Discovery and Development of Hepatitis C Virus NS5A Replication

Perspective pubs.acs.org/jmc

Discovery and Development of Hepatitis C Virus NS5A Replication Complex Inhibitors Makonen Belema,*,† Omar D. Lopez,† John A. Bender,† Jeffrey L. Romine,† Denis R. St. Laurent,† David R. Langley,§ Julie A. Lemm,‡ Donald R. O’Boyle, II,‡ Jin-Hua Sun,‡ Chunfu Wang,‡ Robert A. Fridell,‡ and Nicholas A. Meanwell*,† †

Department of Discovery Chemistry, ‡Department of Virology Discovery, and §Department of Computer-Assisted Drug Design, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States ABSTRACT: Lead inhibitors that target the function of the hepatitis C virus (HCV) nonstructural 5A (NS5A) protein have been identified by phenotypic screening campaigns using HCV subgenomic replicons. The demonstration of antiviral activity in HCV-infected subjects by the HCV NS5A replication complex inhibitor (RCI) daclatasvir (1) spawned considerable interest in this mechanistic approach. In this Perspective, we summarize the medicinal chemistry studies that led to the discovery of 1 and other chemotypes for which resistance maps to the NS5A protein and provide synopses of the profiles of many of the compounds currently in clinical trials. We also summarize what is currently known about the NS5A protein and the studies using NS5A RCIs and labeled analogues that are helping to illuminate aspects of both protein function and inhibitor interaction. We conclude with a synopsis of the results of notable clinical trials with HCV NS5A RCIs.

■

INTRODUCTION The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein plays a critical role in the virus replication cycle, facilitating both replication of the genomic RNA and assembly of the virion as well as modulating host cell factors in order to create an environment hospitable to the virus.1−7 HCV NS5A has no known enzymatic activity, and despite considerable effort, a coherent understanding of the precise function of this remarkably enigmatic protein in virus replication and host cell modulation remains elusive. However, the potential of NS5A as a target for drug discovery was brought acutely into focus with the disclosure of the clinical efficacy of daclatasvir (1).8,9 This NS5A replication complex inhibitor (RCI) demonstrated a rapid and profound reduction in viremia following the administration of single doses of the drug to genotype 1 (GT-1) HCV-infected subjects naive to therapy. The potency and efficacy of 1 stimulated considerable interest in the design and development of NS5A RCIs while also providing useful tools with which to further explore and understand the role of the protein in virus replication.6,10−14 As a consequence, NS5A inhibitors have emerged as a clinically relevant class of HCV therapeutic agents with the potential to be important contributors to direct-acting antiviral (DAA) combination therapies that offer considerable promise to replace the current interferon-dependent drug regimens.15−25

viral protein.26−33 The first reports of inhibitors of HCV replication for which resistance was associated with NS5A claimed a class of iminothiazolidinone derivatives typified by compound 2a.9,34−37 This molecule exhibited an EC50 in a GT1b replicon cell culture assay of 0.57 μM and a CC50 of >50 μM, providing a therapeutic index of over 80. Resistance to 2a was mapped to a Tyr to His substitution at residue 93 (Y93H) of the amino terminus of the NS5A protein, a resistance mutation that has emerged as a hallmark of many NS5A RCIs.9,36,37 The assay that was implemented to identify 2a was dual format in nature, combining a GT-1b HCV subgenomic replicon and a bovine viral diarrhea virus (BVDV) replicon in the same well with both replicating in a human hepatocytederived Huh-7 cell line background.38 A FRET-based NS3/4A protease assay for HCV and a luciferase readout for BVDV provided orthogonal reporters for each virus, while cell viability was assessed by staining the cells with Alamar Blue.38 Compounds were screened at 10 μM, and a key aspect to the design of this assay was that it allowed for the rapid triaging

■

DISCOVERY OF DACLATASVIR The discovery of HCV NS5A RCIs relied upon phenotypic screening using subgenomic GT-1b replicons to interrogate compound collections and identify lead structures targeting this © 2014 American Chemical Society

Special Issue: HCV Therapies Received: November 19, 2013 Published: February 3, 2014 1643

dx.doi.org/10.1021/jm401793m | J. Med. Chem. 2014, 57, 1643−1672

Journal of Medicinal Chemistry

Perspective

that the antiviral activity may be mediated by a degradation product. A bioassay-guided analysis of 2b following incubation in the replicon assay medium isolated two dimeric species 5 (Figure 1) which exhibited potent inhibitory activity in the replicon with EC50 values of 43 and 50 μM), respiratory syncytial virus (RSV), human immunodeficiency virus 1 (HIV-1), or human rhinovirus (HRV) in cell culture at concentrations that were not cytotoxic to the host cell.9,37

Preliminary structure−activity relationships (SARs) associated with 2a revealed that replacing the alanine moiety with proline, as in 3, resulted in an incremental gain in antiviral potency whereas the D-Ala analogue of 2a was over 100-fold less potent, confirming a preference for the natural stereoconfiguration of the amino acid that was discovered with the screening hit.39 Notably, a marked enhancement in inhibitory potency was achieved with the phenylacetamide 2b which exhibited an EC50 of 6 nM in the GT-1b replicon.39 However, as this chemotype was probed further, it became apparent that the benzylic C-5 methine of the iminothiazolidinone of 2b was susceptible to oxidation.39 This was manifested as an oxidative rearrangement that occurred in DMSO solution to give the thiohydantoin 4, a compound devoid of GT-1b HCV inhibitory activity (EC50 >20 μM) (Figure 1).40 Furthermore, it was observed that 2b was unstable when incubated in replicon media under typical assay conditions. However, quite remarkably, the inhibitory activity remained largely unaffected, even when the compound was preincubated in assay medium until essentially undetectable prior to biological evaluation.40 The apparent disconnect between the presence of parental compound and replicon inhibitory activity led to the hypothesis

Figure 1. Proposed chemical reactivity of iminothiazolidinone 2b in the presence of oxygen. 1644



Perspective

the benzylic center of these arylglycine-derived caps was associated with a more potent antiviral effect than the (S)isomer. Moreover, for analogues of 10a a correlation was established between cap basicity and inhibitory potency toward the GT-1a replicon, which suggested that molecules that are more readily protonated at physiological pH may interact with the GT-1a protein through a hydrogen-bonding interaction. This hypothesis envisaged the protonated amine acting as a hydrogen-bond donor to the protein, a pharmacophoric element recapitulated by the NH of the carbamate analogue 10b. The subgenotype inhibitory activities of both 10a and 10b are biased toward GT-1b, with 10a inhibiting GT-1a with an EC50 of ∼8.8 nM and GT-1b with an EC50 of 50 μM).14 However, further studies with this series ultimately led to improvements in the antiviral profile of the chemotype (vide infra).61 The 3-aryl-4-alkynylpyrazole derivative 26 inhibits replication of a GT-1b replicon with an EC50 of 65-fold resistance to 26 when reverseengineered into the parental GT-1b replicon. Although the morpholine analogue 27 was less affected by the Y93H mutation, with just a 6-fold shift in the EC50, analogues 28−30 exhibited EC50 values that were 71- to 105-fold higher in the Y93H replicon than in wild type virus, suggesting that the target of these compounds is the NS5A protein.

functionalities were explored.62 The selection of replicons resistant to 26 followed by cDNA sequencing identified Chart 1

1648



Perspective

Table 2. In Vitro Antiviral Activity Reported for Select HCV NS5A RCIs That Have Entered Clinical Trialsa EC50 (nM)

a

compd

GT-1a

GT-1b

GT-2a (JFH-1)

GT-3a

GT-4a

GT-5a

GT-6a

31 32 33 34 35 PPI-668 GS-5816

0.031 0.004 0.014 0.008 0.0585 0.135 0.013

0.005 0.003 0.005 0.003 0.008 0.016 0.015

20.8 0.003 0.0124 0.024 0.054 0.139 0.009

10.1 0.02 0.0193 0.017 NA 1.256 0.013

0.007 0.003 0.00171 0.002 0.0018 0.052 0.009

NA NA 0.0043 0.037 0.0025 0.039 0.059

NA NA 0.415 NA NA NA 0.007

NA = data not available.

■

CLINICALLY EFFECTIVE HCV NS5A INHIBITORS WITH PAN-GENOTYPE INHIBITORY ACTIVITY The SARs developed for 1 suggested that the essential pharmacophore of this chemotype is a dipeptide mimetic, optimally either a Pro-Val-carbamate or a Pro-phenylglycine motif, projected at each end of a linear central scaffold. Subsequent studies showed that the dipeptide elements can be deployed in either a symmetrical fashion or an unsymmetrical combination and separated by a distance of approximately 15− 20 Å.7 An extensive range of NS5A RCIs have been synthesized that have largely been based on the design of alternative central scaffolds that present variants of the important dipeptide moieties with an appropriate topological spacing.7,10−14 As a testament to the intensity of the interest in NS5A RCIs that developed following the disclosure of the clinical effect of 1, over 150 patent applications claiming NS5A inhibitors have been published by the World Patent Office. The first of these appeared in June 2010, 2.5 years after the applications disclosing the prototypical chemotype were published.63,64 An analysis of representative compounds selected from 35 patent applications filed by 11 pharmaceutical houses that published between February 2008 and March 2011 noted the conservation of the peptide-based cap elements deployed on a variety of scaffolds based on a two-dimensional plot of Tanimoto distance scores.7 Several NS5A RCIs have risen to prominence based on demonstrated clinical efficacy in HCV-infected subjects, while additional molecules are in earlier stages of development (Chart 1). The most advanced compounds are ledipasvir (GS5885, 31),65a,b elbasvir (MK-8742, 32),66 ombitasvir (ABT-267, 33),67 samatasvir (IDX-719, 34),68 GSK2336805 (35),7,69 PPI668 (structure not disclosed), ACH-3102 (structure not disclosed), and GS-5816 (structure not disclosed).65c Notably, 36 appears to be a compound of considerable interest in the PPI-668 series, since it has been prepared on a kilogram scale and optimized formulations have been described.70 These molecules illustrate some of the interesting structural variation that broadens the palindromic pharmacophore defined by 1 and its anilide progenitor 10, with only 33 relying on a symmetrical topology. The remaining molecules embed asymmetry in the core scaffold (32) and the pyrrolidine ring (35) or hybridize imidazole and benzimidazole heterocycles (31, 34, and 36), with 31 extending the asymmetry to differentially substituted pyrrolidines. However, all of these compounds project the two methoxycarbonylvaline cap moieties found in 1 at their termini with the exception of 34, which relies upon the combination of an (S)-methoxycarbonylvaline and an (R)-phenylglycine as the cap elements. The core scaffolds in 31, 32, and 34 are extended compared to that found

in 1, and both 32 and 33 deploy an aromatic ring in the center of the core scaffold that, in the case of 32, is associated with an improved resistance profile (vide infra). Within this series of NS5A inhibitors, 33 presents each of the proline amide moieties in the context of an anilide rather than an imidazole or benzimidazole heterocycle. The antiviral activity reported for each of these compounds in HCV replicons and chimeric replicons is compiled in Table 2 and reveals that all are potent, pan-genotypic inhibitors of HCV replication. There is some variation in profile between the inhibitors that may be a function of the specific replicons and individual conditions employed, precluding a precise direct comparison. The potent in vitro antiviral activity associated with these six compounds has translated into a significant clinical effect on plasma viral RNA titers following administration of single (compound 35) or multiple doses (31−34) of drug to HCV-infected subjects (vide infra). The campaign that led to the discovery of 31 focused on identifying compounds with potent GT-1a inhibition, with the introduction of the fluorene core designed to explore the effect of conformational constraint in the context of 1. However, the fluorenyl methylene that bridges the embedded biphenyl scaffold proved to be labile toward oxidation, a problem solved by substituting this site with two fluorine atoms, a modification that also improved oral exposure compared to a biphenyl prototype. Further structural changes optimized the pyrrolidine amide group with the discovery that, for unsymmetrical compounds, modifications that retained potent antiviral activity required a careful matching of functionality. Thus, the [2,2,1]azabicycloproline mimetic found in 31 was associated with higher antiviral activity when paired with a benzimidazole rather than an imidazole heterocycle. This proline derivative offered improved PK properties eventually leading to the discovery of 31 as the most potent compound in the series.65 The identification of 32 originated in the design of a series of compounds related to the stilbene 19c that sought to take advantage of its structural relationship with 7b.66a The central olefin of 7b was incorporated into a heterocyclic ring, a motif that was subsequently combined with modifications of the proline amide substituents to provide 37 as an early lead. This compound demonstrated potent GT-1b inhibition, EC50 = 4 pM, but was much weaker toward a GT-1a replicon, EC50 = 70 nM. Optimization focused on replacing the two anilide moieties of 37 which led to the synthesis of MK-4882 (38), a compound that exhibited balanced GT-1a and GT-1b inhibitory properties, EC50 values of 9 and 3 pM, respectively.66a The PK properties of 38 revealed low clearance in the rat, dog, and rhesus monkey, with moderate oral bioavailability in the dog (26%) and rat (38%). The antiviral effect of 38 was assessed in three HCVinfected chimpanzees following oral administration of single 1 1649



Perspective

heterocycle core in which the anilides are presumably of importance to the optimal separation between the two important methoxycarbonylvaline moieties.67a Antiviral activity was sensitive to the absolute configuration at the two stereogenic centers of the pyrrolidine with the 2S,5S-isomer of a prototype compound an order of magnitude more potent than the 2R,5R-isomer in a GT-1a replicon. The more potent compounds in this series incorporated lipophilic substituents on the N-phenyl ring, and 33 emerged as a potent antiviral agent that interestingly was poorly orally bioavailable in the rat. However, the oral bioavailability and exposure of 33 were markedly better in the dog and the compound exhibited low clearance and a 4.4 h iv half-life in the monkey. The selection of replicons resistant to 33 identified M28T (8965x), Q30R (800x), Y93C (1675x), Y93H (41382x), and Y93N (66739x) in GT-1a NS5A and L28T (661x), L28M + Y93H (415x), L31F +Y93H (10270x), and L31V + Y93H (12323x) in GT-1b NS5A as the major resistance mutations.67a Inspiration for the design of 35 originated with the spirosubstituted proline 25 that was derived from a series of monomeric NS5A inhibitors.14 Evolution to the bis-imidazolebiphenyl scaffold described with 1 led to the identification of compounds with improved GT-1a inhibitory activity that included 39 (GT-1a EC50 = 1.5 nM; GT-1b EC50 = 0.007 nM).14 A further adjustment to the topology of the spiro ring and additional optimization identified 35 as a compound with potency toward wild-type replicons comparable to that of 1 but with 5- to 10-fold improved potency toward GT-1b replicons incorporating L31V and Y93H resistance mutations. The symmetrical homologue 40 was also prepared and exhibited potent replicon inhibitory activity, with EC50 values of 40 and 10 pM toward GT-1a and GT-1b replicons, respectively.69b

mg/kg doses. All three animals (two had a GT-1a infection, while the third was infected with GT-1b virus) responded to the drug with an average decline in plasma viral load of 2.91 log10 IU/mL by 48 h that subsequently rebounded to baseline. Population sequence analysis of viral RNA recovered from these chimpanzees revealed the presence of resistance mutations in the NS5A sequence in all animals. By 24 h, the GT-1b-infected animal had virus that expressed the Y93H mutation although viral suppression continued to decline until 48 h after dosing. Rebound virus from one of the GT-1ainfected animals contained Q30E and Y93H substitutions which by day 7 was a mixture of wild-type and Y93H virus. In the second GT-1a-infected chimpanzee, which harbored an R155K mutation in the NS3 gene presumably due to prior exposure to an NS3 protease inhibitor, a mixed population of L31M/L was detected at day 10 with only wild-type virus observed at day 28. In a multidose study conducted in two chimpanzees to which 38 was administered daily for 7 days, plasma viral load declined in both animals but viral breakthrough occurred in the GT-1a-infected chimpanzee while on treatment. Sequence analysis detected Q30R and L31M/L mutations in the NS5A gene at day 6 which persisted out to day 10. In the GT-1b-infected chimpanzee, viremia remained suppressed for the duration of the dosing period but sequence analysis 10 days after completing the study revealed the presence of a mixed Y93H/Y population which eventually reverted to wild-type virus. The observation of the rapid emergence of resistant virus and viral breakthrough prompted additional refinement of 38 with the objective of identifying a compound with improved virological properties, particularly toward replicons harboring resistant mutations in NS5A. The introduction of conformational constraint between C-3 of the benzofuran and the 2phenyl substituent represented a key step in this direction but required further adjustment to the topology of this new linker element. This was accomplished by replacing the benzofuran with an indole and installing the linker element via the indole N atom rather than the C-3 atom. Although a prototype compound exhibited unacceptable cytotoxicity, the introduction of a phenyl ring to the linker solved this problem and separation of the diastereomers provided 32 as a compound with 10-fold greater potency toward many of the key GT-1a and GT-1b resistant mutants when compared to 38. The PK properties of 32 in preclinical species were predictive of a low clearance compound in humans, with modeling suggesting good potential for a low daily dose.

Achillion Pharmaceuticals has advanced two NS5A inhibitors, designated ACH-2928 and ACH-3102, into clinical trials, and although the structures of these molecules have not been disclosed, the sole NS5A patent application from this organization exemplifies molecules with interesting threedimensional core structures that include the cyclophane 41 and ferrocene 42.71 ACH-2928 exhibits potent GT-1b replicon inhibition but is over 50-fold weaker toward a GT-1a replicon (Table 3). However, while this compound has demonstrated efficacy in a phase 1b study, development appears to have been halted in favor of ACH-3102, an NS5A inhibitor with more evenly balanced GT-1a and GT-1b inhibition and activity toward several viruses that exhibit resistance to 1 (Table 3).72,73 Enanta Pharmaceuticals has advanced EDP-239 into clinical trials in collaboration with Novartis Pharmaceuticals. Although the structure of this compound has not been disclosed, the alkyne derivatives 43−45 have been specifically identified as

The NS5A replication complex inhibiting pharmacophore in 33 is deployed symmetrically on a trans-substituted pyrrolidine 1650



Perspective

Table 3. In Vitro Antiviral Activity Reported for the HCV NS5A RCIs ACH-2928 and ACH-3102a EC50 (nM)

a

compd

GT-1a

GT-1b

GT-2a

GT-3a

GT-4a

GT-5a

GT-6a

ACH-2928 ACH-3102

0.161 0.026

0.0029 0.005

0.218 0.021

0.103 NA

NA NA

NA NA

0.048 NA

NA = data not available.

exhibited promising potency in a GT-1b replicon with an EC50 of 0.2 nM, a GT-2a replicon was less sensitive, EC50 = 26 nM, while a GT-1a replicon was quite unresponsive to the compound with an EC50 of >226 nM. It is noted, however, that the topology of the thiazole in 47 is different from that of 46, preventing a direct comparison.66 Nevertheless, the general applicability of the thiazole as an imidazole or amide mimetic in pan-genotype NS5A RCIs is questionable and its performance in 46 cannot be fully understood in the absence of a more complete antiviral profile that includes data on the efficacy of this compound in GT-1a and other replicons.76

compounds that interact in vitro with prominent HCV NS3 protease and NS5B polymerase inhibitors in an additive or synergistic fashion.74,75 In addition, it was noted that these compounds exhibit additive inhibition profiles when used in conjunction with a cyclosporine analogue and interferon α in HCV replicons.

HCV NS5A RCIs that incorporate silicon, an element that continues to attract attention in drug design, have appeared in the patent literature.77a,81−83 The NS5A RCI 48 incorporates a silaproline moiety and is claimed to potently inhibit GT-1a and GT-1b replicons with EC50 values of 16 and 3 pM, respectively.82 This compound inhibited chimeric GT-2a (JFH), GT-3, and GT-4a replicons with EC50 values of 27, 350, and 20 pM, respectively, while a GT-2b replicon and GT1a L31V and Y93H were less sensitive with respective EC50 values of 48, 16, and 46 nM.82 In the rat, dog, and monkey, 48 was orally bioavailable with F of 28%, 8.7%, and 11.7% and effective half-lives of 7.6, 18, and 17 h, respectively. Silicon has also been explored as the bridging element between the biphenyl scaffold, with the dibenzo[b,d]silole 49 being a successful representative of this concept that can be recognized as an isostere of the fluorene ring found in 31. In GT-1a and GT-1b replicons, 49 is a potent antiviral agent with EC90 values of 76 and 43 pM, respectively, but the compound is sensitive to the Y93H mutation in a GT-1a replicon where an EC90 of 811 nM was noted.83 Hybrid structures that combine elements of 1 with either 18 or 19a−c have been prepared that may capitalize on subtle differences in the mode of binding of the different chemotypes.84 The 1H-naphtho[1,2-d]imidazole 50 and imidazole 51 are representative examples that are claimed to exhibit EC50 values of 10 μM), was less capable of isolating NS5Acontaining complexes, indicative of the specificity of the interaction. Of particular interest, incubating 52 with replicons harboring the Y93H mutation and eluting the lysate over streptavidin beads also led to the enrichment of NS5A.102 This result indicates that the development of resistance in NS5A is not necessarily associated with a complete expulsion of an inhibitor from the replication complex but rather that the mutant protein is able to restore its function in the presence of a bound molecule. As a consequence, RCI binding to NS5A is a necessary but insufficient event for the expression of inhibitory activity in replicons.

Additional insight into the interaction of RCIs with the NS5A protein was gleaned by selecting for resistant GT-1b replicons using 56 (EC50 = 100 nM) and 57 (EC50 = 60 nM), which are derivatives of 13a. These compounds were selected based on the concept that 56 would rely more upon the bisimidazolyl-biphenyl core for drug−target interactions, while 57, part of a family of compounds that exhibit GT-1b inhibition but that are generally poorly active toward a GT-1a replicon, would be anticipated to be more dependent on the phenylglycine cap element for its activity.39,102,147 Although a priori both compounds exhibited poor activity toward a replicon harboring the Y93H mutation, EC50 >3.3 μM, 56 selected for L31V of NS5A along with Q54R and Q54L while the more cap-biased 57 selected for the Y93H mutation in a GT-1b replicon. These results suggest that the core element interacts preferentially with a region encompassing L31 and Q54 while the cap element is proximal to Y93.102 Notably, 13a could be modeled into the NS5A dimer in a fashion that reflected this topology of interaction. In this study, 13a was docked into a full length homology model of the GT-1b Con1 strain domain I dimer constructed from the X-ray structure of the 25−215 sequence and the amphipathic helix anchor domain whose structure was determined by NMR.101,102,143 The model predicts that 13a binds symmetrically across the dimer interface, with the hydrophobic biphenyl core lying over the Phe37 residues which, in turn, stack over the Pro58 residues. The hydrophobic residues Phe36, Phe35, Val34, and Gly33 flank the core of 13a, and residues Pro32 and Leu31 lay symmetrically over the core and between the phenylglycine cap moieties while the Leu28 and Phe29 residues pack against the pyrrolidine rings and phenylglycine elements, respectively. The Arg30 residues point away from the inhibitor, which allows this loop to adopt a conformation that positions the hydrophobic side chains of loop residues against 13a. The polar residues Tyr93 and Gln62 are positioned so that the imidazole NHs project into the electron rich π-cloud of Tyr93 while the side chains of Gln62 can establish a hydrogen-bond interaction with the imidazole ring N atoms. The Gln54 residues are situated near the pyrrolidine rings of 13a and define the length of the inhibitor binding site. This model places 13a in direct contact with all of the resistance mutations except H58D in the GT-1a strain, which is one amino acid layer below the hydrophobic biphenyl core (Figure 6A and Figure 6B). In this complex the inhibitor

Recent results using azides 54 (GT-1b EC50 = 7 nM) and 55 (GT-1b EC50 = 90 nM) have provided additional evidence for NS5A RCIs interacting directly with monomeric, dimeric, and possibly higher-order multimeric forms of the NS5A protein in HCV replicon cells in the presence or absence of an inhibitory effect on HCV replication.102 Photolysis with ultraviolet light of GT-1b replicon cells exposed to a tritiated version of 54 resulted in the labeling of the NS5A protein, while no labeling 1656



Perspective

observed in cell culture studies remains an important but difficult challenge. A biochemical system of this type would be invaluable for determining if NS5A RCIs disrupt essential function(s) that are dependent upon associations with host or viral proteins performing a necessary but elusive task related to the HCV replication cycle. Adding further to the mechanistic intrigue, a more recent development in the understanding of the mode of action of NS5A RCIs has been derived from modeling the clinical effects of 1 on viral load, which falls very rapidly following administration of 10 or 100 mg doses of the drug to HCVinfected subjects.156 A standard model of HCV dynamics or linear regression analysis of the clinical effect of 1 suggested that the t1/2 of HCV was ∼0.7 h, markedly shorter than earlier estimates based on interferon α therapy which had estimated the t1/2 to be ∼2.7 h. This discrepancy was resolved by analyzing viral dynamics in the context of virus replication and virion assembly or secretion. The model resulting from this detailed analysis predicted that 1 exerted a dual inhibitory effect on the virus, blocking virion production or secretion in addition to RNA replication with 99.8% and 99% effectiveness, respectively. This model afforded an estimate of HCV halflife of ∼45 min, much closer to the observed value. In vitro experiments with infectious GT-2a JFH-1 virus that compared the effects of 1 and a nucleoside-based NS5B inhibitor on RNA replication and virion production supported this hypothesis. The rate of decline of intracellular HCV RNA was similar for both compounds, consistent with these mechanistically distinct inhibitors blocking virus replication, as can be observed in replicons. However, extracellular virus titers declined at different rates, with the effect of 1 observed immediately and rapidly while the effect of the nucleoside inhibitor was slower and occurred in parallel with the inhibition of viral RNA replication. NS5A has been implicated in virion assembly with effects mapped to domain III, distant from the putative site of action of 1 in domain I.111,157,158 However, these data suggest that the effects of 1 on the function of the NS5A protein may be more complex than inhibiting replication complex activity and extend to interfering with virion assembly by an as yet undefined mechanism.

caps are proximal to the Y93H residues and the interacting elements are summarized in Figure 6A.102

Figure 6. Residues of HCV NS5A predicted to be in close contact with RCI 13a based on modeling studies and a model of 13a bound into a full length homology model of GT-1b NS5A domain I. (A) Residues predicted to be in close contact with 13a based on modeling studies.102 (B) Model of 13a (gray) bound into the full length homology model of genotype 1b Con1 strain NS5A domain I. Subdomain IA is colored purple. Subdomain IB is green. The class 1 proline motif is colored blue, and the amphipathic α-helical Nterminus is colored red. The yellow colored residues L28, L31, Q54, and Y93 are sites of resistance to some RCIs in some strains. Zinc ions are colored blue.

■

CLINICAL TRIALS WITH HCV NS5A INHIBITORS Clinical trials with HCV NS5A RCIs have had a significant impact on the field of exploratory HCV therapeutics with several molecules entering clinical development. With inherently high in vitro potency and broad genotype inhibitory properties that typically translate into good clinical efficacy, it is anticipated that compounds from this mechanistic class will be an important contributor to the combinations of DAA treatment options, particularly when used in conjunction with other pan-genotypic HCV inhibitors.16−21,159−161 The phase 1 single ascending dose (SAD) clinical trial of 1 in HCV-infected subjects provided validation of the clinical relevance of targeting HCV NS5A, revealing a profile of a rapid and profound reduction in viremia that has become characteristic of the class.9 In GT-1-infected subjects, plasma viral load declined by 1.8, 3.2, and 3.3 log10 IU/mL measured 24 h following doses of 1, 10, and 100 mg of 1, respectively (Table 5). Notably, the effect of the 100 mg dose persisted for 144 h, with one of the two subjects achieving HCV RNA levels that were below the 25 IU/ mL lower limit of quantitation while the other subject’s viral load at nadir was measured at 35 IU/mL.

While the affinity-tagged inhibitors 52−55 provide useful tools to help probe the mechanism of action of NS5A RCIs and to study the role(s) of NS5A in HCV replication, they are still limited by the need to study NS5A proteins expressed in HCV replicon cells. The development of a system in which in vitro produced NS5A proteins can duplicate the effect of RCIs 1657



Perspective

amino acid substitution.175 In the 30 mg b.i.d. cohort, the two GT-1b-infected subjects maintained control over viremia until the end of the dosing period, with viral load depressed by over 5 log10 IU/mL, while the two subjects infected with GT-1a virus experienced rebound, with Q30E detected as the major variant at day 14 in one while a consensus Q30H variant was found in the other at all time points, including at baseline prior to entry into the study. A Y93H variant was also present in this subject at a significant level of the population at all time points tested, suggesting a linkage at baseline.174 Follow-up samples from selected subjects were assessed at time points up to six months after dosing and revealed the persistent presence of the major resistance mutations.176 The presence of mutations resistant to 1 at baseline was explored more broadly in two studies.177,178 Analysis of the NS5A sequences of 78 HIV-HCV co-infected subjects presenting at a Spanish clinic and scrutiny of 635 sequences in the Los Alamos database found no pre-existing M28T, Q30H/R, L31F/M/V, P32L, or Y93C/H/N resistance mutations in GT-1a or GT-3 viruses, while a methionine at 31 is the WT residue for GT-4.177 The double L31M/Y93H mutant was found in 7% of GT-1b while 13% of GT-4 sequences also expressed Y93H and all GT-1b and GT-4 sequences expressed M28L and L30R changes, although the significance of these is unknown. In 294 GT-1b-infected Japanese patients who were naive to DAA agents, 33 (11.2%) harbored mutations with resistance to 1, a cohort comprising eight subjects expressing L31M, none with L31V, and 24 with a Y93H change, while one subject expressed the double L31M/ Y93H mutant.178 The results from these early clinical trials with 1 indicated very clearly that combination with either pegylated interferon α (Peg-IFNα) and ribavirin or DAA agents with orthogonal resistance patterns would be essential in order to completely control HCV replication for the period of dosing required to effect a cure, currently estimated at 8−12 weeks.169,179−187 In the initial step in this direction for NS5A inhibitors, 1 was evaluated in a phase 2 clinical trial in which it was administered to subjects naive to therapy at doses of 3, 10, and 60 mg q.d. in conjunction with Peg-IFNα and ribavirin for 48 weeks in cohorts of 12 patients, with placebo as the comparator.184 In this trial, the 10 and 60 mg q.d. doses of 1 were associated with an 83% sustained virological response rate at 24 weeks (SVR24) following the cessation of therapy while the 3 mg dose was inferior, affording SVR24 in 5 of 12 patients (42%) which compared with a 25% SVR24 rate in the placebo arm. The phase 2 clinical experience with 1 in combination with Peg-IFNα and ribavirin has encompassed 1110 patients with a safety profile comparable to that associated with Peg-IFNα and ribavirin.184 Phase 2b studies are focused on evaluating shorter dosing regimens in cohorts comprising GT-1/4 and GT-2/3 infected subjects, while phase 3 studies of this regimen are ongoing using a 60 mg dose of 1.186−188 A more ambitious clinical trial that provided the first evidence that HCV-infected subjects could be cured of their infection without resorting to the use of Peg-IFNα and ribavirin combined 1 with the HCV NS3 protease inhibitor asunaprevir (58).189 This study specifically recruited subjects who were GT-1 null responders, individuals who had failed Peg-IFNα and ribavirin therapy and are considered some of the more difficult patients to treat, and subjected them to 24 weeks of therapy. A cohort of 11 subjects received the dual DAA therapy (1 administered at a dose of 60 mg q.d. and 58 at a dose of 600 mg

Table 5. Mean Reductions in HCV RNA after Single Oral Doses of 1 Administered to HCV-Infected Subjects dose (mg) genotype: 1a/1b mean HCV RNA decline at 24 h (IU/mL) mean maximal HCV RNA decline (IU/mL)

1

10

100

6/0 1.8 log10

3/2 3.2 log10

2/3 3.3 log10 3.6 log10

The rapid fall in viremia following administration of 1 has been attributed to a dual effect of the drug on viral replication and virion assembly as discussed earlier.156 The initial decline in viral load is associated with an effect on virion production that is followed by a second phase attributed to the inhibitory effect of the compound on virus replication. This profile contrasts with that of NS3 protease and NS5B inhibitors for which the reduction in viral load exhibits a slower onset, reflecting a role restricted to inhibition of virus replication.162,163 In GT-1a-infected subjects, sequencing of the virus at 24 and 144 h after dosing detected M28T, Q30R/H, and L31M variants while for GT-1b-infected individuals, Y93H was prominent, observations suggesting that the potent and effective suppression of wild-type virus allows resistant viruses to thrive and ultimately dominate. This scenario is to be anticipated based on the high virus replication rate in vivo, estimated to be in excess of 1 trillion virions per day, and an error prone RNA polymerase. The RNA polymerase has been suggested to incorporate between 0.1 and 1 incorrect nucleotides per RNA synthesized, with the more conservative error rate predicting production of more than 100 billion particles per day with one mutation and over 5 billion virions with two mutations.164−169 As a consequence, monotherapeutic treatment of HCV typically leads to the rapid emergence of resistance, and this was the observed outcome when 1 was administered to subjects chronically infected with GT-1 virus at doses of 1, 10, 30, 60, and 100 mg q.d. and 30 mg b.i.d. for 14 days.170−175 Viral load fell rapidly in all cohorts, with a mean maximal effect of 2.8 log10 IU/mL in the 1 mg dosing group and 4.1 log10 IU/mL in the 30 mg b.i.d. group, but most subjects experienced breakthrough.173 Sequencing of the viruses at 1, 2, 4, 7, and 14 days after dosing identified major changes at M28T/A/V, Q30H/R/K/E, L31M/V, and Y93H/ N/C in the GT-1a-infected subjects and L31M/V and Y93H/C in those infected with GT-1b virus.174 These mutations were also observed in the corresponding replicons when passaged in the presence of escalating concentrations of 1.174 Indeed, these studies establish the in vitro replicon system as a highly effective tool with which to identify clinically relevant mutations associated with resistance to NS5A RCIs.148,149,173,174 In the 60 mg q.d. cohort, all four of which were infected with GT-1a virus, several linked double mutants that exhibited high levels of resistance to 1 in vitro were characterized in subjects experiencing breakthrough. This included a Q30H/Y93H variant observed in one patient and mixtures of Q30E and Y93N isolated from two subjects, while a fourth subject with virus breakthrough had a Q30R variant that emerged within 12 h of drug dosing despite plasma levels that were over 15-fold above the replicon EC50 of 7 nM for this mutant.174 This anomaly was resolved when it was determined that this subject had a baseline E62D polymorphism in NS5A that markedly increased the level of resistance conferred to 1 by the Q30R 1658



Perspective

of therapy with Peg-IFNα and ribavirin and 22 subjects who were intolerant to or were deemed medically ineligible for this therapy. Eighteen of these patients were naive to therapy and were considered ineligible for Peg-IFNα/ribavirin therapy because of several anticipated difficulties including advanced age, cytopenia, depression, and hypertension.196 The results from the earlier study were combined for analysis from which a total of 36 patients completed the 24-week dosing regimen of dual therapy. Two patients in the null responder group discontinued therapy and the 19 that completed the drug regimen achieved SVR24, a 90% virological response rate. In the ineligible cohort, four patients discontinued treatment but remained in the study and 14 achieved SVR24, a 64% virological response, with three patients suffering viral breakthrough while four relapsed after treatment.196−198 Although an analysis of response with respect to IL28B genotype revealed a slightly higher response at weeks 2, 3, and 4 in those with the CC genotype, the proportion of CC and CT genotypes achieving SVR24 was similar. In the seven patients who failed therapy, which occurred after all patients had achieved undetectable levels of HCV RNA for extended periods of time, variants with resistance mutations to both drugs were detected at failure, with L31M and Y93H in NS5A and D168A or D168V in NS3 observed in the viral breakthroughs and L31V/M and Y93H in NS5A and D168A/V in NS3 in the relapsers.196,197 Phase 2a clinical studies of 1 in combination with sofosbuvir (59), an NS5B-inhibiting nucleotide prodrug, and with and without ribavirin have been conducted in GT-1, GT-2, and GT3-infected individuals.188,199−201 This trial, which included GT1 patients who have failed previous therapy with Peg-IFNα and ribavirin in conjunction with the protease inhibitors telaprevir or boceprevir, demonstrated high SVR24 rates following 12 or 24 weeks of therapy. Forty-one patients who had failed PegIFNα, ribavirin, and a protease inhibitor, of which 40 had a non-CC IL28B genotype and the majority were infected with GT-1a virus, were administered 1 (60 mg q.d.) and 59 (400 mg q.d.) for 24 weeks with the result that all subjects achieved SVR4 and all but one patient achieved SVR12. The single patient who failed to achieve SVR12 did not report for viral load evaluation at the appointed time but had undetectable levels of viral RNA 24 weeks after the final drug dose. SVR12 rates of 95−100% were observed in the GT-1 cohort administered therapy for 12 weeks, while patients infected with GT-2 and GT-3 achieved SVR12 rates of 86−100%.188 A liver transplant patient with severe recurrent GT-1b HCV infection who had failed prior therapy with Peg-IFNα and ribavirin was administered a 24-week course of therapy with a combination of daily doses of 1 and 59 initiated 8 months after transplant and achieved SVR12.202 Augmentation of the combination of 1 and 58 in GT-1infected patients by the addition of a third agent, the nonnucleoside NS5B polymerase inhibitor BMS-791325 (60), has been explored in a phase 2 clinical trial with the initial results showing promise.203,204 The combination of 1 (60 mg q.d.), 58 (200 mg b.i.d.), and 60 (75 mg or 150 mg b.i.d.) was evaluated in 66 treatment-naive GT-1-infected subjects without cirrhosis, with the drug combination administered for 12 or 24 weeks. In the cohort receiving 75 mg of 60, 94% achieved SVR12 and SVR24 after 12 weeks of therapy while the figures were 94% and 88% for SVR12 and SVR24, respectively, after 24 weeks of dosing.204 In the group administered 150 mg of 60, 89% achieved SVR12 and SVR24 after 12 weeks of therapy while 94% achieved SVR12 and SVR24 after 24 weeks of dosing. In this

b.i.d.) with the comparator arm comprising a cohort of 10 patients who received the dual DAA regimen in conjunction with Peg-IFNα and ribavirin. All 10 patients receiving quadruple therapy achieved SVR12 of which one patient exhibited detectable plasma viral RNA at 24 weeks after treatment but was undetectable 35 days later. SVR48 was achieved by 90% of these subjects, with one patient who had detectable levels of virus at this point found to be free of detectable viral RNA in plasma 13 days later. The dual DAA therapy cohort consisted of two GT-1b- and nine GT-1ainfected patients from which both GT-1b- and two of the GT1a-infected subjects achieved SVR12 and SVR24 while one GT1a patient had undetectable viral RNA in plasma at the end of treatment but subsequently relapsed. The six GT-1a-infected subjects who experienced viral breakthrough on therapy, which occurred as early as week 3 in one patient, were administered Peg-IFNα and ribavirin, and although all exhibited an initial response, this antiviral effect was not durable.189 Sequencing of the viruses from the patients experiencing breakthrough identified the selection of resistance mutations in both the NS3 (R155K and D168A/E/T/V/Y) and NS5A (Q30R, L31M/V, and Y93C/N) proteins as the likely underlying cause of the failure of therapy. While the result of this trial was viewed as “a watershed moment in the treatment of hepatitis C”, it highlighted the need for a third agent to be added to this regimen if GT-1a-infected patients were to be successfully treated with a drug combination that included 1 and 58.189,190

However, the results of this pioneering trial provided confidence to study the dual combination of DAAs in HCVinfected Japanese patients who are predominantly infected with GT-1b virus (∼75% of Japanese infections are GT-1b with the remainder mostly GT-2, while GT-1a is of low prevalence).191−196 In the first of these trials, 1 (60 mg q.d.) and 58 (600 mg b.i.d. initially but subsequently reduced to 200 mg b.i.d.) were administered for 24 weeks to 10 GT-1b-infected patients who were documented as null responders to Peg-IFNα and ribavirin.195 Eight of the patients were determined to be of the IL28B CT genotype that is associated with poor response to Peg-IFNα, while the remaining two were typed as the potentially responsive CC genotype. Nine patients completed the 24-week course of therapy, and all achieved SVR12 and SVR24. The remaining patient experienced an increase in bilirubin levels to grade 4 after 2 weeks of drug dosing and discontinued therapy. At the time of discontinuation, plasma HCV RNA levels in this patient were measured at 1.8 log10 IU/ mL, well below the >5 log10 IU/mL observed at baseline; however, at follow-up visits at 2, 3, 4, 13, and 24 weeks after ending drug treatment, viral RNA was below the level of detection, data consistent with the current definition of cure.191 This trial was subsequently broadened to enroll an additional 11 patients who had a documented null response to 12 weeks 1659



Perspective

population, while one GT-1b patient expressed Y93H to the extent of 1.3%. However, these patients experienced viral load reductions of 1.9 log10 and 2.3 log10 IU/mL, respectively. The resistance mutations were only detected by deep sequencing, and the data suggested that the presence of minority populations of resistant virus can lead to a reduced response to monotherapy if the mutant exhibits a high level of resistance. In the GT-1a cohort, the most common mutant was Q30R, predominant to the extent of 89% at the 30 mg dose but observed with a lower prevalence of 75% at the 90 mg dose where the increased selective pressure led to the emergence of the more resistant Y93C and Y93H mutants.206 In a phase 2a trial examining the combination of 31 (90 mg q.d.), 59 (400 mg q.d.), and ribavirin (1000 or 1200 mg per day based on weight as a divided dose) in treatment-naive HCV-infected subjects without cirrhosis, all 25 patients achieved SVR12 following 12 weeks of therapy.207 The clinical campaign with 31 is extensive, and the compound has been well-tolerated by over 1000 patients in phase 2 clinical trials.208 An open label phase 2 clinical trial has evaluated a fixed dose combination of 31 (90 mg q.d.) and 59 (400 mg q.d.) with and without ribavirin in treatment-naive and treatment-experienced patients who were randomized into two groups based on the presence or absence of cirrhosis.207b This study evaluated the drug combinations in three cohorts without cirrhosis with the dual combination administered for 8 and 12 weeks while a third arm included ribavirin in a dosing regimen that lasted for 8 weeks. In the cirrhotic patients, the two arms evaluated the dual combination with and without ribavirin for 12 weeks. All patients in the two ribavirin-containing regimens and 95% of the patients in the three dual regimen arms achieved SVR12.207b This clinical trial represents the first successful demonstration of efficacy of DAA agents in cirrhotic patients and is the first to show high SVR rates following an 8-week treatment regimen. A phase 1 clinical trial of 35 examined single doses of 10, 30, and 60 mg administered to healthy subjects and multiple doses of 10 and 30 mg for 7 days and 75 mg for 14 days.209 The PK analysis indicated that the plasma AUC and Cmax increased in a dose-proportional fashion after single doses, with the median tmax occurring at 1.5−3 h, while the mean t1/2 ranged from 7 to 10 h, and coadministration of drug with a meal was not associated with a significant effect on exposure.209 Antiviral efficacy was established in HCV-infected subjects administered single doses of drug, with the mean HCV RNA reductions measured at 24 h postdose compiled in Table 7. Viral load decreased in a dose-dependent fashion with the largest effect observed with the two highest doses of 60 and 120 mg, which correlated with the higher plasma exposure at these doses. The majority of the patients were infected with GT-1a virus with just one GT-1b-infected patient in each of the 10 and 60 mg doses, but there were no obvious differences in the effect of genotype on antiviral response, although the study numbers in this early trial are quite small.209 Two metabolites of 35, designated M10 (61) and M11 (62), were detected at low concentrations in the plasma and urine of normal healthy volunteers administered both single and repeat

study, two patients experienced viral breakthrough and one relapsed while there were no deaths or discontinuations due to serious adverse events and no adverse events attributed to the treatment regimen.

Phase 1 and 2a clinical data have also been reported for 31 with efficacy initially established in a multiple ascending dose study conducted in GT-1-infected subjects administered doses of 1, 3, 10, 30, and 90 mg of drug q.d. for 3 days.205,206 Drug exposure in plasma exhibited close to linear increases in response to dose escalation, with Cmax occurring between 4 and 6 h after drug administration, and concentrations of 31 in plasma at all doses higher than 1 mg exceeded the protein binding-adjusted EC90 for the least sensitive GT-1a virus at 24 h. The half-life of 31 in plasma ranged from 22 to 50 h, supportive of a q.d. dosing paradigm. Viral load in the efficacy study fell by >3 log10 IU/mL in all patient groups with the exception of the 1 mg dose cohort, and although GT-1a- and GT-1b-infected subjects exhibited similar responses to the drug, suppression of viral load in GT-1b-infected subjects was more sustained (Table 6). Population sequencing at baseline for this cohort revealed that five patients harbored pre-existing mutations associated with resistance to NS5A inhibitors with four of these GT-1a infections from which two responded with 95%), with both metabolites amounting to 1 log10 IU/mL while the GT-1a group showed no statistical difference in levels of plasma HCV RNA when compared to the placebo control. At the 350 mg dose, HCV RNA declined by a maximum of 2.8 log10 IU/mL but four GT-1b-infected subjects experienced no significant effect in response to the drug as did all of the GT-1a patients in this cohort. The GT-3a cohort received 233 mg of 18 on a t.i.d. dosing schedule with no demonstrable effect on HCV RNA levels in plasma.10 Collectively, these data reflect a failure to achieve sustained plasma levels of drug above the protein binding-adjusted EC50 toward the individual genotypes (Table 12).

Table 10. Antiviral Effects of PPI-668 Administered to HCV GT-1 Infected Subjects q.d. for 3 Daysa dose (mg) mean maximum reduction in HCV RNA (IU/mL)

40

80

160

240

3.22 log10

3.54 log10

3.48 log10

3.74 log10

a

Note that these data do not include viral load measurements from patients with known resistant mutations at baseline and that were analyzed separately.

viremia. Ongoing clinical trials with PPI-668 are evaluating the compound in conjunction with the NS3 protease inhibitor faldaprevir and the non-nucleoside NS5B inhibitor deleobuvir to enhance the therapeutic effect of this dual combination which exhibited SVR12 rates of 3.5 log10 IU/mL in all cohorts and viremia did not return to baseline until day 15 after dosing (Table 11). More specifically, one subject in the 300 mg

Table 12. Cmax and Cmin for 18 after Oral Dosing of the Drug to Subjects Infected with GT-1 HCV

no.

mean maximum reduction in HCV RNA (IU/mL)

25 50 150 300 placebo

6 4 4 4 5

4.04 log10 3.78 log10 3.52 log10 3.93 log10 −0.68 log10

Cmax (day 1) (ng/mL)

Cmin (day 1) (ng/mL)

90 t.i.d. 233 t.i.d. 350 b.i.d.

97.8 276.0 373.5

10.1 45.2 56.4

■

EPILOGUE The discovery and development of HCV NS5A RCIs provide a compelling contemporary example of the value of phenotypic screening to identify mechanistically novel and clinically relevant antiviral agents.28,29 HCV NS5A is an enigmatic protein that plays critical roles in virus replication and propagation but has no demonstrable enzymatic activity or specific biochemical effect that would facilitate the design and implementation of the kind of screen that is used to interrogate more conventional drug targets. Indeed, even at this point, compounds that interfere with the function(s) of HCV NS5A can only be characterized by the use of cell-based screens. The demonstration of potent clinical efficacy with 1 and its pangenotype inhibitory properties has led to the emergence of this class of HCV inhibitor as an important component of DAA drug combinations, many of which are currently undergoing clinical evaluation. Because HCV replication involves only RNA intermediates and there is no archiving of the virus genome in the host cell chromosomes, HCV is a curable disease. The HCV NS5A RCI 1 in combination with the protease inhibitor 58 was a critical component of the two-drug regimen that established that DAA agents can cure GT-1b HCV infection in the absence of immune modulation. These two compounds form the basis of the first request for approval of an interferonand ribavirin-free drug regimen for the treatment of HCV GT1b infection with a regulatory filing in Japan that was announced on November 2, 2013. There is considerable anticipation for the availability of additional effective drug combinations for the treatment of HCV that will seek marketing approval over the coming years. For the first time in history, we stand at the threshold of being able to cure a chronic infection with therapy based on small molecule DAA agents.

Table 11. Mean Maximum Reduction in HCV RNA after Oral Administration of Single Doses of ACH-3102 to Subjects Infected with HCV GT-1 dose (mg)

dose (mg)

dose group had an L31M mutation at baseline but experienced a 3.4 log10 IU/mL decline in viral load, which contrasts with the poor efficacy seen with 31 when this mutation was present. A second subject with a Y93C mutation responded with a 4.4 log10 IU/mL decline in plasma viral RNA. In a phase 1b trial with 32 conducted with GT-1 and GT-3 HCV-infected subjects, plasma viral RNA declined rapidly following oral administration of doses ranging from 5 to 50 mg q.d. for 5 days to GT-1 patients with a mean maximal effect of 3.7−5.1 log10 IU/mL.221,222 The GT-3-infected patients received higher doses of 32 that ranged from 10 to 100 mg q.d. for 5 days, a regimen that was associated with a 3.4 log10 IU/mL mean maximal reduction in plasma viral RNA. M28V and Y93H resistance-associated mutations were detected at baseline in two GT-1-infected subjects, but both responded with a reduction in viral load of >3 log10 IU/mL.222 Postdosing population sequence analysis revealed the presence of M28A/ T/V, Q30H/R, L31F/V/I/M, and Y93C/H/N/R mutations in GT-1-infected patients at the end of treatment that persisted through the follow-up period, while A30E/K/T, L31F/I, and Y93C/H/R were found in GT-3-infected subjects. The nonsymmetrical NS5A inhibitor 18, which demonstrated a strong inhibitory profile biased very much toward GT1b inhibition (EC50 = 7 nM, compared to an EC50 toward GT1a of 1.24 μM), was evaluated in subjects infected with GT-1a, GT-1b, and GT-3a virus in a multiple ascending dose study

■

AUTHOR INFORMATION

Corresponding Authors

*M.B.: phone, 203-677-6928; e-mail, makonen.belema@bms. com. 1662



Perspective

*N.A.M.: phone, 203-677-6679; e-mail, nicholas.meanwell@ bms.com.

Professors Lawrence H. Hurley and David E. Thurston. He received his Ph.D. in Computational Chemistry in 1993 from Wesleyan University, CT, under the supervision of Professor David L. Beveridge. David’s technical skills include molecular modeling, synthetic chemistry, and molecular biophysics, and he has contributed to numerous programs in the oncology, virology, and neuroscience therapeutic areas. His primary areas of interest are molecular dynamics, homology modeling, force field development, protein design, and nanotechnology.

Notes

The authors declare no competing financial interest. Biographies Makonen Belema received a B.A. in Chemistry from Swarthmore College, PA, and a Ph.D. in Synthetic Organic Chemistry from Yale University, CT, under the mentorship of Professor F. E. Ziegler before joining Bristol-Myers Squibb in 1996. Early in his industrial career, he worked on a retinoid and an IKK project as part of a broader effort directed at the treatment of dermatological and immunological disorders, respectively. His work over the past decade has focused on the exploration of novel HCV inhibitors targeting the NS5A protein. He is a key contributor to the discovery of daclatasvir.

Julie A. Lemm obtained her Ph.D. in Molecular Genetics and Biochemistry from Washington University School of Medicine, St. Louis, MO, working in the laboratory of Professor Charles Rice where she focused on understanding the temporal regulation of alphavirus and flavivirus polyprotein processing and RNA replication. She is currently in the Department of Virology at Bristol-Myers Squibb, focusing on the discovery and development of antiviral therapeutics. Her research interests include understanding and modulating the replication of positive strand RNA viruses including such important human pathogens as hepatitis C virus and Dengue virus.

Omar D. Lopez studied Chemistry at the National University of Mar del Plata, Argentina, receiving his B.S. degree in 1991. He subsequently attended graduate school at Washington University in St. Louis, MO, where he obtained his Ph.D. in Chemistry (1998) under the supervision of Dr. Scott Gilbertson. His graduate research focused on the use of organoiron complexes in organic synthesis. In 2000, he joined Bristol-Myers Squibb after a postdoctoral appointment with Dr. Stephen Martin at the University of Texas in Austin, where he studied new routes for the synthesis of C-arylglycosides. He is currently part of the Virology Department at Bristol-Myers Squibb and has been involved in HCV-related research for the past 10 years.

Donald R. O’Boyle, II, earned his B.S. degree in Biochemistry at The Pennsylvania State University at State College, PA. Following graduation, he joined Nigel Frasers’ laboratory at the Wistar Institute in Philadelphia to study herpesvirus latency prior to joining the virology drug discovery effort at Bristol-Myers Squibb in 1987. Since then he has been involved with a number of diverse projects directed at the use of biochemical and cell-based assays to identify antiviral compounds against herpesvirus, bovine viral diarrhea virus, and hepatitis C virus. His current studies are focused on studying the inhibitory effect of combinations of NS5A inhibitors including daclatasvir on hepatitis C virus.

John A. Bender received his B.S. degree in Organic Chemistry from North Dakota State University under the supervision of Prof. Mukund Sibi and then went on to the University of Utah where he earned a Ph.D. investigating [4 + 4]-photocycloadditions and interrupted Nazarov cyclizations under Prof. Frederick West. After completing NIH postdoctoral research at the University of Texas, Austin, under the guidance of Prof. Stephen Martin, he accepted a position as a medicinal chemist at Bristol-Myers Squibb in 2000. Presently he is a Principal Scientist at Bristol-Myers Squibb where he has worked on multiple HIV and HCV antiviral projects. His primary research interests remain at the interface of synthetic organic and medicinal chemistry.

Jin-Hua Sun studied Biology at the Nanjing Normal University, Biochemistry for his M.S. degree at Chinese Academy of Science, China, and Molecular Cellular Biology for his second M.S. degree at Oregon State University. In 1993, Jin-Hua joined the lab headed by Dr. Cheng Kao in the Biology Department of Indiana University and started to work on positive-strand RNA viruses and focused on studying RNA replication complexes. In 1997, he took a position in the Virology Department at Bristol-Myers Squibb where he has continued to work positive-strand RNA viruses. He is member of the HCV team at Bristol-Myers Squibb that discovered and developed daclatasvir.

Jeffrey L. Romine obtained his Ph.D. under the direction of Professor Leo Paquette at The Ohio State University and did postdoctoral studies under Professor Albert I. Meyers at Colorado State University. His is presently a Senior Principal Scientist at Bristol-Myers Squibb where he has worked since 1989, publishing most notably on studies of Maxi-K ion channel openers for the treatment of stroke and the discovery of NS5A inhibitors for the treatment hepatitis C virus.

Chunfu Wang studied Preventive Medicine at China Medical University, China, from 1984 to 1989. He started his Ph.D. program at Kanazawa Medical University, Japan, focusing on the control of innate immunity by HCV core−RNA interactions through the PKR pathway (in vitro studies) from 1998 to 2001. He joined the Department of Virology, University of Texas Southwestern Medical Center as a postdoctoral researcher from 2001 to 2006 where his research interests were focused on host−virus interactions that regulate HCV replication and innate immunity versus HCV replication. He joined the Virology Department at Bristol-Myers Squibb in 2006. He is a member of HCV team at Bristol-Myers Squibb that discovered and developed daclatasvir, and his current research interests include the study of the resistance profile and resistance barrier for HCV NS5A replication complex inhibitors.

Denis R. St. Laurent studied Chemistry at the University of Connecticut and received his B.S. degree in 1983. He then joined the Department of Chemistry at The Ohio State University working with Prof. Leo A. Paquette and obtaining his M.S. degree in 1988 where his research involved studies directed toward the total syntheses of magellanine and paniculatine, two naturally occurring lycopodium alkaloids. Since 1988, Denis has contributed to drug discovery in several therapeutic areas at Bristol-Myers Squibb as a member of Discovery Chemistry. Since 2001, Denis’ interests have been focused on the design of drug candidates to target viral infections and he was a part of the team that discovered daclatasvir.

Robert A. Fridell received a Ph.D. in Biology from the University of North Carolina at Chapel Hill where he worked with Lillie Searles on transcriptional and splicing regulation in Drosophila melanogaster. His research as a Postdoctoral Fellow in Bryan Cullen’s laboratory of at Duke University, NC, primarily focused on interactions of host cell factors with the Tat and Rev regulatory proteins of HIV-1. Following

David R. Langley joined Bristol-Myers Squibb in 1986 and is currently Group Leader of the CADD group in Wallingford, CT. He received a B.S. degree in Chemistry in 1984 from the University of Texas at Austin with Professor Stephen F. Martin and remained there to complete a M.S. degree in Medicinal Chemistry in 1986 with 1663



Perspective

Diseases, San Francisco, CA, October 31−November 4, 2008; Abstract LB12. (9) Gao, M.; Nettles, R. E.; Belema, M.; Snyder, L. B.; Nguyen, V. N.; Fridell, R. A.; Serrano-Wu, M. H.; Langley, D. R.; Sun, J.-H.; O’Boyle, D. R., II; Lemm, J. A.; Wang, C.; Knipe, J. O.; Chien, C.; Colonno, R. J.; Grasela, D. M.; Meanwell, N. A.; Hamann, L. G. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 2010, 465, 96−100. (10) Najarro, P.; Mathews, N.; Cockerill, S. NS5A inhibitors. In Hepatitis C Viruses; Tan, S.-L., Ed.; Horizon Bioscience: Wymondham, U.K., 2006; pp 271−292. (11) Schmitz, U.; Tan, S.-L. NS5Afrom obscurity to new target for HCV therapy. Recent Pat. Anti-Infect. Drug Discovery 2008, 3, 77−92. (12) Holler, T. P.; Parkinson, T.; Pryde, D. C. Targeting the nonstructural proteins of hepatitis C virus: beyond hepatitis C virus protease and polymerase. Expert Opin. Drug Discovery 2009, 4, 293− 314. (13) Belda, O.; Targett-Adams, P. Small molecule inhibitors of the hepatitis C virus-encoded NS5A protein. Virus Res. 2012, 170, 1−14. (14) Hamatake, R.; Maynard, A.; Kazmierski, W. M. HCV inhibition mediated through the non-structural protein 5A (NS5A) replication complex. Annu. Rep. Med. Chem. 2012, 47, 331−345. (15) Gish, R. G.; Meanwell, N. A. The NS5A replication complex inhibitorsdifference makers? Clin. Liver Dis. 2011, 15, 627−639. (16) Liang, T. J.; Ghany, M. G. Current and future therapies for hepatitis C virus infection. N. Engl. J. Med. 2013, 368, 1907−1917. (17) Dabbouseh, N. M.; Jensen, D. M. Future therapies for chronic hepatitis C. Nat. Rev. Gastroenterol. Hepatol. 2013, 10, 268−276. (18) Aghemo, A.; De Francesco, R. New horizons in hepatitis C antiviral therapy with direct-acting antivirals. Hepatology 2013, 58, 428−438. (19) Asselah, T.; Marcellin, P. Interferon-free therapy with direct acting antivirals for HCV. Liver Int. 2013, 33 (Suppl. 1), 93−104. (20) Shah, N.; Pierce, T.; Kowdley, K. V. Review of direct-acting antiviral agents for the treatment of chronic hepatitis C. Expert Opin. Invest. Drugs 2013, 22, 1107−1121. (21) Kanda, T.; Yokosuka, O.; Omata, M. Treatment of hepatitis C virus infection in the future. Clin. Transl. Med. 2013, 2, 9−16. (22) Pawlotsky, J.-M. NS5A inhibitors in the treatment of hepatitis C. J. Hepatol. 2013, 59, 375−382. (23) Lok, A. S.-F. HCV NS5A inhibitors in development. Clin. Liver Dis. 2013, 17, 111−121. (24) Lange, C. M.; Jacobson, I. M.; Rice, C. M.; Zeuzem, S. Emerging therapies for the treatment of hepatitis C. EMBO Mol. Med. 2013, 5, 1−12. (25) Manns, M. P.; von Hahn, T. Novel therapies for hepatitis C one pill fits all? Nat. Rev. Drug Discov. 2013, 12, 595−610. (26) Seeger, C. Salient molecular features of hepatitis C virus revealed. Trends Microbiol. 2005, 13, 528−534. (27) Green, N.; Ott, R. D.; Isaacs, R. J.; Fang, H. Cell-based assays to identify inhibitors of viral disease. Expert Opin. Drug Discovery 2008, 3, 671−676. (28) Keller, T. H.; Shi, P.-Y.; Wang, Q.-Y. Anti-infectives: Can cellular screening deliver? Curr. Opin. Chem. Biol. 2011, 15, 529−533. (29) Swinney, D. C.; Anthony, J. How were new medicines discovered? Nat. Rev. Drug Discovery 2011, 10, 507−519. (30) Lohmann, V.; Körner, F.; Koch, J. O.; Herian, U.; Theilmann, L.; Bartenschlager, R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 1999, 285, 110−113. (31) Bartenschlager, R. Hepatitis C virus replicons: potential role for drug development. Nat. Rev. Drug Discovery 2002, 1, 911−916. (32) Bartenschlager, R. The hepatitis C virus replicon system: from basic research to clinical application. J. Hepatol. 2005, 43, 210−216. (33) Taylor, D. R. Evolution of cell culture systems for HCV. Antiviral Ther. 2013, 18, 523−530. (34) Gao, M.; Lemm, J. A.; O’Boyle, D. R.; Nower, P.; Rigat, K., Sun, J.-H. Hepatitis C virus assays. World Patent Application WO-2004/ 015131, February 19, 2004.

his postdoctoral work, he joined the National institutes on Deafness and Other Communication Disorders as a Senior Staff Fellow where he worked with Thomas Friedman on the identification and characterization of genes associated with hereditary hearing loss. He is currently in his 15th year at Bristol-Myers Squibb where he is a Principal Scientist in Virology working on HCV and HIV-1 drug discovery. Nicholas A. Meanwell received his Ph.D. degree from the University of Sheffield, Sheffield, England, with Dr. D. Neville Jones and conducted postdoctoral studies at Wayne State University, Detroit, MI, in collaboration with Professor Carl R. Johnson. He joined BristolMyers Squibb in 1982 where he is currently Executive Director of Discovery Chemistry with responsibility for the discovery and optimization of new therapies for the treatment of viral diseases. His team has advanced clinical candidates in several areas of antiviral drug discovery, including inhibitors of respiratory syncytial virus fusion peptide 6-helix bundle function (BMY-433771), inhibitors of HIV-1 attachment (BMS-663068/BMS-626529), HCV NS3 protease (asunaprevir), HCV NS5A (daclatasvir), and HCV NS5B (BMS-791325).

■

ABBREVIATIONS USED

■

REFERENCES

ADME, absorption, distribution, metabolism and excretion; BVDV, bovine viral diarrhea virus; b.i.d., bis in die (twice a day); CC50, concentration at which cytotoxicity is half maximal; cDNA, complementary deoxyribonucleic acid; DAA, directacting antiviral; EC50, concentration at which inhibition is half maximal; FRET, fluorescence resonance energy transfer; GST, glutathione S-transferase; GT, genotype; HCV, hepatitis C virus; IU/mL, international units per milliliter; NHV, normal healthy volunteer; NS, nonstructural; Peg-IFNα, pegylated interferon α; PK, pharmacokinetic; q.d., quaque die (once per day); RCI, replication complex inhibitor; RNA, ribonucleic acid; SAD, single ascending dose; SAR, structure−activity relationship; SVR, sustained virological response

(1) Macdonald, A.; Harris, M. Hepatitis C virus NS5A: tales of a promiscuous protein. J. Gen. Virol. 2004, 85, 2485−2502. (2) Appel, N.; Schaller, T.; Penin, F.; Bartenschlager, R. From structure to function: new insights into hepatitis C virus RNA replication. J. Biol. Chem. 2006, 281, 9833−9836. (3) He, Y.; Staschke, K. A.; Tan, S.-L. HCV NS5A: a multifunctional regulator of cellular pathway and virus replication. In Hepatitis C Viruses; Tan, S.-L., Ed., Horizon Bioscience: Wymondham, U.K. 2006; pp 267−292. (4) Szabo, G. Hepatitis C virus NS5A proteina master regulator? Gastroenterology 2006, 130, 996−999. (5) Huang, Y.; Staschke, K.; De Francesco, R.; Tan, S.-L. Phosphorylation of hepatitis C virus NS5A nonstructural protein: a new paradigm for phosphorylation-dependent viral RNA replication? Virology 2007, 364, 1−9. (6) Poenisch, M.; Bartenschlager, R. New insights into structure and replication of the hepatitis C virus and clinical implications. Semin. Liver Dis. 2010, 50, 333−347. (7) Cordek, D. G.; Bechtel, J. T.; Maynard, A. T.; Kazmierski, W. M.; Cameron, C. E. Targeting the NS5A protein of HCV: an emerging option. Drugs Future 2011, 36, 691−711. (8) Nettles, R.; Chien, C.; Chung, E.; Persson, A.; Gao, M.; Belema, M.; Meanwell, N. A.; DeMicco, M.; Marbury, T. C.; Goldwater, R.; Northup, P.; Coumbis, J.; Kraft, W. K.; Charlton, M. R.; LopezTalavera, J. C.; Grasela, D. M. 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. Presented at the Annual Meeting of the American Association for the Study of Liver 1664



Perspective

(35) Colonno, R.; Lemm, J. A.; O’Boyle, D. R.; Gao, M.; Romine, J. L.; Martin, S. W., Snyder, L. B.; Serrano-Wu, M.; Deshpande, M.; Whitehouse, D. Combination pharmaceutical agents as inhibitors of HCV replication. World Patent Application WO-2004/014313, February 19, 2004. (36) Romine, J. L.; Martin, S. W.; Snyder, L. B.; Serrano-Wu, M.; Deshpande, M.; Whitehouse, D.; Lemm, J.; O’Boyle, D. R.; Gao, M.; Colonno, R. Iminothiazolidinones as inhibitors of HCV replication. World Patent Application WO-2004/014852, February 19, 2004. (37) Lemm, J. A.; O’Boyle, D. R., II; Liu, M.; Nower, P. T.; Colonno, R.; Deshpande, M. S.; Snyder, L. B.; Martin, S. W.; St. Laurent, D. R.; Serrano-Wu, M. H.; Romine, J. L.; Meanwell, N. A.; Gao, M. Identification of hepatitis C virus NS5A inhibitors. J. Virol. 2010, 84, 482−491. (38) O’Boyle, D. R., II; Nower, P. T.; Lemm, J. A.; Valera, L.; Sun, J.H.; Rigat, K.; Colonno, R.; Gao, M. Development of a cell-based highthroughput specificity screen using a hepatitis C virus-bovine viral diarrhea virus dual replicon assay. Antimicrob. Agents Chemother. 2005, 49, 1346−1353. (39) Romine, J. L.; St. Laurent, D. R.; Leet, J. E.; Martin, S. W.; Serrano-Wu, M. H.; Yang, F.; Gao, M.; O’Boyle, D. R., II; Lemm, J. A.; Sun, J.-H.; Nower, P. T.; Huang, X.; Deshpande, M. S.; Meanwell, N. A.; Snyder, L. B. Inhibitors of HCV NS5A: from iminothiazolidinones to symmetrical stilbenes. ACS Med. Chem. Lett. 2011, 2, 224−229. (40) Lemm, J. A.; Leet, J. E.; O’Boyle, D. R., II; Romine, J. L.; Huang, X. S.; Schroeder, D. R.; Alberts, J.; Cantone, J. L.; Sun, J.-H.; Nower, P. T.; Martin, S. W.; Serrano-Wu, M. H.; Meanwell, N. A.; Snyder, L. B.; Gao, M. Discovery of potent hepatitis C virus NS5A inhibitors with dimeric structures. Antimicrob. Agents Chemother. 2011, 55, 3795− 3802. (41) Viehe, H. G.; Janousek, Z.; Merenyi, R. The captodative effect. Acc. Chem. Res. 1985, 18, 148−154. (42) (a) Kugler-Steigmeier, M. E.; Friederich, U.; Graf, U.; Lutz, W. K.; Maier, P.; Schlatter, C. Genotoxicity of aniline derivatives in various short-term tests. Mutat. Res. 1989, 211, 279−289. (b) Benigni, R.; Giuliani, A.; Franke, R.; Gruska, A. Quantitative structure−activity relationships of mutagenic and carcinogenic aromatic amines. Chem. Rev. 2000, 100, 3697−3714. (c) Shamovsky, I.; Ripa, L.; Börjesson, L.; Mee, C.; Nordén, B.; Hansen, P.; Hasselgren, C.; O’Donovan, M.; Sjö, P. Explanation for main features of structure−genotoxicity relationships of aromatic amines by theoretical studies of their activation pathways in CYP1A2. J. Am. Chem. Soc. 2011, 133, 16168−16185. (d) Garcia, G. X.; Larsen, S. W.; Pye, C.; Galbreath, M.; Isovitsch, R.; Fradinger, E. A. The functional group on (E)-4,4′-disubstituted stilbenes influences toxicity and antioxidative activity in differentiated PC-12 cells. Bioorg. Med. Chem. Lett. 2013, 23, 6355−6359. (43) St. Laurent, D. R.; Belema, M.; Gao, M.; Goodrich, J.; Kakarla, R.; Knipe, J. O.; Lemm, J. A.; Liu, M.; Lopez, O. D.; Nguyen, V. N.; Nower, P. T.; O’Boyle, D., II; Qiu, Y.; Romine, J. L.; Serrano-Wu, M. H.; Sun, J.-H.; Valera, L.; Yang, F.; Yang, X.; Meanwell, N. A.; Snyder, L. B. HCV NS5A replication complex inhibitors. Part 2: Investigation of stilbene prolinamides. Bioorg. Med. Chem. Lett. 2012, 22, 6063− 6066. (44) Lopez, O. D.; Nguyen, V. N.; St. Laurent, D. R.; Belema, M.; Serrano-Wu, M. H.; Goodrich, J. T.; Yang, F.; Qiu, Y.; Ripka, A. S.; Nower, P. T.; Valera, L.; Liu, M.; O’Boyle, D. R., II; Sun, J.-H.; Fridell, R. A.; Lemm, J. A.; Gao, M.; Good, A. C.; Meanwell, N. A.; Snyder, L. B. HCV NS5A replication complex inhibitors. Part 3: Discovery of potent analogs with distinct core topologies. Bioorg. Med. Chem. Lett. 2013, 23, 779−784. (45) St. Laurent, D. R.; Serrano-Wu, M. H.; Belema, M.; Ding, M.; Fang, H.; Gao, M.; Goodrich, J. T.; Krause, R. G.; Lemm, J. A.; Liu, M.; Lopez, O. D.; Nguyen, V. N.; Nower, P. T.; O’Boyle, D. R., II; Pearce, B. C.; Romine, J. L.; Valera, L.; Sun, J.-H.; Wang, Y.-K.; Yang, F.; Yang, X.; Meanwell, N. A.; Snyder, L. B. HCV NS5A replication complex inhibitors. Part 4: Optimization for genotype 1a replicon inhibitory activity. J. Med. Chem. 2014, DOI: 10.1021/jm301796k. (46) Belema, M.; Nguyen, V. N.; St. Laurent, D. R.; Lopez, O. D.; Qiu, Y.; Good, A. C.; Nower, P. T.; Valera, L.; O’Boyle, D. R., II; Sun,

J.-H.; Fridell, R. A.; Lemm, J. A.; Gao, M.; Knipe, J. O.; Meanwell, N. A.; Snyder, L. B. HCV NS5A replication complex inhibitors. Part 5: Discovery of potent and pan-genotypic HCV NS5A replication complex inhibitors. Bioorg. Med. Chem. Lett. 2013, 23, 4428−4435. (47) Belema, M.; Nguyen, V. N.; Romine, J. L.; St. Laurent, D. R.; Lopez, O. D.; Goodrich, J.; Nower, P. T.; O’Boyle, D. R., II; Lemm, J. A.; Fridell, R. A.; Gao, M.; Fang, H.; Krause, R. G.; Wang, Y.-K.; Oliver, A. J.; Good, A. C.; Knipe, J. O.; Meanwell, N. A.; Snyder, L. B. Hepatitis C virus NS5A replication complex inhibitors. Part 6: The discovery of a novel and highly potent biarylimidazole chemotype with inhibitory activity toward genotype 1a and 1b replicons. J. Med. Chem. 2014, DOI: 10.1021/jm4016203. (48) Belema, M.; Nguyen, V. N.; Bachand, C.; Deon, D. H.; Goodrich, J. T.; Lavoie, R.; James, C. A.; Lopez, O. D.; Martel, A.; Romine, J. L.; Ruediger, E. H.; Snyder, L. B.; St. Laurent, D. R.; Good, A. C.; Langley, D. R.; Adams, S. P.; Cantor, G. H.; Chimalakonda, A.; Fura, A.; Knipe, J. O.; Nower, P. T.; O’Boyle, D. R., II; Lemm, J. A.; Fridell, R. A.; Colonno, R. J.; Gao, M.; Meanwell, N. A.; Hamann, L. G. Hepatitis C virus NS5A replication complex inhibitors: the discovery of daclatasvir. J. Med. Chem. 2014, DOI: 10.1021/ jm401836p. (49) (a) Lovering, F.; Bikker, J.; Humblet, C. Escape from the flatland: increasing saturation as an approach to improving clinical success. J. Med. Chem. 2009, 52, 6752. (b) Ritchie, T. J.; Macdonald, S. J. F. The impact of aromatic ring count on compound developability Are too many aromatic rings a liability in drug design? Drug Discovery Today 2009, 14, 1011−1020. (c) Meanwell, N. A. Improving drug candidates by design: a focus on physicochemical properties as a means of improving compound disposition and safety. Chem. Res. Toxicol. 2011, 24, 1420−1456. (50) Wang, C.; Jia, L.; Huang, H.; Qiu, D.; Valera, L.; Huang, X.; Sun, J.-H.; Nower, P. T.; O’Boyle, D. R., II; Gao, M.; Fridell, R. A. In vitro activity of BMS-790052 on hepatitis C virus genotype 4a NS5A. Antimicrob. Agents Chemother. 2012, 56, 1588−1590. (51) Wang, C.; Valera, L.; Jia, L.; Kirk, M. J.; Gao, M.; Fridell, R. A. In vitro activity of daclatasvir on hepatitis C virus genotype 3 NS5A. Antimicrob. Agents Chemother. 2013, 57, 611−613. (52) (a) Lindenbach, B. D.; Evans, M. J.; Syder, A. J.; Wölk, B.; Tellinghuisen, T. L; Liu, C. C.; Maruyama, T.; Hynes, R. O.; Burton, D. R.; McKeating, J. A.; Rice, C. M. Complete replication of hepatitis C virus in cell culture. Science 2005, 309, 623−626. (b) Zhong, J.; Gastaminza, P.; Cheng, G.; Kapadia, S.; Kato, T.; Burton, D. R.; Wieland, S. F.; Uprichard, S. L.; Wakita, T.; Chisari, F. V. Robust hepatitis C virus infection in vitro. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9294−9299. (c) Wakita, T.; Pietschmann, T.; Kato, T.; Date, T.; Miyamoto, M.; Zhao, Z.; Murthy, K.; Habermann, A.; Kraeusslich, H.G.; Mizokami, M.; Bartenschlager, R.; Liang, T. J. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 2005, 11, 791−796. (53) (a) Tiberghien, N.; Lumley, J.; Reynolds, K.; Angell, R. M.; Matthews, N.; Cockerill, G. S.; Barnes, M. C. Pyrazolopyrimidines as anti-hepatitis C agents, their preparation, pharmaceutical compositions and use. World Patent Application WO-2005/047288, May 26, 2005. (b) Spencer, K.; Dennison, H.; Matthews, N.; Barnes, M.; Chana, S. Preparation of morpholinylanilino quinazoline derivatives for use as antiviral agents. World Patent Application WO-2005/105761, November 10, 2005. (c) Cockerill, G. S.; Flack, S. S.; Mathews, N.; Salter, J. I. Preparation of quinazoline derivatives as antiviral agents. World Patent Application WO-2006/079833, August 3, 2006. (d) Mathews, N.; Thomas, A. J. F.; Spencer, K. C.; Tiberghien, N.; Pilkington, C. J.; Jennens, L.; Chana, S.; Fraser, I. J. Preparation of triazolylanilinoquinazolines as antivirals. World Patent Application WO-2007/080401, July 19, 2007. (54) Bianco, A.; Reghellin, V.; Donnici, L.; Fenu, S.; Alvarez, R.; Baruffa, C.; Peri, F.; Pagani, M.; Abrignani, S.; Neddermann, P.; De Francesco, R. Metabolism of phosphatidylinositol 4-kinase IIIαdependent PI4P is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral activity. PLoS Pathog. 2012, 8, e1002576. 1665



Perspective

(55) (a) Bishé, B.; Syed, G.; Siddiqui, A. Phosphoinositides in the hepatitis C virus life cycle. Viruses 2012, 4, 2340−2358. (b) AltanBonnet, N.; Balla, T. Phosphatidylinositol 4-kinases: hostages harnessed to build panviral replication platforms. Trends Biochem. Sci. 2012, 37, 293−302. (56) (a) Wheelhouse, C. J.; Thomas, A. J. F.; Bushnell, D. J.; Lumley, J.; Salter, J. I.; Carter, M. C.; Mathews, N.; Pilkington, C. J.; Angell, R. M. Biphenyl derivatives and their use in treating hepatitis C. World Patent Application WO-2007/031791, March 22, 2007. (b) Lumley, J.; Salter, J. I.; Carter, M. C.; Mathews, N.; Pilkington, C.; Thomas, A. J. F.; Fraser, I. Biphenyl derivatives and their use in treating hepatitis C. World Patent Application WO-2007/138242, December 6, 2007. (c) Carter, M. C.; Mathews, N. Novel biphenyl compounds useful for the treatment of hepatitis C. World Patent Application WO-2010/ 094977, August 26, 2010. (d) Carter, M. C.; Cockerill, S.; Flack, S. S.; Wheelhouse, C. J. Heterocyclic derivatives and their use in treating hepatitis C. World Patent Application WO-2009/034390, March 19, 2009. (57) Conte, I.; Giuliano, C.; Ercolani, C.; Narjes, F.; Koch, U.; Rowley, M.; Altamura, S.; De Francesco, R.; Neddermann, P.; Migliaccio, G.; Stansfield, I. Synthesis and SAR of piperazinyl-Nphenylbenzamides as inhibitors of hepatitis C virus RNA replication in cell culture. Bioorg. Med. Chem. Lett. 2009, 19, 1779−1783. (58) Krueger, A. C.; Madigan, D. L.; Beno, D. W.; Betebenner, D. A.; Carrick, R.; Green, B. E.; He, W.; Liu, D.; Maring, C. J.; McDaniel, K. F.; Mo, H.; Molla, A.; Motter, C. E.; Pilot-Matias, T. J.; Tufano, M. D.; Kempf, D. J. Novel hepatitis C virus replicon inhibitors: synthesis and structure−activity relationships of fused pyrimidine derivatives. Bioorg. Med. Chem. Lett. 2012, 22, 2212−2215. (59) (a) DeGoey, D. A.; Betebenner, D. A; Grampovnik, D. J.; Liu, D.; Pratt, J. K.; Tufano, M. D.; He, W.; Krishnan, P.; Pilot-Matias, T. J.; Marsh, K. C.; Molla, A.; Kempf, D. J.; Maring, C. J. Discovery of pyrido[2,3-d]pyrimidine-based inhibitors of HCV NS5A. Bioorg. Med. Chem. Lett. 2013, 23, 3627−3630. (b) Betebenner, D. A.; DeGoey, D. A.; Maring, C. J.; Krueger, A. C.; Iwasaki, N.; Rockway, T. W.; Cooper, C. S.; Anderson, D. D.; Donner, P. L.; Green, B. E.; Kempf, D. J.; Liu, D.; McDaniel, K. F.; Madigan, D. L.; Motter, C. E.; Pratt, J. K.; Shanley, J. P.; Tufano, M. D.; Wagner, R.; Zhang, R.; Molla, A.; Mo, H.; Pilot-Matias, T. J.; Masse, S. V. L.; Carrick, R. J.; He, W.; Lu, L.; Grampovnik, D. J. Preparation of naphthyridines and pyridopyrimidines as antiviral compounds for treatment of HCV infections. World Patent Application WO-2007/076034, July 5, 2007. (c) Rockway, T. W.; Betebenner, D. A.; Krueger, A. C.; Iwasaki, N.; Cooper, C. S.; Anderson, D. D.; Kempf, D. J.; Madigan, D. L.; Motter, C. E.; Shanley, J. P.; Tufano, M. D.; Wagner, R.; Zhang, R.; Molla, A.; Mo, H.; Pilot-Matias, T.; Masse, S. V. L.; Carrick, R. J.; He, W.; Lu, L. Preparation of 1,6- and 1,8-naphthyridines as antiviral compounds for treatment of HCV infections. World Patent Application WO-2007/ 076035, July 5, 2007. (d) Betebenner, D. A.; DeGoey, D. A.; Maring, C. L.; Krueger, A. C.; Iwasaki, N.; Rockway, T. W.; Cooper, C. S.; Anderson, D. D.; Donner, P. L.; Green, B. E.; Kempf, D. J.; Liu, D.; McDaniel, K. F.; Madigan, D. L.; Motter, C. E.; Pratt, J. K.; Shanley, J. P.; Tufano, M. D.; Wagner, R.; Zhang, R.; Molla, A.; Mo, H.; PilotMatias, T. J.; Masse, S. V. L.; Carrick, R. J.; He, W.; Lu, L.; Grampovnik, D. J. Preparation of fused bicyclic heterocycles, particularly substituted 4-aminophenylpyrido[2,3-d]pyrimidines, as anti-viral compounds for treatment of HCV infections. World Patent Application WO-2007/081517, July 19, 2007. (60) (a) Schmitz, F. U.; Roberts, C. D.; Abadi, A. D. M.; Griffith, R. C.; Leivers, M. R. N-(6-Membered aromatic ring)-amido anti-viral compounds. World Patent Application WO-2007/070556, June 21, 2007. (b) Schmitz, F. U.; Roberts, C. D.; Abadi, A. D. M.; Griffith, R. C.; Leivers, M. R.; Slobodov, I.; Rai, R. N-Heterocyclylpyrrolidine carboxamide derivatives, processes for preparing them, pharmaceutical compositions containing them, and their use as antiviral agents. World Patent Application WO-2007/070600, June 21, 2007. (c) Leivers, M. R.; Schmitz, F. U.; Griffith, R. C.; Roberts, C. D.; Dehghani Mohammad Abadi, A.; Chan, S. A.; Rai, R.; Slobodov, I.; Ton, T. L.

Amido antiviral compounds. World Patent Application WO-2008/ 064218, May 29, 2008. (61) Rai, R.; Schmitz, F. U.; Roberts, C. D.; Slobodov, I.; Leivers, M. R. Anti-viral inhibitors and methods of use. World Patent Application WO-2008/154601, December 18, 2008. (62) Li, G.; Fathi, R.; Yang, Z.; Liao, Y.; Zhu, Q.; Lam, A.; Sandrasagra, A.; Nawoschik, K.; Cho, H.-J.; Cao, J.; Wu, R.; Wobbe, C. R. Compounds and methods for treatment of HCV. World Patent Application WO-2008/048589, April 24, 2008. (63) (a) Bachand, C.; Belema, M.; Deon, D. H.; Good, A. C.; Goodrich, J.; James, C. A.; Lavoie, R.; Lopez, O. D.; Martel, A.; Meanwell, N. A.; Nguyen, V. N.; Romine, J. L.; Ruediger, E. H.; Snyder, L. B.; St. Laurent, D. R.; Yang, F.; Langley, D. R.; Wang, G.; Hamann, L. G. Hepatitis C virus inhibitors. World Patent Application WO-2008/021927, February 21, 2008. (b) Bachand, C.; Belema, M.; Deon, D. H.; Good, A. C.; Goodrich, J.; James, C. A.; Lavoie, R.; Lopez, O. D.; Martel, A.; Meanwell, N. A.; Nguyen, V. N.; Romine, J. L.; Ruediger, E. H.; Snyder, L. B.; St. Laurent, D. R.; Yang, F.; Langley, D. R.; Wang, G. Hamann, L. G. Hepatitis C virus inhibitors. World Patent Application WO-2008/021928, February 21, 2008. (c) Bachand, C.; Belema, M.; Deon, D. H.; Good, A. C.; Goodrich, J.; James, C. A.; Lavoie, R.; Lopez, O. D.; Martel, A.; Meanwell, N. A.; Nguyen, V. N.; Romine, J. L.; Ruediger, E. H.; Snyder, L. B.; St. Laurent, D. R.; Yang, F.; Langley, D. R.; Wang, G.; Hamann, L. G. Hepatitis C virus inhibitors. World Patent Application WO-2008/021936, February 21, 2008. (64) (a) Li, L.; Zhong, M. Inhibitors of HCV NS5A. World Patent Application WO-2010/065668, June 10, 2010. (b) Zhong, M.; Li, L. Inhibitors of HCV NS5A. World Patent Application WO-2010/ 065674, June 10, 2010. (c) Li, L.; Zhong, M. Inhibitors of HCV NS5A. World Patent Application WO-2010/065681, June 10, 2010. (65) (a) Link, J. O.; Taylor, J. G.; Xu, L.; Mitchell, M.; Guo, H.; Liu, H.; Kato, D.; Kirschberg, T.; Sun, J.; Squires, N.; Parrish, J.; Keller, T.; Yang, Z.-Y.; Yang, C.; Matles, M.; Wang, Y.; Wang, K.; Cheng, G.; Tian, Y.; Mogalian, E.; Mondou, E.; Cornpropst, M.; Perry, J.; Desai, M. C. The discovery of ledipasvir (GS-5885), a potent once-daily oral NS5A inhibitor for the treatment of hepatitis C virus infection. J. Med. Chem. 2014, DOI: 10.1021/jm401499g. (b) Link, J. O.; Bannister, R.; Beilke, L. D.; Cheng, G.; Cornpropst, M.; Corsa, A.; Dowdy, E.; Guo, H.; Kato, D.; Kirschberg, T.; Liu, H.; Mitchell, M.; Matles, M.; Mogalian, E.; Mondou, E.; Ohmstede, C.; Peng, B.; Scott, R. W.; Findlay, J. W.; Chittick, G. E.; Wang, F.; Alianti, J.; Sun, J.; Taylor, J.; Tian, Y.; Xu, L.; Yang, C.; Yuen, G. J.; Wang, K.; Eisenberg, E. J. Nonclinical profile and phase 1 results in healthy volunteers for the novel and potent HCV NS5A inhibitor GS-5885. Hepatology 2010, 52, 1215A (Abstract 1883). (c) Cheng, G.; Yu, M.; Peng, B.; Lee, Y.-J.; Trejo-Martin, A.; Gong, R.; Bush, C.; Worth, A.; Nash, M.; Chan, K.; Yang, H.; Beran, R.; Tian, Y.; Perry, J.; Taylor, J.; Yang, C.; Paulson, M.; Delaney, W.; Link, J. O. GS-5816, a second generation HCV NS5A inhibitor with potent antiviral activity, broad genotypic coverage and a high resistance barrier. J. Hepatol. 2013, 55, S484−S485 (Abstract 1191). (66) (a) Coburn, C. A.; Meinke, P. T.; Chang, W.; Fandozzi, C. M.; Graham, D. J.; Hu, B.; Huang, Q.; Kargman, S.; Kozlowski, J.; Liu, R.; McCauley, J. A.; Nomeir, A. A.; Soll, R. M.; Vacca, J. P.; Wang, D.; Wu, H.; Zhong, B.; Olsen, D. B.; Ludmerer, S. W. Discovery of MK-8742: an HCV NS5A inhibitor with broad genotype activity. ChemMedChem 2013, 8, 1930−1940. (b) Kozlowski, J. A.; Rosenblum, S. B.; Coburn, C. A.; Shankar, B. B.; Anilkumar, G.; Chen, L.; Dwyer, M. P.; Jiang, Y.; Keertikar, K. M.; Lavey, B. J.; Selyutin, O. B.; Tong, L.; Wong, M.; Yang, D.-Y.; Yu, W.; Zhou, G.; Wu, H.; Hu, B.; Zhong, B.; Sun, F.; Ji, T.; Shen, C. Preparation of tetracyclic indole derivatives for treatment of HCV infection. World Patent Application WO-2012/040923, April 5, 2012. (c) Kozlowski, J. A.; Rosenblum, S. B.; Coburn, C. A.; Shankar, B. B.; Anilkumar, G. N.; Chen, L.; Dwyer, M. P.; Jiang, Y.; Keertikar, K. M.; Lavey, B. J.; Selyutin, O. B.; Tong, L.; Wong, M.; Yang, D.-Y.; Yu, W.; Zhou, G.; Wu, H.; Hu, B.; Zhong, B.; Sun, F.; Ji, T.; Shen, C.; Rizvi, R.; Zeng, Q. Preparation of tetracyclicindole 1666



Perspective

derivatives and analogs for use in treatment of hepatitis C virus infection. World Patent Application WO-2012/041014, April 5, 2012. (67) (a) DeGoey, D. A.; Randolph, J. T.; Liu, D.; Pratt, J.; Hutchins, C.; Donner, P.; Krueger, A. C.; Matulenko, M.; Patel, S.; Motter, C. E.; Nelson, L.; Keddy, R.; Tufano, M.; Caspi, D. D.; Krishnan, P.; Mistry, N.; Koev, G.; Reisch, T. J.; Mondal, R.; Pilot-Matias, T.; Gao, Y.; Beno, D. W. A.; Maring, C. J.; Molla, A.; Dumas, E.; Campbell, A.; Williams, L.; Collins, C.; Wagner, R.; Kati, W. M. Discovery of ABT-267, a pangenotypic inhibitor of HCV NS5A. J. Med. Chem. 2014, DOI: 10.1021/jm401398x. (b) DeGoey, D. A.; Kati, W. M.; Hutchins, C. W.; Donner, P. L.; Krueger, A. C.; Randolph, J. T.; Motter, C. E.; Nelson, L. T.; Patel, S. V.; Matulenko, M. A.; Keddy, R. G.; Jinkerson, T. K.; Soltwedel, T. N.; Hutchinson, D. K.; Flentge, C. A.; Wagner, R.; Maring, C. J.; Tufano, M. D.; Betebenner, D. A.; Rockway, T. W.; Liu, D.; Pratt, J. K.; Lavin, M. J.; Sarris, K.; Woller, K. R.; Wagaw, S. H.; Califano, J. C.; Li, W. Anti-viral compounds. World Patent Application WO-2010/144646. December 16, 2010. (68) (a) Dousson, C. B.; Dukhan, D.; Parsy, C. Cl.; Pierra, C.; Alexandre, F.-R.; Brandt, G.; Da Costa, D.; Rahali, H.; Paparin, J.-L.; Derock, M.; Convard, T.; Surleraux, D. Preparation of 5,5-fused arylenes or heteroarylenes end-capped with amino acid and peptide derivatives as hepatitis C virus inhibitors. World Patent Application WO-2011/075615, June 23, 2011. (b) Dousson, C. B.; Dukhan, D.; Parsy, C. C.; Pierra, C.; Alexandre, F.-R.; Brandt, G.; Da Costa, D.; Rahali, H.; Paparin, J.-L.; Derock, M.; Convard, T.; Surleraux, D.; Bilello, J. P. Preparation of 5,5-fused arylenes or heteroarylenes endcapped with amino acid and peptide derivatives useful in treatment and prevention of drug resistant hepatitis C infection. World Patent Application WO-2012/135581, October 4, 2012. (c) Dousson, C. B.; La Colla, M.; Chapron, C.; Bilello, J. P.; McCarville, J. F.; Seifer, M.; Parsy, C.; Dukhan, D.; Pierra, C.; Surleraux, D.; Standring, D. N. Idenix NS5A HCV replication inhibitors with low picomolar, pangenotypic in vitro antiviral activity. J. Hepatol. 2011, 54, S326−S327 (Abstract 815). (69) (a) Walker, J.; Crosby, R.; Wang, A.; Woldu, E.; Vamathevan, J.; Voitenleitner, C.; You, S.; Remlinger, K.; Duan, M.; Kazmierski, W.; Hamatake, R. Preclinical characterization of GSK2336805, a novel inhibitor of hepatitis C virus replication that selects for resistance in NS5A. Antimicrob. Agents Chemother. 2014, 58, 38−47. (b) Chen, P.; Couch, R.; Duan, M.; Grimes, R. M.; Kazmierski, W. M.; Norton, B. A.; Tallant, M. Preparation of biphenyl compounds end capped with amino acids or peptide derivatives for treating HCV infection. World Patent Application WO-2011/028596, March 10, 2011. (70) (a) Lorimer, K.; Li, L.; Zhong, M.; Muchnick, A. Solid forms comprising inhibitors of HCV NS5A, compositions thereof, and uses therewith. World Patent Application WO-2013/123092, August 22, 2013. (b) Li, L.; Zhong, M. Inhibitors of HCV NS5A. World Patent Application WO-2010/065681, June 10, 2010. (71) Wiles, J. A.; Wang, Q.; Hashimoto, A.; Pais, G.; Chen, D.; Wang, X.; Gadhachanda, V.; Phadke, A.; Deshpande, M. Substituted aliphanes, cyclophanes, heteraphanes, heterophanes, hetero-heteraphanes and metallocenes useful for treating HCV infections. World Patent Application WO-2012/166716, December 6, 2012. (72) (a) Huang, M.; Yang, G.; Patel, D.; Zhao, Y.; Fabrycki, J.; Marlor, C.; Rivera, J.; Stauber, K.; Gadhachanda, V.; Wiles, J.; Hashimoto, A.; Chen, D.; Wang, Q.; Pais, G.; Wang, X.; Deshpande, M.; Phadke, A. ACH-2928: a novel highly potent HCV NS5A inhibitor with favorable preclinical characteristics. J. Hepatol. 2011, 54 (Suppl. 1), S479 (Abstract 1212). (b) Vince, B.; Lawitz, E.; Searle, S.; Marbury, T.; Robison, H.; Robarge, L.; Olek, E. Novel NS5A inhibitor ACH-2928 phase 1 results in HCV GT-1 patients. J. Hepatol. 2012, 56 (Suppl. 2), S448−S481 (Abstract 1211). (73) Wang, G.; Wiles, J.; Patel, D.; Zhao, Y.; Fabrycki, J.; Weinheimer, S.; Marlor, C.; Rivera, J.; Wang, Q.; Gadhachanda, V.; Hashimoto, A.; Chen, D.; Pais, G.; Wang, X.; Deshpande, M.; Stauber, K.; Huang, M.; Phadke, A. Preclinical characteristics of ACH-3102: a novel HCV NS5A inhibitor with improved potency against genotype 1a virus and variants resistant to 1st generation of NS5A inhibitors. J. Hepatol. 2012, 56, S330 (Abstract 845). Available at http://www.

achillion.com/resourcefiles/it_1380671390/ACHN_ACH-3102_ Preclinical_EASL12.PDF (accessed January 12, 2014). (74) Or, Y. S.; Owens, C. M.; Brasher, B.; Qiu, Y.-L.; Jiang, L. Combination pharmaceutical agents as inhibitors of HCV replication. World Patent Application WO-2011/109037, September 9, 2011. (75) Proof of concept study for safety and efficacy of EDP239 in hepatitis C subjects. ClinicalTrials.gov: http://clinicaltrials.gov/show/ NCT01856426 (accessed January 12, 2014). (76) Lin, H.-M.; Wang, J.-C.; Hu, H.-S.; Wu, P.-S.; Wang, W.-H.; Wu, S.-Y.; Yang, C.-C.; Yeh, T.-K.; Hsu, T.-A.; Jiaang, W.-T.; Chao, Y.-S.; Chern, J.-H.; Yueh, A. Resistance studies of a dithiazol analogue, DBPR110, as a potential hepatitis C virus inhibitor in replicon systems. Antimicrob. Agents Chemother. 2013, 57, 723−733. (77) (a) Meanwell, N. A. Synopsis of some recent tactical application of bioisosteres in drug design. J. Med. Chem. 2011, 54, 2529−2591. (b) Dudkin, V. Y. Bioisosteric equivalence of five-membered heterocycles. Chem. Heterocycl. Compd. 2012, 48, 27−32. (78) (a) Honda, T.; Tajima, H.; Kaneko, Y.; Ban, M.; Inaba, T.; Takeno, Y.; Okamoto, K.; Aono, H. Conformation−activity relationship on novel 4-pyridylmethylthio derivatives with antiangiogenic activity. Bioorg. Med. Chem. Lett. 2008, 18, 2939−2943. (b) Burling, F. T.; Goldstein, B. M. A database study of nonbonded intramolecular sulfur-nucleophile contacts. Acta Crystallogr. 1993, B49, 738−744. (c) Murray, J. S.; Lane, P.; Politzer, P. Simultaneous σ-hole and hydrogen bonding by sulfur- and selenium-containing heterocycles. Int. J. Quantum Chem. 2008, 108, 2770−2781. (79) (a) Wilcken, R.; Zimmermann, M. O.; Lange, A.; Joerger, A. C.; Boeckler, F. M. Principles and applications of halogen bonding in medicinal chemistry and chemical biology. J. Med. Chem. 2013, 56, 1363−1388. (b) Lu, Y.; Shi, T.; Wang, Y.; Yang, H.; Yan, X.; Luo, X.; Jiang, H.; Zhu, W. Halogen bondinga novel interaction for rational drug design? J. Med. Chem. 2009, 52, 2854−2862. (c) Lu, Y.; Liu, Y.; Xu, Z.; Li, H.; Liu, H.; Zhu, W. Halogen bonding for rational drug design and new drug discovery. Expert Opin. Drug Discovery 2012, 7, 375−383. (d) Parisini, E.; Metrangolo, P.; Pilati, T.; Resnati, G.; Terraneo, G. Halogen bonding in halocarbon−protein complexes: a structural survey. Chem. Soc. Rev. 2011, 40, 2267−2278. (e) Riley, K. E.; Hobza, P. Strength and character of halogen bonds in protein− ligand complexes. Cryst. Growth Des. 2011, 11, 4272−4278. (80) (a) Iwaoka, M.; Takemoto, S.; Okada, M.; Tomoda, S. Weak nonbonded S···X (X = O, N, and S) interactions in proteins. Statistical and theoretical studies. Bull. Chem. Soc. Jpn. 2002, 75, 1611−1625. (b) Iwaoka, M.; Takemoto, S.; Tomoda, S. Statistical and theoretical investigations on the directionality of nonbonded S···O interactions. Implications for molecular design and protein engineering. J. Am. Chem. Soc. 2002, 124, 10613−10620. (c) Iwaoka, M.; Isozumi, N. Hypervalent nonbonded interactions of a divalent sulfur atom. Implications in protein architecture and the functions. Molecules 2012, 17, 7266−7283. (81) (a) Franz, A. K.; Wilson, S. O. Organosilicon molecules with medicinal applications. J. Med. Chem. 2013, 56, 388−405. (b) Gately, S.; West, R. Novel therapeutics with enhanced biological activity generated by the strategic introduction of silicon isosteres into known drug scaffolds. Drug Dev. Res. 2007, 68, 156−163. (c) Franz, A. K. The synthesis of biologically active organosilicon small molecules. Curr. Opin. Drug Discovery Dev. 2007, 10, 654−671. (d) Bains, W.; Tacke, R. Silicon chemistry as a novel source of chemical diversity in drug design. Curr. Opin. Drug Discovery Dev. 2003, 6, 526−543. (82) Nair, A. G.; Keertikar, K. M.; Kim, S. H.; Kozlowski, J. A.; Rosenblum, S.; Selyutin, O. B.; Wong, M.; Yu, W.; Zeng, Q. Fused tricyclic silyl compounds and methods of use thereof for the treatment of viral diseases. World Patent Application WO-2011/112429, September 15, 2011. (83) Dwyer, M. P.; Keertikar, K. M.; Zeng, Q.; Mazzola, R. D., Jr.; Yu, W.; Tang, H.; Kim, S. H.; Tong, L.; Rosenblum, S. B.; Kozlowski, J. A.; Nair, A. G. Silyl-containing heterocyclic compounds and methods of use thereof for the treatment of viral diseases. World Patent Application WO-2013/039876, March 21, 2013. 1667



Perspective

(84) (a) McKinnell, R. M.; Long, D. L.; van Orden, L. J.; Jiang, L.; Loo, M.; Saito, D. R.; Zipfel, S.; Stangeland, E. L.; Lepack, K.; Ogawa, G.; Huang, X.; Zhang, W. Novel inhibitors of hepatitis C virus. World Patent Application WO-2012/061552, May 10, 2012. (b) Long, D. L.; McKinnell, R. M.; Jiang, L.; Loo, M.; Lepack, K.; van Orden, L. J.; Ogawa, G.; Huang, X.; Zhang, W. Rod-like hepatitis C virus inhibitors containing fragment {2-[4-(biphenyl-4-yl)-1H-imidazo-2-yl]pyrrolidine-1-carbonylmethyl}amine. World Patent Application WO2013/067267, May 10, 2013. (c) Long, D. L.; McKinnell, R. M.; van Orden, L. J.; Ogawa, G.; Wilton, D. Piperazine-piperidine compounds as hepatitis C virus inhibitors. World Patent Application WO-2013/ 163262, October 31, 2013. (d) McKinnell, R. M.; Long, D. L. Hepatitis C virus inhibitors. World Patent Application WO-2013/ 163270, October 31, 2013. (e) Thalladi, V. R.; Nzerem, J.; Huang, X.; Zhang, W. Crystalline form of a pyridyl-piperazinyl hepatitis C virus inhibitor. World Patent Application WO-2013/165796, November 7, 2013. (85) Gordon, C. P.; Keller, P. A. Control of hepatitis C: a medicinal chemistry perspective. J. Med. Chem. 2005, 48, 1−20. (86) Lindenbach, B. D.; Rice, C. M. Unravelling hepatitis C virus replication from genome to function. Nature 2005, 436, 933−938. (87) Qureshi, S. A. Hepatitis C virusbiology, host evasion strategies, and promising new therapies on the horizon. Med. Res. Rev. 2007, 27, 353−373. (88) Moradpour, D.; Penin, F.; Rice, C. M. Replication of hepatitis C virus. Nat. Rev. Microbiol. 2007, 5, 453−463. (89) Joyce, M. A.; Tyrrell, D. L. The cell biology of hepatitis C virus. Microbes Infect. 2010, 12, 263−271. (90) Scheel, T. K. H.; Rice, C. M. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Nat. Med. 2013, 19, 837−849. (91) Bartenschlager, R.; Lohmann, V.; Penin, F. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat. Rev. Microbiol. 2013, 11, 482−496. (92) Tellinghuisen, T. L.; Marcotrigiano, J.; Gorbalenya, A. E.; Rice, C. M. The NS5A protein of hepatitis C virus is a zinc metalloprotein. J. Biol. Chem. 2004, 279, 48576−48587. (93) Pietschmann, T.; Lohmann, V.; Rutter, G.; Kurpanek, K.; Bartenschlager, R. Characterization of cell lines carrying self-replicating hepatitis C virus RNAs. J. Virol. 2001, 75, 1252−1264. (94) Gao, L.; Aizaki, H.; He, J. W.; Lai, M. M. Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft. J. Virol. 2004, 78, 3480−3488. (95) Liang, Y.; Ye, H.; Kang, C. B.; Yoon, H. S. Domain 2 of nonstructural protein 5A (NS5A) of hepatitis C virus is natively unfolded. Biochemistry 2007, 46, 11550−11558. (96) Hanoulle, X.; Badillo, A.; Verdegem, D.; Penin, F.; Lippens, G. The domain 2 of the HCV NS5A protein is intrinsically unstructured. Protein Pept. Lett. 2010, 17, 1012−1018. (97) Hanoulle, X.; Verdegem, D.; Badillo, A.; Wieruszeski, J. M.; Penin, F.; Lippens, G. Domain 3 of non-structural protein 5A from hepatitis C virus is natively unfolded. Biochem. Biophys. Res. Commun. 2009, 381, 634−638. (98) Blight, K. J.; Kolykhalov, A. A.; Rice, C. M. Efficient initiation of HCV RNA replication in cell culture. Science 2000, 290, 1972−1974. (99) Moradpour, D.; Evans, M. J.; Gosert, R.; Yuan, Z.; Blum, H. E.; Goff, S. P.; Lindenbach, B. D.; Rice, C. M. Insertion of green fluorescent protein into nonstructural protein 5A allows direct visualization of functional hepatitis C virus replication complexes. J. Virol. 2004, 78, 7400−7409. (100) Fridell, R. A.; Valera, L.; Qiu, D.; Kirk, M. J.; Wang, C.; Gao, M. Intragenic complementation of hepatitis C virus NS5A RNA replication-defective alleles. J. Virol. 2013, 87, 2320−2329. (101) Brass, V.; Bieck, E.; Montserret, R.; Wolk, B.; Hellings, J. A.; Blum, H. E.; Penin, F.; Moradpour, D. An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J. Biol. Chem. 2002, 277, 8130−8139.

(102) O’Boyle, D. R., II; Sun, J.-H.; Nower, P. T.; Lemm, J. A.; Fridell, R. A.; Wang, C.; Romine, J. L.; Belema, M.; Nguyen, V. N.; St. Laurent, D. R.; Serrano-Wu, M.; Snyder, L. B.; Meanwell, N. A.; Langley, D. R.; Gao, M. Characterizations of HCV NS5A replication complex inhibitors. Virology 2013, 444, 343−354. (103) Targett-Adams, P.; Graham, E. J. S.; Middleton, J.; Palmer, A.; Shaw, S. M.; Lavender, H.; Brain, P.; Tran, T. D.; Jones, L. H.; Wakenhut, F.; Stammen, B.; Pryde, D.; Pickford, C.; Westby, M. Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action. J. Virol. 2011, 85, 6353−6368. (104) Huang, L.; Hwang, J.; Sharma, S. D.; Hargittai, M. R. S.; Chen, Y.; Arnold, J. J.; Raney, K. D.; Cameron, C. E. Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein. J. Biol. Chem. 2005, 280, 36417−36428. (105) Hwang, J.; Huang, L.; Cordek, D. G.; Vaughan, R.; Reynolds, S. L.; Kihara, G.; Raney, K. D.; Kao, C. C.; Cameron, C. E. Hepatitis C virus nonstructural protein 5A: biochemical characterization of a novel structural class of RNA-binding proteins. J. Virol. 2010, 84, 12480− 12491. (106) Foster, T. L.; Belyaeva, T.; Stonehouse, N. J.; Pearson, A. R.; Harris, M. All three domains of the hepatitis C virus nonstructural NS5A protein contribute to RNA binding. J. Virol. 2010, 84, 9267− 9277. (107) Kaneko, T.; Tanji, Y.; Satoh, S.; Hijikata, M.; Asabe, S.; Kimura, K.; Shimotohno, K. Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome. Biochem. Biophys. Res. Commun. 1994, 205, 320−326. (108) Tanji, Y.; Kaneko, T.; Satoh, S.; Shimotohno, K. Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A. J. Virol. 1995, 69, 3980−3986. (109) Neddermann, P.; Quintavalle, M.; Di Pietro, C.; Clementi, A.; Cerretani, M.; Altamura, S.; Bartholomew, L.; De Francesco, R. Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture. J. Virol. 2004, 78, 13306−13314. (110) Cun, W.; Jiang, J.; Luo, G. The C-terminal alpha-helix domain of apolipoprotein E is required for interaction with nonstructural protein 5A and assembly of hepatitis C virus. J. Virol. 2010, 84, 11532−11541. (111) Appel, N.; Zayas, M.; Miller, S.; Krijnse-Locker, J.; Schaller, T.; Friebe, P.; Kallis, S.; Engel, U.; Bartenschlager, R. Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog. 2008, 4, e1000035. (112) Shirota, Y.; Luo, H.; Qin, W. P.; Kaneko, S.; Yamashita, T.; Kobayashi, K.; Murakami, S. Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRp) NS5B and modulates RNA-dependent RNA polymerase activity. J. Biol. Chem. 2002, 277, 11149−11155. (113) Tripathi, L. P.; Kambara, H.; Chen, Y.-A.; Nishimura, Y.; Moriishi, K.; Okamoto, T.; Morita, E.; Abe, T.; Mori, T.; Matsuura, Y.; Mizuguchi, K. Understanding the biological context of NS5A-host interactions in HCV infection: a network-based approach. J. Proteome Res. 2013, 12, 2537−2551. (114) Giménez-Barcons, M.; Franco, S.; Suárez, Y.; Forns, X.; Ampurdanès, S.; Puig-Basagoiti, F.; Sánchez-Fueyo, A.; Barrera, J. M.; Llovet, J. M.; Bruix, J.; Sánchez-Tapias, J. M.; Rodés, J.; Saiz, J. C. High amino acid variability within the NS5A of hepatitis C virus (HCV) is associated with hepatocellular carcinoma in patients with HCV-1brelated cirrhosis. Hepatology 2001, 34, 158−167. (115) Anzola, M.; Burgos, J. J. Hepatocellular carcinoma: molecular interactions between hepatitis C virus and p53 in hepatocarcinogenesis. Expert Rev. Mol. Med. 2003, 5, 1−16. (116) Enomoto, N.; Sakuma, I.; Asahina, Y.; Kurosaki, M.; Murakami, T.; Yamamoto, C.; Izumi, N.; Marumo, F.; Sato, C. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J. Clin. Invest. 1995, 96, 224−230. 1668



Perspective

(117) Hofmann, W. P.; Zeuzem, S.; Sarrazin, C. Hepatitis C virusrelated resistance mechanisms to interferon alpha-based antiviral therapy. J. Clin. Virol. 2005, 32, 86−91. (118) Tsai, Y. H.; Kuang, W. F.; Lu, T. Y.; Kao, J. H.; Lai, M. Y.; Liu, C. J.; Chen, P. J.; Hwang, L. H. The non-structural 5A protein of hepatitis C virus exhibits genotypic differences in interferon antagonism. J. Hepatol. 2008, 49, 899−907. (119) El-Shamy, A.; Nagano-Fujii, M.; Sasase, N.; Imoto, S.; Kim, S. R.; Hotta, H. Sequence variation in hepatitis C virus nonstructural protein 5A predicts clinical outcome of pegylated interferon/ribavirin combination therapy. Hepatology 2008, 48, 38−47. (120) Ullah, S.; Rehman, H. U.; Idrees, M. Mutations in the NS5A gene are associated with response to interferon + ribavirin combination therapy in patients with chronic hepatitis C virus 3a infection. Eur. J. Gastroenterol. Hepatol. 2013, 25, 1146−1151. (121) Tan, S. L.; Katze, M. G. How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A. Virology 2001, 284, 1−12. (122) Brillet, R.; Penin, F.; Hezode, C.; Chouteau, P.; Dhumeaux, D.; Pawlotsky, J. M. The nonstructural 5A protein of hepatitis C virus genotype 1b does not contain an interferon sensitivity-determining region. J. Infect. Dis. 2007, 195, 432−441. (123) Gale, M. J., Jr.; Korth, M. J.; Tang, N. M.; Tan, S.-L.; Hopkins, D. A.; Dever, T. E.; Polyak, S. J.; Gretch, D. R.; Katze, M. G. Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 1997, 230, 217−227. (124) Gale, M., Jr.; Blakely, C. M.; Kwieciszewski, B.; Tan, S.-L.; Dossett, M.; Tang, N. M.; Korth, M. J.; Polyak, S. J.; Gretch, D. R.; Katze, M. G. Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation. Mol. Cell. Biol. 1998, 18, 5208−5218. (125) Kim, J.; Lee, D.; Choe, J. Hepatitis C virus NS5A protein is phosphorylated by casein kinase II. Biochem. Biophys. Res. Commun. 1999, 257, 777−781. (126) Tellinghuisen, T. L.; Foss, K. L.; Treadaway, J. C.; Rice, C. M. Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein. J. Virol. 2008, 82, 1073− 1083. (127) Arima, N.; Kao, C. Y.; Licht, T.; Padmanabhan, R.; Sasaguri, Y.; Padmanabhan, R. Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A. J. Biol. Chem. 2001, 276, 12675−12684. (128) De Chassey, B.; Navratil, V.; Tafforeau, L.; Hiet, M. S.; AublinGex, A.; Agaugue, S.; Meiffren, G.; Pradezynski, F.; Faria, B. F.; Chantier, T.; Le Breton, M.; Pellet, J.; Davoust, N.; Mangeot, P. E.; Chaboud, A.; Penin, F.; Jacob, Y.; Vidalain, P. O.; Vidal, M.; André, P.; Rabourdin-Combe, C.; Lotteau, V. Hepatitis C virus infection protein network. Mol. Syst. Biol. 2008, 4, 230. (129) Hamamoto, I.; Nishimura, Y.; Okamoto, T.; Aizaki, H.; Liu, M.; Mori, Y.; Abe, T.; Suzuki, T.; Lai, M. M.; Miyamura, T.; Moriishi, K.; Matsuura, Y. Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B. J. Virol. 2005, 79, 13473−13482. (130) Hanoulle, X.; Badillo, A.; Wieruszeski, J. M.; Verdegem, D.; Landrieu, I.; Bartenschlager, R.; Penin, F.; Lippens, G. Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B. J. Biol. Chem. 2009, 284, 13589−13601. (131) Foster, T. L.; Gallay, P.; Stonehouse, N. J.; Harris, M. Cyclophilin A interacts with domain II of hepatitis C virus NS5A and stimulates RNA binding in an isomerase-dependent manner. J. Virol. 2011, 85, 7460−7464. (132) Campas, C.; Castaner, R. DEBIO-025: cyclophilin inhibitor treatment of HCV infection. Drugs Future 2008, 33, 1012−1017. (133) Watashi, K. Alisporivir, a cyclosporin derivative that selectively inhibits cyclophilin, for the treatment of HCV infection. Curr. Opin. Invest. Drugs 2010, 11, 213−224. (134) Gallay, P. A.; Lin, K. Profile of alisporivir and its potential in the treatment of hepatitis C. Drug Des. Dev. Ther. 2013, 7, 105−115.

(135) Flisiak, R.; Dumont, J. M.; Crabbé, R. Cyclophilin inhibitors in hepatitis C viral infection. Expert Opin. Invest. Drugs 2007, 16, 1345− 1354. (136) Gallay, P. A. Cyclophilin inhibitors: a novel class of promising host-targeting anti-HCV agents. Immunol. Res. 2012, 52, 200−210. (137) Lin, K.; Gallay, P. Curing a viral infection by targeting the host: the example of cyclophilin inhibitors. Antiviral Res. 2013, 99, 68−77. (138) Peel, M.; Scribner, A. Cyclophilin inhibitors as antiviral agents. Bioorg. Med. Chem. Lett. 2013, 23, 4485−4492. (139) Randall, G.; Panis, M.; Cooper, J. D.; Tellinghuisen, T. L.; Sukhodolets, K. E.; Pfeffer, S.; Landthaler, M.; Landgraf, P.; Kan, S.; Lindenbach, B. D.; Chien, M.; Weir, D. B.; Russo, J. J.; Ju, J.; Brownstein, M. J.; Sheridan, R.; Sander, C.; Zavolan, M.; Tuschl, T.; Rice, C. M. Cellular cofactors affecting hepatitis C virus infection and replication. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12884−12889. (140) Hepatitis C virus protein interaction database. http://apps. sanbi.ac.za/hcvpro/Search.php (accessed January 12, 2014). (141) Pichlmair, A.; Kandasamy, K.; Alvisi, G.; Mulhern, O.; Sacco, R.; Habjan, M.; Binder, M.; Stefanovic, A.; Eberle, C.-A.; Goncalves, A.; Bürckstümmer, T.; C. Müller, A.; Fauster, A.; Holze, C.; Lindsten, K.; Goodbourn, S.; Kochs, G.; Weber, F.; Bartenschlager, R.; Bowie, A. G.; Bennett, K. L.; Colinge, J.; Superti-Furga, G. Viral immune modulators perturb the human molecular network by common and unique strategies. Nature 2012, 487, 486−490. (142) Dolan, P. T.; Zhang, C.; Khadka, S.; Arumugaswami, V.; Vangeloff, A. D.; Heaton, N. S.; Sahasrabudhe, S.; Randall, G.; Sun, R.; LaCount, D. J. Identification and comparative analysis of hepatitis C virus−host cell protein interactions. Mol. BioSyst. 2013, 9, 3199−3209. (143) Tellinghuisen, T. L.; Marcotrigiano, J.; Rice, C. M. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 2005, 435, 374−379. (144) Love, R. A.; Brodsky, O.; Hickey, M. J.; Wells, P. A.; Cronin, C. N. Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus. J. Virol. 2009, 83, 4395−4403. (145) Dimitrova, M.; Imbert, I.; Kieny, M. P.; Schuster, C. Protein− protein interactions between hepatitis C virus nonstructural proteins. J. Virol. 2003, 77, 5401−5414. (146) Lim, P. J.; Chatterju, U.; Cordek, D.; Sharma, S. D.; GarciaRivera, J. A.; Cameron, C. E.; Lin, K.; Targett-Adams, P.; Gallay, P. A. Correlation between NS5A dimerization and HCV replication. J. Biol. Chem. 2012, 287, 30861−30873. (147) Amblard, F.; Zhang, H.; Zhou, L.; Shi, J.; Bobeck, D. R.; Nettles, J. H.; Chavre, S.; McBrayer, T. R.; Tharnish, P.; Whitaker, T.; Coats, S. J.; Schinazi, R. F. Synthesis and evaluation of non-dimeric HCV NS5A inhibitors. Bioorg. Med. Chem. Lett. 2013, 23, 2031−2034. (148) Fridell, R. A.; Qiu, D.; Wang, C.; Valera, L.; Gao, M. Resistance analysis of the hepatitis C virus NS5A inhibitor BMS-790052 in an in vitro replicon system. Antimicrob. Agents Chemother. 2010, 54, 3641− 3650. (149) Wang, C.; Huang, H.; Valera, L.; Sun, J.-H.; O’Boyle, D. R.; Nower, P. T.; Jia, L.; Qiu, D.; Huang, X.; Altaf, A.; Gao, M.; Fridell, R. A. Hepatitis C virus RNA elimination and development of resistance in replicon cells treated with BMS-790052. Antimicrob. Agents Chemother. 2012, 56, 1350−1358. (150) Fridell, R. A.; Qiu, D.; Valera, L.; Wang, C.; Rose, R. E.; Gao, M. Distinct functions of NS5A in hepatitis C virus RNA replication uncovered by studies with the NS5A inhibitor BMS-790052. J. Virol. 2011, 85, 7312−7320. (151) Lemm, J. A.; O’Boyle, D., II; Liu, M.; Nower, P. T.; Colonno, R.; Deshpande, M. S.; Snyder, L. B.; Martin, S. W.; St. Laurent, D. R.; Serrano-Wu, M. H.; Romine, J. L.; Meanwell, N. A.; Gao, M. Identification of hepatitis C virus NS5A inhibitors. J. Virol. 2010, 84, 482−491. (152) Lee, C.; Ma, H.; Hang, J. Q.; Leveque, V.; Sklan, E. H.; Elazar, M.; Klumpp, K.; Glenn, J. S. The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A nonstructural viral protein. Virology 2011, 414, 10−18. (153) Qiu, D.; Lemm, J. A.; O’Boyle, D. R.; Sun, J.-H.; Nower, P. T.; Nguyen, V.; Hamann, L. G.; Snyder, L. B.; Deon, D. H.; Ruediger, E.; 1669



Perspective

Meanwell, N. A.; Belema, M.; Gao, M.; Fridell, R. A. The effects of NS5A inhibitors on NS5A phosphorylation, polyprotein processing and localization. J. Gen. Virol. 2011, 92, 2502−2511. (154) Quintavalle, M.; Sambucini, S.; Di Pietro, C.; De Francesco, R.; Neddermann, P. The α-isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation. J. Virol. 2006, 80, 11305−11312. (155) Quintavalle, M.; Sambucini, S.; Summa, V.; Orsatti, L.; Talamo, F.; De Francesco, R.; Neddermann, P. Hepatitis C virus NS5A is a direct substrate of casein kinase Iα, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors. J. Biol. Chem. 2007, 282, 5536−5544. (156) Guedj, J.; Dahari, H.; Rong, L.; Sansone, N. D.; Nettles, R. E.; Cotler, S. J.; Layden, T. J.; Uprichard, S. L.; Perelson, A. S. Modeling shows that the NS5A inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis C virus half-life. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 3991−3996. (157) Tellinghuisen, T. L.; Foss, K. L.; Treadaway, J. Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS Pathog. 2008, 4, e1000032. (158) Hughes, M.; Griffin, S.; Harris, M. Domain III of NS5A contributes to both RNA replication and assembly of hepatitis C virus particles. J. Gen. Virol 2009, 90, 1329−1334. (159) Pockros, P. J. Interferon-free hepatitis C therapy: How close are we? Drugs 2012, 72, 1825−1831. (160) Doyle, J. S.; Aspinall, E.; Liew, D.; Thompson, A. J.; Hellard, M. E. Current and emerging antiviral treatments for hepatitis C infection. Br. J. Clin. Pharmacol. 2013, 75, 931−943. (161) Assis, D. N.; Lim, J. K. New pharmacotherapy for hepatitis C. Clin. Pharmacol. Ther. 2012, 92, 294−305. (162) Rong, L.; Guedj, J.; Dahari, H.; Coffield, D. J., Jr.; Levi, M.; Smith, P.; Perelson, A. S. Analysis of hepatitis C virus decline during treatment with the protease inhibitor danoprevir using a multiscale model. PLoS Comput. Biol. 2013, 9 (3), e1002959. (163) Guedj, J.; Dahari, H.; Shudo, E.; Smith, P.; Perelson, A. S. Hepatitis C viral kinetics with the nucleoside polymerase inhibitor mericitabine (RG7128). Hepatology 2012, 55, 1030−1037. (164) Neumann, A. U.; Lam, N. P.; Dahari, H.; Gretch, D. R.; Wiley, T. E.; Layden, T. J.; Perelson, A. S. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-α therapy. Science 1998, 282, 103−107. (165) Perelson, A. S.; Herrmann, E.; Micol, F.; Zeuzem, S. New kinetic models for the hepatitis C virus. Hepatology 2005, 42, 749− 754. (166) Shudo, E.; Ribeiro, R. M.; Perelson, A. S. Modeling HCV kinetics under therapy using PK and PD information. Expert Opin. Drug Metab. Toxicol. 2009, 5, 321−332. (167) Rong, L.; Perelson, A. S. Treatment of hepatitis C virus infection with interferon and small molecule direct antivirals: viral kinetics and modeling. Crit. Rev. Immunol. 2010, 30, 131−148. (168) Ribeiro, R. M.; Li, H.; Wang, S.; Stoddard, M. B.; Learn, G. H.; Korber, B. T.; Bhattacharya, T.; Guedj, J.; Parrish, E. H.; Hahn, B. H.; Shaw, G. M.; Perelson, A. S. Quantifying the diversification of hepatitis C virus (HCV) during primary infection: estimates of the in vivo mutation rate. PLoS Pathog. 2012, 8 (8), e1002881. (169) Chatterjee, A.; Guedj, J.; Perelson, A. S. Mathematical modeling of HCV infection: What can it teach us in the era of direct-acting antiviral agents? Antiviral Ther. 2012, 17, 1171−1182. (170) Sarrazin, C.; Kieffer, T. L.; Bartels, D.; Hanzelka, B.; Müh, U.; Welker, M.; Wincheringer, D.; Zhou, Y.; Chu, H.-M.; Lin, C.; Weegink, C.; Reesink, H.; Zeuzem, S.; Kwong, A. D. Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir. Gastroenterology 2007, 132, 1767−1777. (171) Rong, L.; Dahari, H.; Ribeiro, R. M.; Perelson, A. S. Rapid emergence of protease inhibitor resistance in hepatitis C virus. Sci. Transl. Med. 2010, 2, 1−9. (172) Susser, S.; Welsch, C.; Wang, Y.; Zettler, M.; Domingues, F. S.; Karey, U.; Hughes, E.; Ralston, R.; Tong, X.; Herrmann, E.; Zeuzem,

S.; Sarrazin, C. Characterization of resistance to the protease inhibitor boceprevir in hepatitis C virus-infected patients. Hepatology 2009, 50, 1709−1718. (173) Nettles, R. E.; Gao, M.; Bifano, M.; Chung, E.; Persson, A.; Marbury, T. C.; Goldwater, R.; DeMicco, M. P.; Rodriguez-Torres, M.; Vutikullird, A.; Fuentes, E.; Lawitz, E.; Lopez-Talavera, J. C.; Grasela, D. M. Multiple ascending dose study of BMS-790052, a nonstructural protein 5A replication complex inhibitor, in patients infected with hepatitis C virus genotype 1. Hepatology 2011, 54, 1956−1965. (174) Fridell, R. A.; Wang, C.; Sun, J.-H.; O’Boyle, D. R., II; Nower, P.; Valera, L.; Qiu, D.; Roberts, S.; Huang, X.; Kienzle, B.; Bifano, M.; Nettles, R. E.; Gao, M. Genotypic and phenotypic analysis of variants resistant to hepatitis C virus nonstructural protein 5A replication complex inhibitor BMS-790052 in humans: in vitro and in vivo correlations. Hepatology 2011, 54, 1924−1935. (175) Sun, J.-H.; O’Boyle, D. R., II; Zhang, Y.; Wang, C.; Nower, P.; Valera, L.; Roberts, S.; Nettles, R. E.; Fridell, R. A.; Gao, M. Impact of a baseline polymorphism on the emergence of resistance to the hepatitis C virus nonstructural protein 5A replication complex inhibitor, BMS-790052. Hepatology 2012, 55, 1692−1699. (176) Wang, C.; Sun, J.-H.; O’Boyle, D. R., II; Nower, P.; Valera, L.; Roberts, S.; Fridell, R. A.; Gao, M. Persistence of resistant variants in hepatitis C virus-infected patients treated with the NS5A replication complex inhibitor daclatasvir. Antimicrob. Agents Chemother. 2013, 57, 2054−2065. (177) Plaza, Z.; Soriano, V.; Vispo, E.; Del Mar Gonzalez, M.; Barreiro, P.; Seclen, E.; Poveda, E. Prevalence of natural polymorphisms at the HCV NS5A gene associated with resistance to daclatasvir, an NS5A inhibitor. Antiviral Ther. 2012, 17, 921−926. (178) Suzuki, F.; Sezaki, H.; Akuta, N.; Suzuki, Y.; Seko, Y.; Kawamura, Y.; Hosaka, T.; Kobayashi, M.; Saito, S.; Arase, Y.; Ikeda, K.; Kobayashi, M.; Mineta, R.; Watahiki, S.; Miyakawa, Y.; Kumada, H. Prevalence of hepatitis C virus variants resistant to NS3 protease inhibitors or the NS5A inhibitor (BMS-790052) in hepatitis patients with genotype 1b. J. Clin. Virol. 2012, 54, 352−354. (179) Gelman, M. A.; Glenn, J. S. Mixing the right hepatitis C inhibitor cocktail. Trends Mol. Med. 2010, 17, 34−46. (180) Rustgi, V. K. Safety of small molecules in combination with interferon-based therapy for hepatitis C virus. Expert Opin. Drug Safety 2010, 9, 883−892. (181) Kieffer, T. L.; Kwong, A. D.; Picchio, G .R. Viral resistance to specifically targeted antiviral therapies for hepatitis C (STAT-Cs). J. Antimicrob. Chemother. 2010, 65, 202−212. (182) Halfon, P.; Sarrazin, C. Future treatment of chronic hepatitis C with direct acting antivirals: is resistance important? Liver Int. 2012, 32 (Suppl. 1), 79−87. (183) Ferenci, P. Treatment of chronic hepatitis CAre interferons really necessary? Liver Int. 2012, 32 (Suppl. 1), 108−112. (184) Pol, S.; Ghalib, R. H.; Rustgi, V. K.; Martorell, C.; Everson, G. T.; Tatum, H. A.; Hézode, C.; Lim, J. K.; Bronowicki, J.-P.; Abrams, G. A.; Bräu, N.; Morris, D. W.; Thuluvath, P. J.; Reindollar, R. W.; Yin, P. D.; Diva, U.; Hindes, R.; McPhee, F.; Hernandez, D.; Wind-Rotolo, M.; Hughes, E.; Schnittman, S. Daclatasvir for previously untreated chronic hepatitis C genotype-1 infection: a randomised, parallel-group, double-blind, placebo-controlled, dose-finding, phase 2a trial. Lancet Infect. Dis. 2012, 12, 671−677. (185) Jacobson, I.; Dimitrova, D.; Hughes, E. A.; Yin, P. D.; Mendez, P.; Yogendran, L.; Schnittman, S. Safety profile of daclatasvir in combination with peginterferon alfa and ribavirin in 1100 patients with chronic HCV infection treated in phase 2 studies. J. Hepatol. 2013, 58 (Suppl. 1), S489 (Abstract 1203). (186) Hézode, C.; Hirschfield, G. M.; Ghesquiere, W.; Sievert, W.; Rodriguez-Torres, M.; Shafran, S. D.; Thuluvath, P. J.; Tatum, H. A.; Waked, I.; Esmat, G. E.; Lawitz, E.; Rustgi, V. K.; Pol, S.; Weis, N.; Pockros, P.; Bourliere, M.; Serfaty, L.; Vierling, J. M.; Fried, M. W.; Weiland, O.; Brunetto, M. R.; Everson, G. T.; Zeuzem, S.; Kwo, P. Y.; Sulkowski, M. S.; Brau, N.; Wind-Rotolo, M.; Liu, Z.; Hughes, E. A.; Schnittman, S. M.; Yin, P. D. Daclatasvir, an NS5A replication complex inhibitor, combined with peginterferon alfa-2a and ribavirin in 1670



Perspective

treatment-naive HCV-genotype 1 or 4 subjects: phase 2b COMMAND-1 SVR12 results. Hepatology 2012, 56 (Suppl. 4), 553A−554A (Abstract 755). (187) Dore, G. J.; Lawitz, E.; Hézode, C.; Shafran, S. D.; Ramji, A.; Tatum, H. A.; Taliani, G.; Tran, A.; Brunetto, M. R.; Zaltron, S.; Strasser, S. I.; Weis, N.; Ghesquiere, W.; Lee, S. S.; Larrey, D. G.; Pol, S.; Harley, H.; George, J.; Fung, S.; de Ledinghen, V.; Hagens, P.; Cohen, D. S.; Hughes, E. A.; Cooney, E. L. Twelve- or 16-week treatment with daclatasvir combined with peginterferon alfa and ribavirin for hepatitis C virus genotype 2 or 3 infection: Command GT2/3 study. Hepatology 2012, 56 (Suppl. 4), 558A−559A (Abstract 762). (188) Herbst, D. A.; Reddy, K. R. NS5A inhibitor, daclatasvir, for the treatment of chronic hepatitis C virus infection. Expert Opin. Invest. Drugs 2013, 22, 1337−1346. (189) Lok, A. S.; Gardiner, D. F.; Lawitz, E.; Martorell, C.; Everson, G. T.; Ghalib, R.; Reindollar, R.; Rustgi, V.; McPhee, F.; Wind-Rotolo, M.; Persson, A.; Zhu, K.; Dimitrova, D. I.; Eley, T.; Guo, T.; Grasela, D. M.; Pasquinelli, C. Preliminary study of two antiviral agents for hepatitis C genotype 1. N. Engl. J. Med. 2012, 366, 216−224. (190) Chung, R. T. A watershed moment in the treatment of hepatitis C. N. Engl. J. Med. 2012, 366, 273−275. (191) Hayashi, K.; Katano, Y.; Kuzuya, T.; Tachi, Y.; Honda, T.; Ishigami, M.; Itoh, A.; Hirooka, Y.; Ishikawa, T.; Nakano, I.; Urano, F.; Yoshioka, K.; Toyoda, H.; Kumada, T.; Goto, H. Prevalence of hepatitis C virus genotype 1a in Japan and correlation of mutations in the NS5A region and single-nucleotide polymorphism of interleukin28B with the response to combination therapy with pegylatedinterferon-alpha 2b and ribavirin. J. Med. Virol. 2012, 84, 438−444. (192) Pellicelli, A. M.; Romano, M.; Stroffolini, T.; Mazzoni, E.; Mecenate, F.; Monarca, R.; Picardi, A.; Bonaventura, M. E.; Mastropietro, C.; Vignally, P.; Andreoli, A.; Marignani, M.; D’Ambrosio, C.; Miglioresi, L.; Nosotti, L.; Mitidieri, O.; Gentilucci, U. V.; Puoti, C.; Barbaro, G.; Barlattani, A.; Furlan, C.; Barbarini, G.; CLEO Group. HCV genotype 1a shows a better virological response to antiviral therapy than HCV genotype 1b. BMC Gastroenterol. 2012, 12, 162. (193) Takada, N.; Takase, S.; Takada, A.; Date, T. Differences in the hepatitis C virus genotypes in different countries. J. Hepatol. 1993, 17, 277−283. (194) Zein, N. N. Clinical significance of hepatitis C virus genotypes. Clin. Microbiol. Rev. 2000, 13, 223−235. (195) Chayama, K.; Takahashi, S.; Toyota, J.; Karino, Y.; Ikeda, K.; Ishikawa, H.; Watanabe, H.; McPhee, F.; Hughes, E.; Kumada, H. Dual therapy with the nonstructural protein 5A inhibitor, daclatasvir, and the nonstructural protein 3 protease inhibitor, asunaprevir, in hepatitis C virus genotype 1b-infected null responders. Hepatology 2012, 55, 742−748. (196) Suzuki, Y.; Ikeda, K.; Suzuki, F.; Toyota, J.; Karino, Y.; Chayama, K.; Kawakami, Y.; Ishikawa, H.; Watanabe, J.; Hu, W.; Eley, T.; McPhee, F.; Hughes, E.; Kumada, H. Dual oral therapy with daclatasvir and asunaprevir for patients with HCV genotype 1b infection and limited treatment options. J. Hepatol. 2013, 58, 655−662. (197) Karino, Y.; Toyota, J.; Ikeda, K.; Suzuki, F.; Chayama, K.; Kawakami, Y.; Ishikawa, H.; Watanabe, H.; Hernandez, D.; Yu, F.; McPhee, F.; Kumada, H. Characterization of virologic escape in hepatitis C virus genotype 1b patients treated with the direct-acting antivirals daclatasvir and asunaprevir. J. Hepatol. 2013, 58, 646−654. (198) Londoño, M.-C.; Lens, S.; Forms, X. Interferon free regimens for the “difficult-to-treat”: Are we there? J. Hepatol. 2013, 58, 643− 645. (199) (a) Sulkowski, M. S.; Gardiner, D. F.; Rodriguez-Torres, M.; Reddy, K. R.; Hassanein, T.; Jacobson, I. M.; Lawitz, E.; Lok, A. S.; Hinestrosa, F.; Thuluvath, P. J.; Schwartz, H.; Nelson, D. R.; Eley, T.; Wind-Rotolo, M.; Huang, S.-P.; Gao, M.; McPhee, F.; Sherman, D.; Hindes, R.; Symonds, W. T.; Pasquinelli, C.; Grasela, D. M. 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 genotype 1, 2, or 3. Presented at the 63rd Annual Meeting of the American Association for the Study of Liver Diseases, Boston, MA, November 9−13, 2012; Abstract LB-2. (b) Sulkowski, M. S.; Gardiner, D. F.; Rodriguez-Torres, M. K.; Reddy, K. R.; Hassanein, T.; Jacobson, I.; Lawitz, E.; Lok, A. S.; Hinestrosa, F.; Thuluvath, P. J.; Schwartz, H.; Nelson, D. R.; Everson, G. T.; Eley, T.; Wind-Rotolo, M.; Huang, S.-P.; Gao, M.; Hernandez, D.; McPhee, F.; Sherman, D.; Hindes, R.; Symonds, W.; Pasquinelli, C.; Grasela, D. M. Daclatasvir plus sofosbuvir for previously treated or untreated chronic HCV infection. N. Engl. J. Med. 2014, 370, 211−221. (200) Anonymous. Daclatasvir plus sofosbuvir with or without ribavirin yields 100% SVR24 rate in genotype 1 patients who fail telaprevir or boceprevir. Gastroenterol. Hepatol. 2013, 9 (Suppl. 3), 14−15. (201) Sulkowski, M. S.; Gardiner, D. F.; Rodriguez-Torres, M.; Reddy, K. R.; Hassanein, T.; Jacobson, I.; Lawitz, E.; Lok, A. S.; Hinestrosa, F.; Thuluvath, P. J.; Schwartz, H.; Nelson, D. R.; Everson, G. T.; Eley, T.; Wind-Rotolo, M.; Huang, S.-P.; Gao, M.; McPhee, F.; Hernandez, D.; Sherman, D.; Hindes, R.; Symonds, W.; Pasquinelli, C.; Grasela, D. M.; AI444040 Study Group. Sustained virologic response with daclatasvir plus sofosbuvir ± ribavirin (RBV) in chronic HCV genotype (gt) 1-infected patients who previously failed telaprevir (TVR) or boceprevir (BOC). J. Hepatol. 2013, 58 (Suppl. 1), S570 (Abstract 1417). (202) Fontana, R. J.; Bifano, M.; Hindes, R.; Symonds, W. T.; Dimitrova, D. First ever successful use of daclatasvir and GS-7977, an interferon-free oral regimen, in a liver transplant recipient with severe recurrent hepatitis C. Hepatology 2012, 56, 524A (Abstract 694). (203) Everson, G. T.; Sims, K. D.; Rodriguez-Torres, M.; Hézode, C.; Lawitz, E.; Bourlière, M.; Loustaud-Ratti, V.; Rustgi, V.; Schwartz, H.; Tatum, H.; Marcellin, P.; Pol, S.; Thuluvath, P. J.; Eley, T.; Wang, X.; Huang, S.-P.; McPhee, F.; Wind-Rotolo, M.; Chung, E.; Pasquinelli, C.; Grasela, D. M.; Gardiner, D. F. Interim analysis of an interferon (IFN)- and ribavirin (RBV)-free regimen of daclatasvir (DCV), asunaprevir (ASV), and BMS-791325 in treatment-naive, hepatitis C virus genotype 1-infected patients. J. Hepatol. 2013, 58 (Suppl. 1), S573 (Abstract 1423). (204) Everson, G. T.; Sims, K. D.; Rodriguez-Torres, M.; Hézode, C.; Lawitz, E.; Bourlière, M.; Loustaud-Ratti, V.; Rustgi, V.; Schwartz, H.; Tatum, H.; Marcellin, P.; Pol, S.; Thuluvath, P. J.; Eley, T.; Wang, X.; Huang, S.-P.; McPhee, F.; Wind-Rotolo, M.; Chung, E.; Pasquinelli, C.; Grasela, D. M.; Gardiner, D. F. Efficacy of an interferon- and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 in treatment-naive patients with HCV genotype 1 infection. Gastroenterology 2014, 146, 420−429. (205) Lawitz, E. J.; Gruener, D.; Hill, J. M.; Marbury, T.; Moorehead, L.; Mathias, A.; Cheng, G.; Link, J. O.; Wong, K. A.; Mo, H.; McHutchison, J. G.; Brainard, D. M. A phase 1, randomized, placebocontrolled, 3-day, dose-ranging study of GS-5885, an NS5A inhibitor, in patients with genotype 1 hepatitis C. J. Hepatol. 2012, 57, 24−31. (206) Wong, K. A.; Worth, A.; Martin, R.; Svarovskaia, E.; Brainard, D. M.; Lawitz, E.; Miller, M. D.; Mo, H. Characterization of hepatitis C virus resistance from a multiple dose clinical trial of the novel NS5A inhibitor GS-5885. Antimicrob. Agents Chemother. 2013, 57, 6333− 6340. (207) (a) Gane, E. J.; Stedman, C. A.; Hyland, R. H.; Ding, X.; Pang, P. S.; Symonds, W. T.; McHutchison, J. G. ELECTRON: all-oral sofosbuvir-based 12-week regimens for the treatment of chronic HCV GT 1 infection. J. Hepatol. 2013, 58 (Suppl. 1), S6−S7 (Abstract 14). (b) Lawitz, E.; Poordad, F. F.; Pang, P. S.; Hyland. R. H.; Ding, X.; Mo, H.; Symonds, W. T.; McHutchison, J. G.; Membreno, F. Sofosbuvir and ledipasvir fixed-dose combination with and without ribavirin in treatment-naive and previously treated patients with genotype 1 hepatitis C virus infection (LONESTAR): an open label, randomised, phase 2 trial. Lancet 2013, DOI: 10.1016/S01406736(13)62121-2. (208) Everson, G. T.; Lawitz, E.; Thompson, A. J.; Sulkowski, M. S.; Zhu, Y.; Brainard, D. M.; Mendelson, L.; McHutchison, J. G.; Pang, P. S.; Yang, J. C.; Marcellin, P.; Afdhal, N. The NS5A inhibitor GS-5885 1671



Perspective

is safe and well-tolerated in over 1000 patients treated in phase 2 studies. Hepatology 2012, 56, 572A (Abstract 783). (209) Wilfret, J.; Walker, K.; Adkison, L.; Jones, Y.; Lou, J.; Gan, S.; Castellino, C.; Moseley, J.; Horton, M.; de Serres, A.; Culp, I.; Goljer, W.; Spreen, W. Safety, tolerability, pharmacokinetics, and antiviral activity of GSK2336805, an inhibitor of hepatitis C virus NS5A, in healthy subjects and subjects chronically infected with genotype 1 hepatitis C virus. Antimicrob. Agents Chemother. 2013, 57, 5037−5044. (210) Wilfret, D.; Adkison, K. K.; Jones, L.; Lou, Y.; Gan, J.; Spreen, W. An echocardiogram study to evaluate the effect of the novel hepatitis C virus NS5A inhibitor GSK2336805 on cardiac contractility in healthy subjects. Antimicrob. Agents Chemother. 2013, 57, 5141− 5143. (211) Gardner, S.; Cutrell, A.; Elko-Simms, C.; Adkison, K.; Hamatake, R.; Walker, J.; Rodriguez-Torres, M.; Hong, Z. A doubleblind, randomized, placebo-controlled study to assess the safety, antiviral activity, and pharmacokinetics of GSK2336805 when given as monotherapy and in combination with peginterferon alfa-2a and ribavirin in hepatitis C virus genotype 1-infected treatment-naive subjects. Liver Int. 2013, DOI: 10.1111/liv.12334. (212) (a) Zhou, X. J.; Vince, B.; Dubuc Patrick, G.; Chen, J.; Pietropaolo, K.; Sullivan-Bólyai, J. Z. Mayers, D. Safety, pharmacokinetic and preliminary antiviral activity results from a first-in-human study of IDX719, a pan-genotypic novel HCV NS5A inhibitor. Presented at the 7th International Workshop on Clinical Pharmacology of Hepatitis Therapy, June 27, 2012. Available at http://www. idenix.com/hcv/IDX719_HCVClinPharmMtg_ FINAL%206%2027%2012.pdf (accessed January 12, 2014). (b) Vince, B.; Hill, J. M.; Lawitz, E. J.; O’Riordan, W.; Webster, L. R.; Gruener, D. M.; Mofsen, R. S.; Murillo, A.; Donovan, E.; Chen, J.; McCarville, J. F.; Sullivan-Bólyai, J. Z.; Mayers, D.; Zhou, X.-J. A randomized, doubleblind, multiple-dose study of the pan-genotypic NS5A inhibitor samatasvir in patients infected with hepatitis C virus genotype 1, 2, 3 or 4. J. Hepatol. 2014, in press, DOI: 10.1016/j.jhep.2014.01.003. (213) Mayers, D. L.; Vince, B.; Hill, J. M.; Lawitz, E.; O’Riordan, W. D.; Webster, L. R.; Gruener, D.; Mofsen, R. S.; Murillo, A.; Donovan, E. F.; Chen, J.; Sullivan-Bolyai, J.; Zhou, X.-J. IDX719, HCV NS5A inhibitor, demonstrates pan-genotypic activity after three days of monotherapy in genotype 1, 2, 3 or 4 HCV-infected subjects. Hepatology 2012, 56, 1020A (Abstract 1174). (214) Dousson, C. B. Next generation HCV NS5A inhibitor: in vitro antiviral optimization for pan-genotypic activity and preclinical profile. Presented at the 26th International Conference on Antiviral Research, San Francisco, CA, May 11−15, 2013. Available at http://www.idenix. com/hcv/ Idenix%20NS5A%20Presentation%20ICAR%20May%202013%20FI NAL.pdf (accessed January 12, 2014). (215) Dumas, E. O.; Lawal, A.; Menon, R. M.; Podsadecki, T.; Awni, W.; Dutta, S.; Williams, L. Pharmacokinetics, safety and tolerability of the HCV NS5A inhibitor ABT-267 following single and multiple doses in healthy adult volunteers. J. Hepatol. 2011, 54 (Suppl.1), S475−S476. (216) Lawitz, E.; Marbury, T.; Campbell, A.; Dumas, E.; Pilot-Matias, T.; Krishnan, P.; Setze, C.; Xie, W.; Podsadecki, T.; Bernstein, B.; Williams, L. Safety and antiviral activity of ABT-267, a novel NS5A inhibitor, during 3-day monotherapy: first study in HCV genotype-1 ̈ subjects. J. Hepatol. 2012, 56 (Suppl. (GT1)-infected treatment-naive 2), S469−S470. (217) Kowdley, K. V.; Lawitz, E.; Poordad, F.; Cohen, D. E.; Nelson, D. R.; Zeuzem, S.; Everson, G. T.; Kwo, P.; Foster, G. R.; Sulkowski, M.; Xie, W.; Pilot-Matias, T.; Liossis, G.; Larsen, L.; Khatri, A.; Podsadecki, T.; Bernstein, B. 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 naive patients and 93% in prior null responders with HCV genotype 1 infection. Hepatology 2012, 56 (Suppl. S1), 1515A (Abstract LB-01). (218) Lalezari, J. P.; Farrell, G. C.; Shah, P. S.; Schwab, C.; Walsh, D.; Vig, P.; Brown, N. A.; Ruby, E.; Halfon, S.; Colonno, R.; Li, L.; Johnston, B.; Wargin, W.; Gane, E. J. PPI-668, a potent new pan-

genotypic HCV NS5A inhibitor: phase 1 efficacy and safety. Hepatology 2012, 56 (Suppl. S1), 1065A−1066A (Abstract 1874). (219) Zeuzem, S.; Soriano, V.; Asselah, T.; Bronowicki, J.-P.; Lohse, A. W.; Müllhaupt, B.; Schuchmann, M.; Bourlière, M.; Buti, M.; Roberts, S. K.; Gane, E. J.; Stern, J. O.; Vinisko, R.; Kukolj, G.; Gallivan, J.-P.; Böcher, W. O.; Mensa, F. J. Faldaprevir and deleobuvir for HCV genotype 1 infection. N. Engl. J. Med. 2013, 369, 630−639. (220) Muir, A.; Hill, J.; Lawitz, E.; Marbury, T.; Robarge, L.; Robison, H.; Hui, J.; Huang, M.; Agarwal, A.; Perelson, A.; Deshpande, M.; Kocinsky, H. ACH-3102, a second generation NS5A inhibitor demonstrates potent antiviral activity in patients with genotype 1a infection despite the presence of baseline NS5A-resistant variants. J. Hepatol. 2013, 58 (Suppl.1), S360 (Abstract 876). Available at http:// www.achillion.com/PL/pdf/ACH-3102_Ph1_HCVstudy_EASL2013. pdf (accessed January 12, 2014). (221) Yeh, W. W.; Lipardi, C.; Jumes, P.; De Lepeleire, I. M.; Van Den Bulk, N.; Caro, L.; Huang, X.; Mangin, E.; Nachbar, R. B.; Gane, E. J.; Papa, S.; Ghiavii, N.; Wagner, F. D.; Butterton, J. R. MK-8742, a HCV NS5A inhibitor with a broad spectrum of HCV genotypic activity, demonstrates potent antiviral activity in genotype-1 and -3 HCV-infected patients. Hepatology 2013, 58 (Suppl. S1), 438A−439A (Abstract 479). (222) Liu, R.; Curry, S.; McMonagle, P.; Nachbar, R. B.; Pak, I.; Jumes, P.; Ludmerer, S.; Asante-Appiah, E.; Hazuda, D.; Yeh, W. W.; Howe, A. Y. Resistance analysis of genotype-1 and -3 HCV-infected patients receiving MK-8742, a HCV NS5A inhibitor with potent antiviral activity. Hepatology 2013, 58 (Suppl. S1), 435A (Abstract 472).

1672


Discovery and Development of Hepatitis C Virus NS5A Replication

Recommend Documents