EDITORIALS 4. Hintermann E, Holdener M, Bayer M, et al. Epitope spreading of the anti-CYP2D6 antibody response in patients with autoimmune hepatitis and in the CYP2D6 mouse model. J Autoimmun 2011;37:242–253. 5. de Boer YS, van Gerven NMF, Zwiers A, et al. Genomewide association study identifies variants associated with autoimmune hepatitis type 1. Gastroenterology 2014; 147:443–452. 6. Longhi MS, Liberal R, Holder B, et al. Inhibition of interleukin-17 promotes differentiation of CD25(-) cells into stable T regulatory cells in patients with autoimmune hepatitis. Gastroenterology 2012;142: 1526–1535. 7. Grant CR, Liberal R, Holder BS, et al. Dysfunctional CD39(POS) regulatory T cells and aberrant control of Thelper type 17 cells in autoimmune hepatitis. Hepatology 2014;59:1007–1015. 8. Bonito AJ, Aloman C, Fiel MI, et al. Medullary thymic epithelial cell depletion leads to autoimmune hepatitis. J Clin Invest 2013;123:3510–3524. 9. Cheng MH, Anderson MS. Monogenic autoimmunity. Annu Rev Immunol 2012;30:393–427. 10. Mells GF, Kaser A, Karlsen TH. Novel insights into autoimmune liver diseases provided by genome-wide association studies. J Autoimmun 2013;46:41–54. 11. Hirschfield GM, Liu X, Xu C, et al. Primary biliary cirrhosis associated with HLA, IL12A, and IL12RB2 variants. N Engl J Med 2009;360:2544–2555. 12. Karlsen TH, Franke A, Melum E, et al. Genome-wide association analysis in primary sclerosing cholangitis. Gastroenterology 2010;138:1102–1111. 13. Hindorff LA, Sethupathy P, Junkins HA, et al. Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci U S A 2009;106:9362–9367.

14. Orru V, Steri M, Sole G, et al. Genetic variants regulating immune cell levels in health and disease. Cell 2013; 155:242–256. 15. Liu JZ, Hov JR, Folseraas T, et al. Dense genotyping of immune-related disease regions identifies nine new risk loci for primary sclerosing cholangitis. Nat Genet 2013; 45:670–675. 16. Zhernakova A, van Diemen CC, Wijmenga C. Detecting shared pathogenesis from the shared genetics of immune-related diseases. Nat Rev Genet 2009;10:43–55. 17. Price P, Witt C, Allcock R, et al. The genetic basis for the association of the 8.1 ancestral haplotype (A1, B8, DR3) with multiple immunopathological diseases. Immunol Rev 1999;167:257–274. 18. Sollid LM, Thorsby E. HLA susceptibility genes in celiac disease: genetic mapping and role in pathogenesis. Gastroenterology 1993;105:910–922. 19. Blomhoff A, Olsson M, Johansson S, et al. Linkage disequilibrium and haplotype blocks in the MHC vary in an HLA haplotype specific manner assessed mainly by DRB1*03 and DRB1*04 haplotypes. Genes Immun 2006; 7:130–140. 20. Sollid LM, Jabri B. Triggers and drivers of autoimmunity: lessons from coeliac disease. Nat Rev Immunol 2013; 13:294–302.

Reprint requests Address requests for reprints to: G.M. Hirschfield, Centre for Liver Research, University of Birmingham, Wolfson Drive, Birmingham, B15 2TT, UK. e-mail: g.hirschfi[email protected]. Conflicts of interest The authors disclose no conflicts. © 2014 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2014.06.020

HCV NS5A Inhibitors: The Devil Is in the Details See “Kinetic analyses reveal potent and early blockade of hepatitis C virus assembly by NS5A inhibitors,” by McGivern DR, Masaki T, Williford S, et al, on page 453.

T

he development of anti-hepatitis C virus (HCV) therapy has evolved very rapidly in the last decade. The necessity of developing new antiviral candidates was a result of suboptimal response rates and severe side effects of interferon (IFN)-based therapies. These major drug development efforts and the subsequent clinical successes have been a direct result of advancements in our understanding of the HCV life cycle, which was brought about by the development of the HCV replicon system1,2 and the HCV infectious clone,3,4 which have served as screening tools for anti-HCV drugs. Although early HCV treatment was not target specific (IFN/ribavirin), the first generation of directly acting antivirals (DAAs) approved for the clinic targeted the

viral protease. The protease inhibitors telaprevir and boceprevir were administered in combination with IFN/ribavirin to increase response rates.5,6 Recently, an improved secondgeneration protease inhibitor has been approved (simeprevir),7 as has a nucleotide analog acting as a chain terminator at the viral polymerase’s active site (sofosbuvir), that enable significant further increases in response rates and shorter treatment courses when used in appropriate combination with other agents that may still include IFN for some genotypes.8 Approved regimens for completely IFN-free therapy for most genotypes will be available at the end of 2014 and will include other DAA classes. The latter include molecules that, unlike these approved DAAs, were not developed with a clear HCV viral protein target in mind. Rather, they emerged from random screening efforts against cells harboring HCV replicons. The most potent class of compounds discovered in this way is the so-called NS5A inhibitors (daclatasvir [DCV], ledipasvir, ombitasvir, and MK-8742 are a few examples), with DCV being the first such inhibitor to be discovered.9 Although DCV’s exact mechanism of action (MOA) was not 273

EDITORIALS clear, it showed very high potency, inhibiting HCV replication in the low picomolar concentration (EC50 of 4–20 pM for genotype 1).9 The first clue that NS5A was DCV’s target emerged from analyzing the location of HCV replicon mutations that conferred resistance to DCV, because they were found to map to domain I of NS5A.10,11 Further studies

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indicated that DCV and its derivatives can directly bind NS5A,9,12 strengthening the notion that this viral protein is indeed DCV’s target. Studies into the MOA of DCV suggested that NS5A inhibitors inhibit HCV replication with kinetics similar to that of protease inhibitors and that DCV perturbs the function of new replication complexes13 and prevents

EDITORIALS the proper localization of NS5A into functional HCV replication complexes.14 Further analysis of the activity of NS5A inhibitors using an infectious clone showed a distinct inhibition of virus production at an unknown stage of particle production.15 There remain, however, open questions regarding the exact MOA of this class of drugs. Given this background, McGivern et al16 in this issue of Gastroenterology sought to further address the MOA of the NS5A inhibitors by performing an elaborate quantitative analysis of the kinetics of NS5A inhibitors inhibition compared with those observed with other classes of DAAs, using assays designed to probe specific aspects of the viral life cycle: Viral replication, production of infectious virus and viral polyprotein synthesis, steady-state abundance of viral RNA, virus particle assembly and active/nascent viral RNA synthesis (see McGivern et al, Figures 1, 2, 3, and 4, respectively). They used a wide range of NS5A inhibitor concentrations (ledipasvir, DCV, and MK-8742) that include concentrations within the range of the drugs’ EC50s in a viral system that matches the genotype against which these inhibitors were optimized (genotype 1). Key findings included that compared with inhibitors of the HCV protease and polymerase, NS5A inhibitors were much slower in the onset of RNA replication inhibition (see McGivern et al, Figures 1 and 2), whereas the effect of the drug on viral particle production is rapid and nearly complete within 3 hours (McGivern et al, Figure 2). Moreover, perhaps the most intriguing finding is their observation that the effect on viral particle production is exerted at an early stage of assembly (McGivern et al, Figure 4) and not at a later stage of the particle production process. The effect of NS5A inhibitors on RNA replication is intriguing in that these inhibitors seem to inhibit nascent RNA replication but not ongoing RNA replication. This is most strikingly seen by their observation that, although picomolar concentrations of NS5A inhibitors are sufficient to eradicate RNA replication and polyprotein production by day 3, only modest inhibition of RNA synthesis was observed at earlier time points—even if dramatically higher (eg, micromolar) concentrations of NS5A inhibitors were used (McGivern et al, Figures 1 and 2). The authors concluded that inhibition of RNA replication is achieved by inhibiting newly formed replication complexes and not preformed ones (McGivern et al, Figure 3). The inability of NS5A inhibitors to target RNA replication within preexisting replication complexes may thus help to account

for the slower decline in overall RNA synthesis observed at early time points after NS5A inhibitors addition. NS5A is among the most intriguing proteins encoded by the HCV genome. Although NS5A has no known enzymatic activity, this protein has been implicated in many aspects of the HCV life cycle, has been shown to interact with many host cell proteins and pathways,17,18 and a large number of these interacting partner proteins have been demonstrated to be essential for HCV RNA replication. This suggests that NS5A is a key regulator at multiple stages of the HCV life cycle, that different host cell proteins interact with NS5A at each of these stages, and it should not be surprising that a given small molecule inhibitor of NS5A can have differential effects on these various stages. The work by McGivern et al is an important step in elucidating the NS5A inhibitors class of inhibitors’ MOA, unfolding another layer in the mystery of NS5A activity and its roles in the viral life cycle. Several questions, however, remain unresolved, including why the NS5A inhibitors are so much more potent than other classes of DAAs, and the molecular details of how that is achieved. Transdominant inhibition, wherein one “poisoned” NS5A monomer within a large complex of NS5A proteins can disrupt the function of the entire complex immediately comes to mind. Interestingly, Love et al19 hypothesized that the two described dimeric structures of NS5A19,20 can be present in the cell, and that an NS5A monomer can interact with its counterparts via two different surfaces to form a superhelical polymer. Such a polymer could set the stage for trans-dominant inhibition, wherein DCV’s inhibition of only one dimer in the chain may affect the whole chain, which could represent the underpinning of DCV’s picomolar EC50. This can also explain why the drug inhibits only newly formed (or maybe the formation of) replication complexes. The polymer hypothesis also suggests that the N-terminal amphipathic helix of NS5A21 in this superhelix oligomer cannot associate with a flat membrane surface. This may explain how NS5A is involved in the formation of the DMV and replication complex.22 In line with this possibility, and based on NS5A structure modeling and the position of resistance mutations within this structure,19,20 as well as the finding by O’Boyle et al12 that DCV can pull down NS5A as a dimer, is the suggestion that the inhibitor binds the dimer form of NS5A. One may consider that binding of the drug may stabilize one dimeric form, thereby preventing a

= Figure 1. HCV life cycle and the potential sites of NS5A inhibitor action. (A) The HCV life cycle. Upon entry (i), the viral genome uncoats (ii) and is translated to generate the viral proteins (iii). The non-structural proteins 4B and 5A modify host membranes to form the sites of HCV replication—termed the membranous webs (iv)–upon which the HCV replication complex is assembled and RNA is synthesized. The HCV RNA and capsid protein then assemble and enter into the ER-lumen along with membranes decorated with the envelope proteins (v). The virus is then transported via the secretory pathway to exit the cell (vi). (B) Possible sites of NS5A inhibitor action. NS5A inhibitors act on newly formed RNA replication complexes (vii) but not on preformed ones (viii). The synthesized RNA is then transported from the replication complex via an unknown mechanism, possibly involving NS5A (ix), to within close proximity of lipid droplets and the RNA assembles with the capsid protein in close proximity to lipid droplets (x). The assembly progresses by association of the RNA-capsid proteins complex with envelope proteins within the ER-lumen. Rapid inhibition of assembly by NS5A inhibitors (as demonstrated by McGivern et al.) is hypothesized to be the result of targeting (ix) and (x), and may also be mediated by negative feedback (xi) triggered by inhibition of RNA replication with resulting diversion of RNA templates towards replication as opposed to assembly. 275

EDITORIALS conformational change of the dimer that is necessary for the HCV life cycle. Another possible explanation for the low NS5A inhibitors concentration necessary to inhibit HCV arises from the finding by McGivern et al16 and others13 that the inhibitor acts on only active newly formed replication complexes, which may represent only a small fraction of the total NS5A. Thus, only a very small amount of NS5A inhibitors might be required for inhibiting this fraction of NS5A. It is not clear, however, how such selectivity of the drug toward a subpopulation of NS5A would be achieved. In the simplest view, NS5A has two independent roles in both RNA replication and particle production (the former being implied by DCV’s inhibition of HCV replication in replicon cells where no particle production can occur). It is also possible, however, that these functions are tightly linked together. In this scenario, DCV’s inhibition of RNA replication triggers a NS5A-mediated immediate shut down of particle assembly (Figure 1). Such a linkage could provide a mechanism for HCV to prevent further packaging of viral genomes and instead divert them to serve as templates to overcome the inhibition of RNA replication. To summarize, the work of McGivern et al has expanded our understanding of the MOA of the new NS5A inhibitors. We still do not fully understand the functions and roles that NS5A has in the viral life cycle. Small molecule inhibitors play a key role in enabling us to understand the function of their target and the NS5A inhibitors are no exception, as demonstrated in this work. We believe that it is only a matter of time before we will be able to take another stab at unfolding the MOA of NS5A inhibitors and NS5A function, and it is not unlikely with the multitude of functions that are associated with NS5A that other classes of inhibitors may be discovered that will shed further light on the protein’s function. MENASHE ELAZAR Department of Medicine Division of Gastroenterology and Hepatology and Department of Microbiology and Immunology Stanford University School of Medicine JEFFREY S. GLENN Department of Medicine Division of Gastroenterology and Hepatology and Department of Microbiology and Immunology Stanford University School of Medicine and Veterans Administration Medical Center Palo Alto, California

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Reprint requests Address requests for reprints to: Jeffrey S. Glenn, Department of Medicine, Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford University, CCSR 3115A, 269 Campus Drive, Stanford, California, 94305-5171. e-mail: [email protected]. Conflicts of interest The authors disclose no conflicts. © 2014 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2014.06.021

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