of new RCs and that preformed RCs are refractory to the inhibitory effects of this class of DAA ( Lee et al., 2011, Qiu et al., 2011 and Targett-Adams et al., 2011). Consistent with this theory, HCV RNA synthesis within purified extracellular RCs is refractory to the inhibitory properties of NS5A-targeting molecules (Lee et al., 2011). Why NS5A protein molecules might not be perpetually accessible to inhibitors is unknown. Conceivably, an inhibitor binding site on NS5A may be revealed discontinuously due to conformational flexibility associated with NS5A as the protein performs particular roles during HCV genome synthesis; perhaps modulated by specific protein-protein interactions. Accordingly, it may only become visible to inhibitors at specific stages during NS5A involvement in HCV RNA replication, e.g. during polyprotein processing and/or formation of RCs.
2.3.3. Redistribution of intracellular NS5A in response to inhibition
In HCV replicon-containing cells, NS5A is sequestered in RCs derived from, and colocalized with, the ER membrane (Gosert et al., 2003 and Targett-Adams et al., 2008). However, in the presence of NS5A inhibitors, subcellular localization of NS5A is altered (Lee et al., 2011, Qiu et al., 2011 and Targett-Adams et al., 2011). Specifically, in replicon-containing cells exposed to NS5A-targeting molecules, NS5A is redistributed from the ER to the surface of LDs (Targett-Adams et al., 2011). Redistribution of NS5A is concomitant with the onset of replication inhibition and is a specific phenotypic response of NS5A protein to NS5A-targeting small molecules (Targett-Adams et al., 2011). Moreover, NS5A inhibitor molecules colocalize with redistributed NS5A protein and a NS5A species encoding a Y93H substitution is refractory to compound-mediated redistribution (Jones et al., 2011 and Targett-Adams et al., 2011). Altered intracellular distribution of NS5A protein in the absence of HCV RNA replication has also been observed following exposure of cells to BMS-790052 (Lee et al., 2011). Redistribution of NS5A protein from ER to LDs in replicon-containing cells is likely not directly responsible for the antiviral properties of NS5A-targeting molecules since NS5A is localized at LDs in virus-infected cells and has an intrinsic capacity to associate with LDs in the absence of other virus-encoded proteins (Miyanari et al., 2007, Shi et al., 2002 and Targett-Adams et al., 2008). The altered localization of NS5A in response to inhibition is probably a phenotypic marker for the MOA of NS5A-targeting molecules in replicon-containing cells and serves to reinforce the link between compound MOA and NS5A. Accordingly, when a compound interaction site becomes accessible on the surface of NS5A in replicon-containing cells, binding of inhibitor may lock or alter the conformation of the protein rendering it incapable of RC incorporation. Inhibited NS5A, no longer sequestered to RCs on the ER, adopts a pattern of localization reminiscent of that when expressed in the absence of other HCV proteins, i.e. the protein is redistributed to the LD surface.
2.3.4. Inhibition by concerted allostery
A unique property of NS5A-targeting molecules is their exquisite potency; single figure picomolar EC50 values for cell-based HCV RNA replication assays are unprecedented for anti-HCV small molecule inhibitors. The approximate molarity of NS proteins within a replicon cell has been estimated to be 0.6-600 nM (Targett-Adams et al., 2011). This is consistent for HCV DAAs such as protease and polymerase inhibitors, which are expected to inhibit their target protein with 1:1 stoichiometry and usually exhibit low nanomolar EC50 values in HCV replicon assays. However, NS5A-targeting compounds are approximately 1000-fold more potent. Therefore, a 'potency paradox' exists in which few molecules of a NS5A-targeting inhibitor can abrogate the function of many more molecules of NS5A protein (Gao, 2010 and Targett-Adams et al., 2011). Consistently with this hypothesis, it has been calculated that only 6 molecules of BMS-790052 per HCV replicon-containing cell are required to elicit an antiviral effect (Gao, 2010). Extrapolated further, this indicates that 1 molecule of BMS-790052 can inhibit the function of 9863 molecules of NS5A protein (Gao, 2010). Thus, NS5A-targeting molecules may exert their inhibitory effects in a dominant-negative manner, e.g. a single molecule of inhibitor could effect a structural distortion of a single NS5A moiety, which is communicated through concerted allostery to many other NS5A molecules connected together in an oligomeric complex (Monod et al., 1965). Alternatively, it is possible that NS5A-targeting molecules can inhibit multiple NS5A-dependent stages in HCV RNA replication that interact synergistically such that inhibitory effects are amplified (Fridell et al., 2011a and Qiu et al., 2011), or NS5A inhibitors may only target NS5A molecules that are actively involved in RNA replication and this number may be lower than anticipated. In a different scenario, the function of large NS5A oligomers may be regulated by a host factor present in limited numbers; compound-mediated inhibition of the interaction formed between NS5A oligomers and the host factor could also explain the apparent dominant-negative MOA exhibited by NS5A-targeting molecules.
2.3.5. Inhibition of NS5A hyperphosphorylation
NS5A is a highly phosphorylated protein and, in cell culture, exists as basally phosphorylated and hyperphosphorylated species (Kaneko et al., 1994). Basal phosphorylation is thought to occur at residues in the central and C-terminal parts of the protein, while several highly conserved serine residues in the central part of the protein are needed for NS5A hyperphosphorylation (Appel et al., 2005 and Tanji et al., 1995). Hyperphosphorylation of NS5A has been proposed to serve as a regulatory mechanism; modulating distinct NS5A functions in the HCV life cycle (Appel et al., 2005, Evans et al., 2004, Huang et al., 2007 and Tellinghuisen et al., 2008a). Curiously, BMS-790052-like molecules have been shown to inhibit hyperphosphorylation of NS5A (Fridell et al., 2011a, Lemm et al., 2010 and Qiu et al., 2011). However, targeting the HCV NS3 protease or certain human kinases can also reduce hyperphosphorylation of NS5A (Lemm et al., 2010 and Quintavalle et al., 2006). Thus, constraining NS5A hyperphosphorylation is not a property specific to the NS5A inhibitor class. Although BMS-790052 can inhibit hyperphosphorylation of NS5A encoded by genotype 1b replicons (Fridell et al., 2011a), reducing the intrinsic capacity for NS5A hyperphosphorylation has been shown to increase levels of replicon RNA synthesis (Lohmann et al., 2003 and Neddermann et al., 2004). Therefore, the link between the antiviral effects of NS5A-targeting molecules and their capacity to inhibit NS5A hyperphosphorylation is not understood. Rather than a causative mechanism underpinning antiviral activity, it is possible that deregulation of NS5A hyperphosphorylation serves as a phenotypic consequence following pharmacological inhibition of NS5A functions (Fridell et al., 2011a and Qiu et al., 2011).
2.3.6. The MOA of NS5A inhibitors
Based upon our current understanding of NS5A function and NS5A inhibitors, the following model can be proposed (Fig. 3). During the HCV life cycle, possibly during polyprotein processing and/or de novo RC formation, a compound-interaction site becomes accessible on the surface of NS5A protein and an inhibitor binds to domain I of NS5A. Binding may result in conformational alteration such that NS5A can no longer be incorporated into RCs, e.g. through abrogation of specific protein-protein interactions involving NS5A or disruption of specific contacts formed with the ER membrane. This structural distortion could be communicated to many other molecules in an oligomeric complex via concerted allostery such that entire NS5A complexes may be rendered non-functional. NS5A then exhibits a variety of phenotypic markers related to inhibition such as an altered cellular distribution and deregulated hyperphosphorylation, which are a consequence of its terminated ability to participate in HCV RNA replication. Specific deregulation of NS5A domain I-related mechanisms that serve to regulate the precise micro-environment required for RC formation and function is, therefore, a possible MOA for this class of HCV inhibitor. Ambiguity concerning the exact MOA of NS5A-targeting molecules has resulted in BMS-790052 often referred to as a 'NS5A replication complex inhibitor' rather than a more definite 'NS5A inhibitor'. Increased understanding of the role of NS5A in the HCV life cycle and the availability of further molecular tools, such as new inhibitors and assays to probe defined NS5A functions, will help further refine our hypotheses regarding the exact MOA for this class of inhibitor.
3. Clinical utility of NS5A-targeting DAAs
NS5A inhibitors exhibit a number of preclinical properties that make them excellent candidates for progression into clinical development. A hallmark feature of this class of inhibitor is retention of picomolar activity against all HCV genotypes; for example, BMS-790052 exhibits replicon EC50 values between 9 pM (for genotype 1b) and 146 pM (for genotype 3a) (Gao et al., 2010). Pan-genotype activity is an attractive quality for an HCV DAA due to the worldwide distribution of HCV genotypes and the potential for patients to be co-infected with more than 1 genotype/subtype (Asselah et al., 2003, Giannini et al., 1999, Matsubara et al., 1996, Schijman et al., 2004 and Toyoda et al., 1998). Importantly, this broad spectrum anti-HCV activity does not incur any penalty in terms of cellular toxicity for a range of cell types (Gao et al., 2010 and Lemm et al., 2010). Since NS5A-targeting molecules represent a novel class of HCV DAA, they can theoretically be partnered with any other DAA mechanistic class to create tailored DAA combination regimens. Preclinical data demonstrating NS5A inhibitors exhibit additive to synergistic antiviral effects when combined with pegIFN and/or RBV, NS3 protease, or NS5B polymerase inhibitors in replicon-containing cells support this notion (Fridell et al., 2010, Gao et al., 2010 and Graham et al., 2011). Further lending credence to the suitability of NS5A inhibitors as components of future DAA cocktails are the observations that other HCV DAAs retain their antiviral activity in BMS-790052-resistant replicon-containing cell lines (Fridell et al., 2010). Thus, NS5A-associated resistance polymorphisms do not confer cross-resistance to other HCV DAAs; a crucial property for components of combination regimens. Together with once-daily dosing potential, NS5A inhibitors have tremendous clinical potential and examples of NS5A-targeting molecules are now found in all stages of clinical development (Table 2).
3.1. Proof-of-concept studies validate NS5A as a therapeutic target
In 2010, a landmark proof-of-concept study was published by BMS, which validated NS5A as a viable therapeutic target for antiviral strategies designed to combat HCV (Gao et al., 2010). This Phase I monotherapy trial evaluated single doses of BMS-790052 in genotype 1-infected HCV patients and just 1 mg of the drug was sufficient to produce a mean 1.8 log10 IU/ml reduction in HCV viral load measured 24 h following administration of the molecule (Gao et al., 2010). Single doses of 10 mg and 100 mg produced greater antiviral effects; at 24 h post-dose, mean plasma HCV RNA levels fell by 3.2 log10 IU/ml and 3.3 log10 IU/ml, respectively (Gao et al., 2010). Furthermore, the 100 mg dose resulted in a remarkable sustained antiviral response for an additional 120 h in two subjects (Gao et al., 2010). The pharmacokinetic properties of BMS-790052 indicated once-daily dosing potential since the compound had a mean plasma elimination half-life of 10-14 h and, after 24 h following single oral doses of 10-100 mg, all subjects had plasma concentrations of the molecule above the 10-fold protein binding-adjusted EC90 for HCV genotypes 1a and 1b (Gao et al., 2010). Importantly, BMS-790052 was safe and well tolerated up to doses of 200 mg and no clinically relevant adverse effects were observed (Gao et al., 2010). These data were later confirmed in a 14-day multiple-ascending dose study using BMS-790052 monotherapy, which demonstrated a 2.8-4.1 log10 IU/ml mean maximum decline in HCV RNA levels from baseline before most patients experienced viral rebound on or before day 7 of treatment (Nettles et al., 2011). This study also revealed preliminary indications that the antiviral efficacy of BMS-790052 was greater in genotype 1b-infected patients compared to genotype 1a-infected patients (Nettles et al., 2011); a finding that was to produce significant results in later trials (Lok et al., 2012b). Further validation of NS5A as a therapeutic target has emerged from subsequent Phase I evaluations of additional NS5A-targeting molecules: Gilead's GS-5885 (Lawitz et al., 2012b), Abbott's ABT-267 (Lawitz et al., 2012a), and Achillion's ACH-2928 (Vince et al., 2012). In each case, the respective drug was administered once-daily for three days to subjects infected with HCV genotype 1. These molecules all produced rapid reductions in HCV RNA levels in blood plasma; median maximal HCV RNA reductions during the dosing period ranged from 3.1 to 3.68 log10 IU/ml (Lawitz et al., 2012a, Lawitz et al., 2012b and Vince et al., 2012). These data indicated that inhibitors of the HCV NS5A protein held considerable promise as direct-acting anti-HCV agents that warranted continued clinical development.
3.2. NS5A inhibitors beyond phase I
HCV DAAs will not likely be used as monotherapy to treat HCV because they rapidly select for resistant virus variants. In fact, NS5A resistance polymorphisms are readily detected in HCV genomes after only 1-3 days monotherapy treatment of HCV genotype 1-infected patients with NS5A-targeting compounds (Gao et al., 2010, Lawitz et al., 2012a and Lawitz et al., 2012b). Beyond proof-of-concept Phase I studies, two avenues of clinical development are possible for NS5A inhibitors. Firstly, following the precedent set by the HCV protease inhibitors Incivek and Victrelis, NS5A inhibitors could be evaluated as an add-on therapy to the traditional SOC for HCV. The advantage of this approach is that the regulatory pathway is clearly defined and the journey to approval and licensing could be relatively straightforward, particularly following completion of successful non-inferiority comparisons. The second approach involves assessing the efficacy of NS5A inhibitors as components of all-oral pegIFN-sparing regimens. The latter strategy serves to cement a future for NS5A-targeting molecules since it is widely anticipated that, in the near future, pegIFN-free regimens will predominate as first-line therapies to treat the majority of HCV-infected patients. In practice, both approaches for the clinical development of NS5A inhibitors are being actively pursued.
3.2.1. NS5A inhibitors with pegIFN/RBV
At least 4 NS5A-targeting molecules are being evaluated in combination with pegIFN/RBV: BMS-790052, GS-5885, ABT-267, and GSK23368005 (Table 1 and Table 2). A Phase II trial of BMS-790052 administered with pegIFN/RBV to treatment-naïve HCV genotype 1-infected patients for 48 weeks achieved SVR24 (sustained virological response 24 weeks after treatment cessation) in 83% of patients compared to 25% (for n = 12) given pegIFN/RBV alone (Pol et al., 2012). Despite the small patients numbers involved, these data compared favorably to SVR24 rates of 68-75% evident following treatment of treatment-naïve patients with pegIFN/RBV plus the HCV protease inhibitors Incivek or Victrelis ( Jacobson et al., 2011 and Poordad et al., 2011), and indicated that BMS-790052 also significantly enhanced the efficacy of pegIFN/RBV (Pol et al., 2012). Preliminary data has also suggested BMS-790052 plus pegIFN/RBV is capable of achieving higher rates of virologic suppression than pegIFN/RBV alone in difficult-to-treat HCV-infected patient populations, e.g. previous pegIFN/RBV partial and non-responders (Ratziu et al., 2012). An interim analysis of the COMMAND-2 study revealed HCV RNA was undetectable following 12 weeks of BMS-790052 plus pegIFN/RBV in 34.1% of previous non-responders (for n = 132) and 56.7% of prior partial responders (for n = 67) compared to 0% (for n = 17) for the control group (pegIFN/RBV alone given to prior partial responders) (Ratziu et al., 2012). Furthermore, when BMS-790052 was administered to HCV genotype 1-infected prior non-responders in combination with pegIFN/RBV and the HCV DAA asunaprevir (BMS-650032; an HCV NS3 protease inhibitor) (Pasquinelli et al., 2012), SVR24 of 90% (for n = 10) was achieved (Lok et al., 2012b). An interim analysis of an additional and expanded study using a similar patient population treated with the same quad regimen revealed consistent findings with over 90% SVR4 (for n = 41) (Lok et al., 2012a). Taken together, these findings highlight two important properties of BMS-790052; firstly, the capacity of the molecule to enhance efficacy of pegIFN/RBV when given to both treatment naïve and previous non/partial responders, and secondly, the potential of the drug to be combined with another HCV DAA. Abbott have also recently disclosed an equally impressive interim analysis of a Phase II trial examining the antiviral efficacy of their NS5A inhibitor, ABT-267, when administered with pegIFN/RBV to HCV genotype 1-infected treatment-naïve patients (Sullivan et al., 2012). Following 12 weeks of combination therapy, 86% of patients (for n = 28) had HCV RNA levels below the limit of detection compared to 22% (for n = 9) of patients that received only pegIFN/RBV (Sullivan et al., 2012). Time will dictate whether this promising early efficacy data will translate to a sustained antiviral response since these patients, having completed 12 weeks of combination therapy, will now be treated for a further 36 weeks with pegIFN/RBV alone (Sullivan et al., 2012).
3.2.2. NS5A inhibitors as components of all-oral pegIFN-free regimens
The availability of all-oral pegIFN-sparing therapies for the majority of HCV-infected patients is likely to become a reality within the next decade. Development of these regimens is driven by the desire to treat patients for a shorter duration with more efficacious and less toxic therapies, and the very promising data emerging from clinical trials evaluating all-oral DAA combination therapies. Furthermore, an estimated 30-60% of patients infected with HCV cannot be treated with current therapies because of medical contraindications to pegIFN/RBV; highlighting the high clinical unmet need for pegIFN-free regimens (Falck-Ytter et al., 2002 and Kanwal et al., 2007). A universal all-oral DAA combination suitable for all HCV infections in every patient population has yet to be identified and, in the short- to medium-term future, multiple regimens tailored to different HCV-infected patient populations are likely to exist. NS5A inhibitors are destined to be components of at least a proportion of these anticipated therapies and are currently being assessed in pegIFN-free combinations with other promising HCV DAAs; NS3 protease inhibitors, nucleoside/tide-based inhibitors of the NS5B polymerase, and non-nucleoside/tide-based inhibitors of the NS5B polymerase (Table 2). In many cases, RBV remains a component of pegIFN-free DAA combinations under evaluation since it has been shown to provide additive antiviral activity in this context (Zeuzem et al., 2012). Hailed as a 'watershed' moment in the treatment of hepatitis C, administration of a BMS-790052/asunaprevir combination for 24-weeks to HCV genotype 1-infected prior pegIFN/RBV non-responders cured 4 subjects providing proof-of-concept for the potential of DAA-only regimens (Lok et al., 2012b). In this study of a small cohort of 11 patients, 36% achieved SVR24; 2 of 9 infected with HCV genotype 1a, and 2 of 2 infected with genotype 1b (Lok et al., 2012b). A separate Phase II study of the same pegIFN-free BMS-790052/asunaprevir combination in HCV genotype 1b-infected prior non-responders built upon this success, and all 9 patients that completed the therapy achieved SVR24 (Chayama et al., 2012). In a larger Phase II trial, this same DAA combination also produced impressive results when used to treat HCV genotype 1b-infected non-responders or those ineligible/intolerant to pegIFN/RBV (Suzuki et al., 2012a). The headline-grabbing statistic here was that 77% (for n = 43) of these difficult-to-treat patients achieved SVR24 following 24 weeks of treatment (Suzuki et al., 2012a). This equated to SVR24 figures of 91% for non-responders (for n = 21) and 64% for the pegIFN/RBV ineligible/intolerant (for n = 22) (Suzuki et al., 2012a). Although the therapy was not associated with a totally clean safety profile in this case (5 serious adverse events were reported) (Suzuki et al., 2012a), 77% SVR24 using a pegIFN/RBV-sparing therapy in these notoriously hard-to-treat patients remains impressive. Therefore, it is not surprising that this DAA combination is now in Phase III development for HCV genotype 1b-infected patients (Table 2).
BMS-790052 is also proving its utility in a pegIFN-free combination composed of GS-7977, Gilead's HCV NS5B nucleotide polymerase inhibitor, with or without RBV in treatment-naïve patients infected with HCV genotypes 1, 2, or 3 (Sulkowski et al., 2012a). An interim analysis of this trial revealed 100% SVR4 rates following 24 weeks of treatment for genotype 1 both with (for n = 14) and without RBV (for n = 15), 100% for genotype 2/3 plus RBV (for n = 14), and 86% for genotype 2/3 minus RBV (for n = 14) (Sulkowski et al., 2012a). This combination is particularly promising since GS-7977 plus RBV is not as effective as first hoped for the treatment of HCV genotype 1-infected prior non-responders (Gane et al., 2012), indicating the drug will need to be combined with at least one other DAA to maximize its effectiveness in difficult-to-treat patient populations. Accordingly, Gilead has recently announced the successful co-formulation of GS-5885 (their own NS5A inhibitor) with GS-7977 in a fixed-dose single-pill for evaluation in HCV genotype 1-infected patients with an anticipated Phase III study to commence late 2012 (Bischofberger, 2012). GS-5885 is also currently under evaluation in a number of other trials in combination with Gilead proprietary HCV DAAs (Table 2), and an interim analysis of a Phase II trial evaluating a quad regimen composed of RBV plus GS-5885, GS-9451 (HCV NS3 protease inhibitor), and GS-9190 (HCV NS5B non-nucleotide polymerase inhibitor) in HCV genotype 1-infected treatment-naïve patients was recently disclosed (Sulkowski et al., 2012b). Total SVR12 following 12 weeks of treatment (in the higher GS-5885 dosing arm) was a respectable 81% (for n = 21), which equated to 77% (for n = 13) and 89% (for n = 8) for genotypes 1a and 1b, respectively (Sulkowski et al., 2012b). After increasing the treatment duration to 24 weeks, total SVR4 was 100% (for n = 21) and, of the subjects analyzed in this cohort thus far, SVR12 was also 100% (for n = 4) (Sulkowski et al., 2012b). These findings taken as a whole reveal pegIFN-free DAA combination regimens containing NS5A inhibitors are capable of achieving high SVR rates in HCV-infected patients with reduced treatment duration and no pegIFN-related toxicity, even in difficult-to-treat patient populations. NS5A inhibitors are quickly becoming particularly useful additions to the HCV antiviral toolbox.
3.3. Development of viral resistance to NS5A inhibitors
NS5A inhibitors are remarkable molecules but they have an Achilles heel. Specifically, current NS5A inhibitors are susceptible to certain drug-resistant virus variants, particularly in patients infected with HCV genotype 1a, which leads to viral breakthrough during treatment (Lawitz et al., 2012b, Lok et al., 2012b, Nettles et al., 2011 and Sullivan et al., 2012). The liability of NS5A-targeting molecules for resistance polymorphisms in HCV genotype 1a is predicted from replicon studies; resistance mutations resulting from single nucleotide substitutions in the NS5A gene of genotype 1a confer greater protection from the antiviral effects of the inhibitor compared to those of genotype 1b (Fridell et al., 2010, Fridell et al., 2011b and Gao et al., 2010). The nature of NS5A resistance polymorphisms identified in replicon studies are the same as those observed in clinical studies, typically substitution at NS5A residues 28, 30, 31, and 93 for genotype 1a and residues 31 and 93 for genotype 1b, thus confirming the utility of the replicon system to predict in vivo virologic behavior ( Fridell et al., 2010, Fridell et al., 2011b, Lawitz et al., 2012a, Lawitz et al., 2012b, Lok et al., 2012b and Sullivan et al., 2012). The impact of HCV genotype 1a resistance was dramatically exemplified in a Phase II clinical trial described by Lok et al., 2012a and Lok et al., 2012b. This study included a cohort of 11 subjects (9 infected with HCV genotype 1a and 2 with genotype 1b) treated for 24 weeks with BMS-790052 and asunaprevir (Lok et al., 2012b). Six of the genotype 1a-infected patients exhibited viral breakthrough on treatment, evident from weeks 3 to 12, and 1 patient relapsed post-treatment (Lok et al., 2012b). Resistance polymorphisms were detected in both HCV NS3 and NS5A genes; for NS5A, Q30R, L31M/V, and Y93C/N substitutions were detected (Lok et al., 2012b). The Y93N substitution in the NS5A protein of genotype 1a is particularly problematic for BMS-790052, and renders the genotype 1a replicon 47,000-fold less susceptible to the antiviral properties of the inhibitor (Fridell et al., 2010). This means that in vivo drug concentrations may not be sufficient to fully inhibit replication of such mutants facilitating selection over wild-type (WT) virus following prolonged drug exposure (Fridell et al., 2011b). Although, the issue of NS5A drug-resistance is less pronounced for genotype 1b, it remains evident ( Fridell et al., 2011b, Lawitz et al., 2012b and Nettles et al., 2011).
HCV drug resistance is problematic because the virus exhibits astounding levels of genetic diversity in chronically infected individuals; driven by a high replicative ability of the virus in the liver (up to a trillion HCV particles can be produced in a chronically infected individual per day) compounded by an error-prone nature of genome synthesis (Guedj et al., 2010). As a result, it is predicted that every possible single and double nucleotide mutations in the virus genome are generated multiple times on a daily basis (Guedj et al., 2010 and Rong et al., 2010). Since replicon species harboring NS5A resistance polymorphisms are not always substantially impaired in terms of replicative fitness compared to their WT counterparts (Fridell et al., 2010 and Gao et al., 2010), HCV genomes encoding resistance-associated polymorphisms in NS5A are readily detected in virus-infected patients at baseline, i.e. pre-treatment (Fridell et al., 2011b, Lawitz et al., 2012b, Nettles et al., 2011, Plaza et al., 2012, Pol et al., 2012 and Suzuki et al., 2012b). This effectively gives the virus a head-start to establishing populations of drug-resistant quasi-species before the patient has even been exposed to an inhibitor of NS5A. Further complicating the problem of resistance, recent data have revealed long-term persistence of NS5A resistance polymorphisms following 3-day monotherapy treatment of patients with GS-5885 (Wong et al., 2012). NS5A resistance polymorphisms, present at baseline or selected during GS-5885 treatment, persisted in 100% and 50% of HCV genotype 1a- and 1b-infected patients respectively 48 weeks following treatment cessation (Wong et al., 2012). The primary NS5A resistance substitution observed in genotype 1b-infected patients (Y93H) decreased over time indicating a reduced replicative capacity in vivo and suggesting this mutant would eventually be replaced by WT virus after a suitable period of time off therapy (Wong et al., 2012). However, L31M, the primary NS5A resistance substitution detected in genotype 1a-infected subjects, did not decrease over time and was present in 90% of patients 48 weeks following the end of therapy (Wong et al., 2012). This finding may relate to the observation that genotype 1a replicons encoding L31M replicate more efficiently than genotype 1b replicons encoding Y93H compared to their respective WT parents (Fridell et al., 2010); suggesting less selective pressure for L31M to be replaced with WT virus in the absence of drug. Therefore, reduction of resistant 1b, but not 1a variants, in long term follow-up may also contribute to the greater propensity for inhibitors of NS5A to control genotype 1b resistance. Nevertheless, a defining feature of current NS5A inhibitors is their low genetic barrier to resistance; single nucleotide polymorphisms in the NS5A gene, often present at baseline, can encode single amino acid substitutions in the NS5A protein that dramatically reduce the sensitivity of HCV to the antiviral effects of NS5A-targeting molecules without incurring large costs to replicative fitness. To mitigate the challenge of resistance, NS5A inhibitors must be combined with other HCV DAAs for maximum therapeutic impact.
4. Future perspectives
How might NS5A inhibitors shape future treatment options for patients infected with HCV? Expedited development strategies suggest that all-oral HCV DAA combinations composed BMS-790052/asunaprevir (Table 2) and GS-5885/GS-7977 (Bischofberger, 2012) could be available to treat a variety of HCV-infected patient populations as early as 2014. RBV may still be required to augment the antiviral efficacy of such combinations; Phase III trial designs for these combinations include RBV ± arms and the outcomes of these evaluations are eagerly anticipated. As components of HCV DAA combinations, NS5A inhibitors will continue contributing towards a welcome drive ultimately leading to the departure of pegIFN-containing therapies.
The current HCV-infected population is aging and the number of patients seeking treatment will increasingly be those who have had the virus for longer periods of time and, thus, will be afflicted with HCV-associated liver diseases such as fibrosis and cirrhosis. Small Phase II trials designed to evaluate efficacy of therapeutic regimens in otherwise 'healthy' HCV-infected patients, i.e. no HIV/HBV coinfection, low/no fibrosis, no cirrhosis, etc. do not represent the majority of patients who will need treating in the future. Treating HCV-infected patients with compensated cirrhosis is challenging since they are often unable to receive pegIFN-based treatment due to contraindications or intolerance. Cirrhotic patients are also particularly susceptible to treatment-related adverse effects. The favorable safety profiles of NS5A inhibitors may indicate that DAA combinations containing these molecules could be particularly suited for HCV-infected patients with liver diseases resulting from HCV infection. Traditionally, HCV-infected patients with cirrhosis have been excluded from clinical trials but this is now beginning to change. For example, Boehringer Ingelheim's SOUND-C2 trial of BI201335 (NS3 protease inhibitor) and BI207127 (NS5B non-nucleoside inhibitor) ± RBV (Soriano et al., 2012), and BMS' trial of BMS-790052 and the NS3 protease inhibitor TMC435 ± RBV (trial numbers NCT01132313 and NCT01628692, respectively) both include cirrhotic patients. Hopefully, we will soon see more studies addressing the medical needs of this important patient population. Also, when evaluating data from clinical trials that exclude cirrhotic patients, we must be mindful that this growing patient population will inevitably fare less well than patients without HCV-associated liver diseases.
Although currently elusive, co-crystallization of NS5A with bound inhibitor, particularly for genotype 1a, could represent a very useful tool to help explain the different susceptibilities of resistance mutations to NS5A-targeting inhibitors in genotypes 1a and 1b. Such information could also be used to apply rationally designed medicinal chemistry strategies to create modified molecules to reduce the impact of resistance polymorphisms. However, despite this lack of knowledge, discovery of new NS5A inhibitors have now been reported that demonstrate impressive preclinical virological profiles (Liu et al., 2012 and Yang et al., 2012). Both Achillion's and Merck's NS5A inhibitors (ACH-3102 and MK-8742, respectively) retain substantial levels of in vitro potency against NS5A resistance polymorphisms that plague early NS5A inhibitors, such as BMS-790052 and GS-5885 ( Liu et al., 2012 and Yang et al., 2012). Accordingly, they can be viewed as 'second generation' NS5A inhibitors and time will tell whether these favorable preclinical profiles will also translate to superior clinical efficacy.
5. Closing remarks
The discovery of NS5A inhibitors and clinical validation of NS5A as a therapeutic target signals a new and exciting era for HCV drug development. Continued research will aim to elucidate the precise MOA of NS5A inhibitors to further our understanding of NS5A functions and facilitate discovery of new molecules with improved properties such as a higher barrier to resistance. Virus resistance plagues HCV drug development and is certainly not specific to only NS5A inhibitors. However, all-oral combination regimens composed of DAA components representing multiple mechanistic classes will have the capability to combat HCV drug resistance and the true benefit of NS5A inhibitors will likely be as components of tailored HCV DAA drug cocktails. Here, they can utilize their exquisite potency to rapidly drive down virus replication within the protection provided by the presence of other HCV DAAs, so that viral resistance to any one DAA component can be suppressed by others present in the same combination. The future is looking promising for the millions of people currently afflicted with HCV; hepatitis C is a disease that we can ultimately cure, and NS5A inhibitors could play a pivotal role.