NS5A inhibitors: A new breakthrough for the treatment of chronic hepatitis C
2010; Elsevier BV; Volume: 54; Issue: 5 Linguagem: Inglês
10.1016/j.jhep.2010.11.033
ISSN1600-0641
Autores Tópico(s)Systemic Lupus Erythematosus Research
ResumoChemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, Serrano-Wu MH, Langley DR, Sun JH, O’Boyle DR 2nd, Lemm JA, Wang C, Knipe JO, Chien C, Colonno RJ, Grasela DM, Meanwell NA, Hamann LG. Nature. 2010;465(7294):96–100. Copyright (2010). Abstract reprinted with permission from Macmillan Publishers Ltd.http://www.ncbi.nlm.nih.gov/pubmed/20410884Abstract: The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people. Current therapy relies upon a combination of pegylated interferon-alpha and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC(50)) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log(10) reduction in mean viral load measured 24h post-dose that was sustained for an additional 120h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, Serrano-Wu MH, Langley DR, Sun JH, O’Boyle DR 2nd, Lemm JA, Wang C, Knipe JO, Chien C, Colonno RJ, Grasela DM, Meanwell NA, Hamann LG. Nature. 2010;465(7294):96–100. Copyright (2010). Abstract reprinted with permission from Macmillan Publishers Ltd. http://www.ncbi.nlm.nih.gov/pubmed/20410884 Abstract: The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people. Current therapy relies upon a combination of pegylated interferon-alpha and ribavirin, a poorly tolerated regimen typically associated with less than 50% sustained virological response rate in those infected with genotype 1 virus. The development of direct-acting antiviral agents to treat HCV has focused predominantly on inhibitors of the viral enzymes NS3 protease and the RNA-dependent RNA polymerase NS5B. Here we describe the profile of BMS-790052, a small molecule inhibitor of the HCV NS5A protein that exhibits picomolar half-maximum effective concentrations (EC(50)) towards replicons expressing a broad range of HCV genotypes and the JFH-1 genotype 2a infectious virus in cell culture. In a phase I clinical trial in patients chronically infected with HCV, administration of a single 100-mg dose of BMS-790052 was associated with a 3.3 log(10) reduction in mean viral load measured 24h post-dose that was sustained for an additional 120h in two patients infected with genotype 1b virus. Genotypic analysis of samples taken at baseline, 24 and 144h post-dose revealed that the major HCV variants observed had substitutions at amino-acid positions identified using the in vitro replicon system. These results provide the first clinical validation of an inhibitor of HCV NS5A, a protein with no known enzymatic function, as an approach to the suppression of virus replication that offers potential as part of a therapeutic regimen based on combinations of HCV inhibitors. Recently, Gao et al. identified a potent HCV inhibitor targeting the non-traditional viral protein NS5A [[1]Gao M. Nettles R.E. Belema M. Snyder L.B. Nguyen V.N. Fridell R.A. et al.Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.Nature. 2010; 465: 96-100Crossref PubMed Scopus (835) Google Scholar]. HCV is an enveloped virus with a 9.6 kb single-stranded RNA genome [[2]Choo Q.L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.Science. 1989; 244: 359-362Crossref PubMed Scopus (6209) Google Scholar], a member of the Flaviviridae family, genus Hepacivirus. The HCV lifecycle begins with virion entry into the cytosol with a complex series of steps including attachment to its specific host cell receptor. The HCV RNA genome serves as a template for viral replication via a negative strand copy and as a messenger RNA for virus production. It is translated into a polyprotein that is cleaved by both host and viral proteases to release the individual enzymes and proteins that mediate virus replication, assembly, and release. All of the HCV enzymes are essential for HCV replication and are, therefore, potential targets for drug discovery. The knowledge of the structures of the NS3 protease and NS5B polymerase has allowed structure-based drug design, leading to the development of inhibitors of these enzymes. Several findings suggest that HCV modulation of IFN induction and signalling attenuates the expression of IFN-stimulated genes, allowing HCV to escape the antiviral actions of the host response [[3]Asselah T. Bièche I. Sabbagh A. Bedossa P. Moreau R. Valla D. et al.Gene expression and hepatitis C virus infection.Gut. 2009; 58: 846-858Crossref PubMed Scopus (91) Google Scholar].The standard of care (SOC) is the association of pegylated interferon (PEG-IFN) plus ribavirin (RBV). Sustained virological response (SVR) defined by an absence of HCV RNA measured at 24 weeks after cessation of treatment and equivalent to viral eradication, is associated with a reduction in the risk of cirrhosis and HCC [[4]Maylin S. Martinot-Peignoux M. Moucari R. Boyer N. Ripault M.P. Cazals-Hatem D. et al.Eradication of hepatitis C virus in patients successfully treated for chronic hepatitis C.Gastroenterology. 2008; 135: 821-829Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar]. It has been recently reported that 12 weeks post-treatment follow-up is as relevant as 24 weeks to define SVR [[5]Martinot-Peignoux M. Stern C. Maylin S. Ripault M.P. Boyer N. Leclere L. et al.Twelve weeks posttreatment follow-up is as relevant as 24 weeks to determine the sustained virologic response in patients with hepatitis C virus receiving pegylated interferon and ribavirin.Hepatology. 2010; 51: 1122-1126Crossref PubMed Scopus (144) Google Scholar]. At present, in genotype 2 or 3 infected patients, SVR rates approach 80%; in genotype 1 SVR rates reach less than 50%. It has been shown that some interferon-stimulated genes are highly expressed in non-responders; thus, pre-activation of the IFN system in these patients appears to limit the effect of IFN antiviral therapy [[6]Asselah T. Bieche I. Narguet S. Sabbagh A. Laurendeau I. Ripault M.P. et al.Liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis C.Gut. 2008; 57: 516-524Crossref PubMed Scopus (192) Google Scholar].New drug therapies such as protease and polymerase inhibitors, designated as direct-acting antivirals (DAAs), are under development [[7]Asselah T. Benhamou Y. Marcellin P. Protease and polymerase inhibitors for the treatment of hepatitis C.Liver Int. 2009; 29: 57-67Crossref PubMed Scopus (72) Google Scholar]. In HCV genotype 1 patients, very promising results have been reported when the protease inhibitors telaprevir or boceprevir are added to the current SOC. The final results of phase III studies have shown that they increase the SVR rates from less than 50% (PEG-IFN plus RBV) to approximately 70% (PEG-IFN plus RBV plus protease inhibitor) [8Sherman KE, Flamm SL, Afdhal NH, Nelson DR, Sulkowski MS, Everson GT, et al. Telaprevir in combination with peginterferon Alfa2a and ribavirin for 24 or 48 weeks in treatment-naïve genotype 1 HCV patients who achieved an extended rapid viral response: final results of Phase 3 ILLUMINATE study. Hepatology 52, AASLD 2010; Abstract LB-2.Google Scholar, 9Jacobson IM, McHutchison JG, Dusheiko GM, Di Bisceglie AM, Reddy R, Bzowej NH, et al. Telaprevir in combination with peginterferon and ribavirin in genotype 1 HCV treatment-naïve patients: final results of Phase 3 ADVANCE study. Hepatology 52, AASLD 2010; Abstract 211.Google Scholar, 10McHutchison J.G. Manns M.P. Muir A.J. Terrault N.A. Jacobson I.M. Afdhal N.H. et al.Telaprevir for previously treated chronic HCV infection.N Engl J Med. 2010; 362: 1292-1303Crossref PubMed Scopus (655) Google Scholar, 11Kwo P.Y. Lawitz E.J. McCone J. Schiff E.R. Vierling J.M. Pound D. et al.Efficacy of boceprevir, an NS3 protease inhibitor, in combination with peginterferon alfa-2b and ribavirin in treatment-naive patients with genotype 1 hepatitis C infection (SPRINT-1): an open-label, randomised, multicentre phase 2 trial.Lancet. 2010; 376: 705-716Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 12Poordad F, McCone J, Bacon BR, Bruno S, Manns MP, Sulkowski MS, et al. Boceprevir combined with peginterferon alfa-2b/ribavirin for treatment-naive patients with hepatitis C virus genotype 1: SPRINT-2 final results. Hepatology 52, AASLD 2010; Abstract LB-4.Google Scholar, 13Bacon BR, Gordon SC, Lawitz E, Marcellin P, Vierling JM, Zeuzem S, et al. HCV RESPOND-2 final results: high sustained virological response among genotype 1 previous non-responders and relapsers to peginterferon/ribavirin when re-treated with boceprevir plus pegintron (peginterferon alfa-2b)/ribavirin. Hepatology 52, AASLD 2010; Abstract 216.Google Scholar]. The near future SOC will consist of a regimen that combines a protease inhibitor with PEG-IFN plus RBV.A particularly interesting perspective would be IFN-sparing regimens that rely solely on DAA combinations. Therefore, the identification and development of new molecules with different mechanisms of action, having additive or synergistic effects, is needed. The goals for a DAA combination should be to increase antiviral efficacy, minimize the emergence of resistance, and limit side effects. The first study of a combination of DAAs in patients was the proof-of-concept INFORM-1 study [[14]Gane E. Roberts S. Stedman C. Angus P.W. Ritchie B. Elston R. et al.Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial.Lancet. 2010; 376: 1467-1475Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar]. In this randomised, placebo-controlled, double-blind trial, 87 genotype 1 infected patients were randomized to receive up to 13 days of either oral combination therapy with RG7227/danoprevir, a NS3/4A protease inhibitor, and RG7128, a nucleoside polymerase inhibitor, or with matched placebos. The median reduction in HCV-RNA from the baseline was 5 log10, which fell below the level of detection in 88% of the patients who received the highest dose of both danoprevir (900 mg bid) and RG7128 (1000 mg bid). No evidence of resistance to either compound was observed during the study and no serious adverse events were reported. The antiviral efficacy was similar in naïve and treatment-experienced patients, including non-responders. In the final cohort of patients who received the highest dose of RG7227 and RG7128, 100% achieved early virological response after 24 weeks of PEG-IFN and RBV treatment. The final SVR results will certainly be interesting.There is a great need for additional HCV antivirals to provide more effective, better-tolerated treatment options that can be used in combination therapy, which will be necessary for building successful treatment regimens. In general, the development of antivirals has focused on targeting viral proteins with known enzymatic activities, such as protease or polymerase that are readily recapitulated in vitro. However, a major breakthrough has recently been achieved that advances HCV inhibition beyond these traditional approaches. Researchers at Bristol-Myers Squibb have now reported that a compound, BMS-790052, with a new mechanism of action led to dramatic reductions in viral load and produced few side effects in a phase I clinical study [[1]Gao M. Nettles R.E. Belema M. Snyder L.B. Nguyen V.N. Fridell R.A. et al.Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.Nature. 2010; 465: 96-100Crossref PubMed Scopus (835) Google Scholar]. BMS-790052 targets NS5A, a HCV non-structural protein that possesses no enzymatic activity but plays a critical role in regulating viral replication and host cell interactions. Structures of NS5A and BMS-790052 are provided in Figs. 1A and 1B. Although the function of HCV NS5A is still poorly understood, the BMS researchers have shown that small molecules targeting this protein are potent and specific inhibitors of viral replication.NS5A is a membrane-associated phosphoprotein present in basally phosphorylated (p56) and hyperphosphorylated (p58) forms [15Holler T.P. Parkinson T. Pryde D.C. Targeting the non-structural proteins of hepatitis C virus: beyond hepatitis C virus protease and polymerase.Expert Opin Drug Discov. 2009; 4: 3Crossref Scopus (16) Google Scholar, 16Love 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: 4395-4403Crossref PubMed Scopus (205) Google Scholar]. It was previously reported that only p58-defective mutants could be complemented in trans, and NS5A is involved in HCV virion production, suggesting that different forms of NS5A exert multiple functions at various stages of the viral life cycle. The N terminus of NS5A (domain I) has been crystallized in alternative dimeric forms and contains both zinc- and RNA-binding domains, properties that have been demonstrated in vitro [[16]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: 4395-4403Crossref PubMed Scopus (205) Google Scholar] (Fig. 1). NS5A has been shown to interact with a number of host proteins and is implicated in interferon resistance in vivo [[15]Holler T.P. Parkinson T. Pryde D.C. Targeting the non-structural proteins of hepatitis C virus: beyond hepatitis C virus protease and polymerase.Expert Opin Drug Discov. 2009; 4: 3Crossref Scopus (16) Google Scholar]. The strategy used by Gao et al. to identify a lead HCV NS5A inhibitor provides a contemporary demonstration of the effectiveness of an approach to drug discovery based on chemical genetics [[17]Stockwell B.R. Exploring biology with small organic molecules.Nature. 2004; 432: 846-854Crossref PubMed Scopus (386) Google Scholar]. Indeed, this methodology is uniquely applicable to targets similar to HCV NS5A for which precise functions are unknown and for which the development of biochemical assays are infeasible. The lead molecule was optimized into the clinical candidate BMS-790052 whose chemical structure is shown in Fig. 1B. This compound is active at picomolar concentrations in vitro towards replicons expressing a broad range of HCV genotypes and acts in an additive to synergistic fashion with interferon and other small molecule antiviral compounds. Mean plasma concentration–time profile of BMS-790052 after single oral administration to healthy subjects is shown in Fig. 2A. The effect of this compound on viral load in HCV-infected subjects following single oral doses of the compound are provided in Fig. 2B.Fig. 2Mean plasma concentration–time profile of BMS-790052. (A) Mean plasma concentration–time profile (time 0–72 h) of BMS-790052 after single oral administration of 1–200 mg of drug to healthy subjects. In a double-blind, placebo-controlled, sequential, single ascending-dose study, eight male or female subjects were randomized within each dose panel (1, 10, 25, 50, 100 and 200 mg) to drug or placebo in a ratio of 3:1. BMS-790052 or placebo was administered in the fasted state. The plasma samples obtained at various times were analysed for BMS-790052 by a validated liquid chromatography tandem mass spectrometry assay. Pharmacokinetic parameter values for individual subjects were derived by non-compartmental methods by a validated pharmacokinetic analysis programme. PBA-EC90 = protein-binding-adjusted EC90 for the individual genotype in a replicon assay. Error bars, standard deviation. From Ref. [[1]Gao M. Nettles R.E. Belema M. Snyder L.B. Nguyen V.N. Fridell R.A. et al.Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.Nature. 2010; 465: 96-100Crossref PubMed Scopus (835) Google Scholar]. (B) Mean change in log10 HCV RNA with 90% confidence intervals after administration of single oral doses of BMS-790052 to HCV-infected patients. In a double-blind, placebo-controlled, sequential, single ascending-dose study, six subjects were randomized within each dose panel (1, 10, 100 mg) to drug or placebo in a ratio of 5:1. BMS-790052 or placebo was administered in the fasted state. Owing to a dosing error, all six subjects received BMS-790052 in the 1 mg panel. One subject in the 10 mg panel withdrew from the study 8 h after administration of the study drug for nondrug-related reasons; HCV RNA data from the subject are included up until the subject withdrew. From Ref. [[1]Gao M. Nettles R.E. Belema M. Snyder L.B. Nguyen V.N. Fridell R.A. et al.Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.Nature. 2010; 465: 96-100Crossref PubMed Scopus (835) Google Scholar].View Large Image Figure ViewerDownload Hi-res image Download (PPT)The resistance profile of BMS-790052 is shown in Table 1 which reveals that, inhibitor sensitivity maps to the N terminus of domain 1 of NS5A [1Gao M. Nettles R.E. Belema M. Snyder L.B. Nguyen V.N. Fridell R.A. et al.Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.Nature. 2010; 465: 96-100Crossref PubMed Scopus (835) Google Scholar, 18Lemm J.A. O’Boyle 2nd, D. Liu M. Nower P.T. Colonno R. Deshpande M.S. et al.Identification of hepatitis C virus NS5A inhibitors.J Virol. 2010; 84: 482-491Crossref PubMed Scopus (170) Google Scholar, 19Fridell 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: 3641-3650Crossref PubMed Scopus (256) Google Scholar]. In addition, the BMS researchers demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle.Table 1Resistance profile of genotype 1a and 1b replicons exposed to BMS-790052 adapted from [18]Lemm J.A. O’Boyle 2nd, D. Liu M. Nower P.T. Colonno R. Deshpande M.S. et al.Identification of hepatitis C virus NS5A inhibitors.J Virol. 2010; 84: 482-491Crossref PubMed Scopus (170) Google Scholar.1Mean ± standard deviation, determined in transient transfection assays (n ⩾3). Open table in a new tab Phase II clinical studies combining BMS-790052 with the NS3 protease inhibitor BMS-650032 are ongoing and interim results have shown that this combination therapy alone or with PEG-IFN and RBV results in undetectable HCV RNA through 12 weeks of therapy in HCV genotype 1 null responders [[20]Lok AS, Gardiner DF, Lawitz E, Martorell C, Everson GT, Ghalib RH, et al. Combination therapy with BMS-790052 and BMS-650032 alone or with pegIFN/RBV results in undetectable HCV RNA through 12 weeks of therapy in HCV genotype 1 null responders. Hepatology 52, AASLD 2010; Abstract LB-8.Google Scholar].At present, several studies of DAA combinations are ongoing (Table 2) [20Lok AS, Gardiner DF, Lawitz E, Martorell C, Everson GT, Ghalib RH, et al. Combination therapy with BMS-790052 and BMS-650032 alone or with pegIFN/RBV results in undetectable HCV RNA through 12 weeks of therapy in HCV genotype 1 null responders. Hepatology 52, AASLD 2010; Abstract LB-8.Google Scholar, 21Zeuzem S, Buggisch P, Agarwal K, Manns MP, Marcellin P, Foster G, et al. Dual, triple, and quadruple combination treatment with a protease inhibitor (GS-9256) and a polymerase inhibitor (GS-9190) alone and in combination with ribavirin (RBV) or PegIFN/RBV for up to 28 days in treatment naïve, genotype 1 HCV subjects. Hepatology 52, AASLD 2010; Abstract LB-1.Google Scholar, 22Zeuzem S, Asselah T, Angus PW, Zarski JP, Larrey D, Mullhaupt B, et al. Strong antiviral activity and safety of IFN-sparing treatment with the protease inhibitor BI 201335, the HCV polymerase inhibitor BI 207127 and ribavirin in patients with chronic hepatitis C. Hepatology 52, AASLD 2010; Abstract LB-7.Google Scholar].Table 2Some 20Lok AS, Gardiner DF, Lawitz E, Martorell C, Everson GT, Ghalib RH, et al. Combination therapy with BMS-790052 and BMS-650032 alone or with pegIFN/RBV results in undetectable HCV RNA through 12 weeks of therapy in HCV genotype 1 null responders. Hepatology 52, AASLD 2010; Abstract LB-8.Google Scholar, 21Zeuzem S, Buggisch P, Agarwal K, Manns MP, Marcellin P, Foster G, et al. Dual, triple, and quadruple combination treatment with a protease inhibitor (GS-9256) and a polymerase inhibitor (GS-9190) alone and in combination with ribavirin (RBV) or PegIFN/RBV for up to 28 days in treatment naïve, genotype 1 HCV subjects. Hepatology 52, AASLD 2010; Abstract LB-1.Google Scholar under development in phase 2 clinical studies. Open table in a new tab In conclusion, clinical proof-of-concept has recently been achieved with NS5A inhibitors, indicating that small molecules targeting a non-traditional viral protein without any known enzymatic activity can also have profound antiviral effects in HCV-infected subjects. Achieving high potency and selectivity against a non-mammalian target, the traditional goal of antiviral medicinal chemistry, has in the past translated into a wider therapeutic index in the clinic. Although preliminary, these data indicate that inhibitors of HCV NS5A offer considerable promise for the treatment of HCV infection. Once new DAAs become available, treatment strategies that combine several drugs with different mechanisms of action could hopefully result in IFN- and/or RBV-sparing regimens. Ongoing studies are directed towards demonstrating that such combinations of DAAs have synergistic antiviral potency, a low risk of resistance with a good safety profile.Conflict of interestThe authors who have taken part in this study have declared a relationship with the manufacturers of the drugs involved. Tarik Asselah has been an investigator or speaker for BMS, Gilead, Merck, Roche, and Tibotec. Recently, Gao et al. identified a potent HCV inhibitor targeting the non-traditional viral protein NS5A [[1]Gao M. Nettles R.E. Belema M. Snyder L.B. Nguyen V.N. Fridell R.A. et al.Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.Nature. 2010; 465: 96-100Crossref PubMed Scopus (835) Google Scholar]. HCV is an enveloped virus with a 9.6 kb single-stranded RNA genome [[2]Choo Q.L. Kuo G. Weiner A.J. Overby L.R. Bradley D.W. Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.Science. 1989; 244: 359-362Crossref PubMed Scopus (6209) Google Scholar], a member of the Flaviviridae family, genus Hepacivirus. The HCV lifecycle begins with virion entry into the cytosol with a complex series of steps including attachment to its specific host cell receptor. The HCV RNA genome serves as a template for viral replication via a negative strand copy and as a messenger RNA for virus production. It is translated into a polyprotein that is cleaved by both host and viral proteases to release the individual enzymes and proteins that mediate virus replication, assembly, and release. All of the HCV enzymes are essential for HCV replication and are, therefore, potential targets for drug discovery. The knowledge of the structures of the NS3 protease and NS5B polymerase has allowed structure-based drug design, leading to the development of inhibitors of these enzymes. Several findings suggest that HCV modulation of IFN induction and signalling attenuates the expression of IFN-stimulated genes, allowing HCV to escape the antiviral actions of the host response [[3]Asselah T. Bièche I. Sabbagh A. Bedossa P. Moreau R. Valla D. et al.Gene expression and hepatitis C virus infection.Gut. 2009; 58: 846-858Crossref PubMed Scopus (91) Google Scholar]. The standard of care (SOC) is the association of pegylated interferon (PEG-IFN) plus ribavirin (RBV). Sustained virological response (SVR) defined by an absence of HCV RNA measured at 24 weeks after cessation of treatment and equivalent to viral eradication, is associated with a reduction in the risk of cirrhosis and HCC [[4]Maylin S. Martinot-Peignoux M. Moucari R. Boyer N. Ripault M.P. Cazals-Hatem D. et al.Eradication of hepatitis C virus in patients successfully treated for chronic hepatitis C.Gastroenterology. 2008; 135: 821-829Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar]. It has been recently reported that 12 weeks post-treatment follow-up is as relevant as 24 weeks to define SVR [[5]Martinot-Peignoux M. Stern C. Maylin S. Ripault M.P. Boyer N. Leclere L. et al.Twelve weeks posttreatment follow-up is as relevant as 24 weeks to determine the sustained virologic response in patients with hepatitis C virus receiving pegylated interferon and ribavirin.Hepatology. 2010; 51: 1122-1126Crossref PubMed Scopus (144) Google Scholar]. At present, in genotype 2 or 3 infected patients, SVR rates approach 80%; in genotype 1 SVR rates reach less than 50%. It has been shown that some interferon-stimulated genes are highly expressed in non-responders; thus, pre-activation of the IFN system in these patients appears to limit the effect of IFN antiviral therapy [[6]Asselah T. Bieche I. Narguet S. Sabbagh A. Laurendeau I. Ripault M.P. et al.Liver gene expression signature to predict response to pegylated interferon plus ribavirin combination therapy in patients with chronic hepatitis C.Gut. 2008; 57: 516-524Crossref PubMed Scopus (192) Google Scholar]. New drug therapies such as protease and polymerase inhibitors, designated as direct-acting antivirals (DAAs), are under development [[7]Asselah T. Benhamou Y. Marcellin P. Protease and polymerase inhibitors for the treatment of hepatitis C.Liver Int. 2009; 29: 57-67Crossref PubMed Scopus (72) Google Scholar]. In HCV genotype 1 patients, very promising results have been reported when the protease inhibitors telaprevir or boceprevir are added to the current SOC. The final results of phase III studies have shown that they increase the SVR rates from less than 50% (PEG-IFN plus RBV) to approximately 70% (PEG-IFN plus RBV plus protease inhibitor) [8Sherman KE, Flamm SL, Afdhal NH, Nelson DR, Sulkowski MS, Everson GT, et al. Telaprevir in combination with peginterferon Alfa2a and ribavirin for 24 or 48 weeks in treatment-naïve genotype 1 HCV patients who achieved an extended rapid viral response: final results of Phase 3 ILLUMINATE study. Hepatology 52, AASLD 2010; Abstract LB-2.Google Scholar, 9Jacobson IM, McHutchison JG, Dusheiko GM, Di Bisceglie AM, Reddy R, Bzowej NH, et al. Telaprevir in combination with peginterferon and ribavirin in genotype 1 HCV treatment-naïve patients: final results of Phase 3 ADVANCE study. Hepatology 52, AASLD 2010; Abstract 211.Google Scholar, 10McHutchison J.G. Manns M.P. Muir A.J. Terrault N.A. Jacobson I.M. Afdhal N.H. et al.Telaprevir for previously treated chronic HCV infection.N Engl J Med. 2010; 362: 1292-1303Crossref PubMed Scopus (655) Google Scholar, 11Kwo P.Y. Lawitz E.J. McCone J. Schiff E.R. Vierling J.M. Pound D. et al.Efficacy of boceprevir, an NS3 protease inhibitor, in combination with peginterferon alfa-2b and ribavirin in treatment-naive patients with genotype 1 hepatitis C infection (SPRINT-1): an open-label, randomised, multicentre phase 2 trial.Lancet. 2010; 376: 705-716Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 12Poordad F, McCone J, Bacon BR, Bruno S, Manns MP, Sulkowski MS, et al. Boceprevir combined with peginterferon alfa-2b/ribavirin for treatment-naive patients with hepatitis C virus genotype 1: SPRINT-2 final results. Hepatology 52, AASLD 2010; Abstract LB-4.Google Scholar, 13Bacon BR, Gordon SC, Lawitz E, Marcellin P, Vierling JM, Zeuzem S, et al. HCV RESPOND-2 final results: high sustained virological response among genotype 1 previous non-responders and relapsers to peginterferon/ribavirin when re-treated with boceprevir plus pegintron (peginterferon alfa-2b)/ribavirin. Hepatology 52, AASLD 2010; Abstract 216.Google Scholar]. The near future SOC will consist of a regimen that combines a protease inhibitor with PEG-IFN plus RBV. A particularly interesting perspective would be IFN-sparing regimens that rely solely on DAA combinations. Therefore, the identification and development of new molecules with different mechanisms of action, having additive or synergistic effects, is needed. The goals for a DAA combination should be to increase antiviral efficacy, minimize the emergence of resistance, and limit side effects. The first study of a combination of DAAs in patients was the proof-of-concept INFORM-1 study [[14]Gane E. Roberts S. Stedman C. Angus P.W. Ritchie B. Elston R. et al.Oral combination therapy with a nucleoside polymerase inhibitor (RG7128) and danoprevir for chronic hepatitis C genotype 1 infection (INFORM-1): a randomised, double-blind, placebo-controlled, dose-escalation trial.Lancet. 2010; 376: 1467-1475Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar]. In this randomised, placebo-controlled, double-blind trial, 87 genotype 1 infected patients were randomized to receive up to 13 days of either oral combination therapy with RG7227/danoprevir, a NS3/4A protease inhibitor, and RG7128, a nucleoside polymerase inhibitor, or with matched placebos. The median reduction in HCV-RNA from the baseline was 5 log10, which fell below the level of detection in 88% of the patients who received the highest dose of both danoprevir (900 mg bid) and RG7128 (1000 mg bid). No evidence of resistance to either compound was observed during the study and no serious adverse events were reported. The antiviral efficacy was similar in naïve and treatment-experienced patients, including non-responders. In the final cohort of patients who received the highest dose of RG7227 and RG7128, 100% achieved early virological response after 24 weeks of PEG-IFN and RBV treatment. The final SVR results will certainly be interesting. There is a great need for additional HCV antivirals to provide more effective, better-tolerated treatment options that can be used in combination therapy, which will be necessary for building successful treatment regimens. In general, the development of antivirals has focused on targeting viral proteins with known enzymatic activities, such as protease or polymerase that are readily recapitulated in vitro. However, a major breakthrough has recently been achieved that advances HCV inhibition beyond these traditional approaches. Researchers at Bristol-Myers Squibb have now reported that a compound, BMS-790052, with a new mechanism of action led to dramatic reductions in viral load and produced few side effects in a phase I clinical study [[1]Gao M. Nettles R.E. Belema M. Snyder L.B. Nguyen V.N. Fridell R.A. et al.Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.Nature. 2010; 465: 96-100Crossref PubMed Scopus (835) Google Scholar]. BMS-790052 targets NS5A, a HCV non-structural protein that possesses no enzymatic activity but plays a critical role in regulating viral replication and host cell interactions. Structures of NS5A and BMS-790052 are provided in Figs. 1A and 1B. Although the function of HCV NS5A is still poorly understood, the BMS researchers have shown that small molecules targeting this protein are potent and specific inhibitors of viral replication. NS5A is a membrane-associated phosphoprotein present in basally phosphorylated (p56) and hyperphosphorylated (p58) forms [15Holler T.P. Parkinson T. Pryde D.C. Targeting the non-structural proteins of hepatitis C virus: beyond hepatitis C virus protease and polymerase.Expert Opin Drug Discov. 2009; 4: 3Crossref Scopus (16) Google Scholar, 16Love 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: 4395-4403Crossref PubMed Scopus (205) Google Scholar]. It was previously reported that only p58-defective mutants could be complemented in trans, and NS5A is involved in HCV virion production, suggesting that different forms of NS5A exert multiple functions at various stages of the viral life cycle. The N terminus of NS5A (domain I) has been crystallized in alternative dimeric forms and contains both zinc- and RNA-binding domains, properties that have been demonstrated in vitro [[16]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: 4395-4403Crossref PubMed Scopus (205) Google Scholar] (Fig. 1). NS5A has been shown to interact with a number of host proteins and is implicated in interferon resistance in vivo [[15]Holler T.P. Parkinson T. Pryde D.C. Targeting the non-structural proteins of hepatitis C virus: beyond hepatitis C virus protease and polymerase.Expert Opin Drug Discov. 2009; 4: 3Crossref Scopus (16) Google Scholar]. The strategy used by Gao et al. to identify a lead HCV NS5A inhibitor provides a contemporary demonstration of the effectiveness of an approach to drug discovery based on chemical genetics [[17]Stockwell B.R. Exploring biology with small organic molecules.Nature. 2004; 432: 846-854Crossref PubMed Scopus (386) Google Scholar]. Indeed, this methodology is uniquely applicable to targets similar to HCV NS5A for which precise functions are unknown and for which the development of biochemical assays are infeasible. The lead molecule was optimized into the clinical candidate BMS-790052 whose chemical structure is shown in Fig. 1B. This compound is active at picomolar concentrations in vitro towards replicons expressing a broad range of HCV genotypes and acts in an additive to synergistic fashion with interferon and other small molecule antiviral compounds. Mean plasma concentration–time profile of BMS-790052 after single oral administration to healthy subjects is shown in Fig. 2A. The effect of this compound on viral load in HCV-infected subjects following single oral doses of the compound are provided in Fig. 2B. The resistance profile of BMS-790052 is shown in Table 1 which reveals that, inhibitor sensitivity maps to the N terminus of domain 1 of NS5A [1Gao M. Nettles R.E. Belema M. Snyder L.B. Nguyen V.N. Fridell R.A. et al.Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.Nature. 2010; 465: 96-100Crossref PubMed Scopus (835) Google Scholar, 18Lemm J.A. O’Boyle 2nd, D. Liu M. Nower P.T. Colonno R. Deshpande M.S. et al.Identification of hepatitis C virus NS5A inhibitors.J Virol. 2010; 84: 482-491Crossref PubMed Scopus (170) Google Scholar, 19Fridell 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: 3641-3650Crossref PubMed Scopus (256) Google Scholar]. In addition, the BMS researchers demonstrated that NS5A inhibitors, as well as an active-site inhibitor that specifically binds NS3 protease, could block the hyperphosphorylation of NS5A, which is believed to play an essential role in the viral life cycle. 1Mean ± standard deviation, determined in transient transfection assays (n ⩾3). Phase II clinical studies combining BMS-790052 with the NS3 protease inhibitor BMS-650032 are ongoing and interim results have shown that this combination therapy alone or with PEG-IFN and RBV results in undetectable HCV RNA through 12 weeks of therapy in HCV genotype 1 null responders [[20]Lok AS, Gardiner DF, Lawitz E, Martorell C, Everson GT, Ghalib RH, et al. Combination therapy with BMS-790052 and BMS-650032 alone or with pegIFN/RBV results in undetectable HCV RNA through 12 weeks of therapy in HCV genotype 1 null responders. Hepatology 52, AASLD 2010; Abstract LB-8.Google Scholar]. At present, several studies of DAA combinations are ongoing (Table 2) [20Lok AS, Gardiner DF, Lawitz E, Martorell C, Everson GT, Ghalib RH, et al. Combination therapy with BMS-790052 and BMS-650032 alone or with pegIFN/RBV results in undetectable HCV RNA through 12 weeks of therapy in HCV genotype 1 null responders. Hepatology 52, AASLD 2010; Abstract LB-8.Google Scholar, 21Zeuzem S, Buggisch P, Agarwal K, Manns MP, Marcellin P, Foster G, et al. Dual, triple, and quadruple combination treatment with a protease inhibitor (GS-9256) and a polymerase inhibitor (GS-9190) alone and in combination with ribavirin (RBV) or PegIFN/RBV for up to 28 days in treatment naïve, genotype 1 HCV subjects. Hepatology 52, AASLD 2010; Abstract LB-1.Google Scholar, 22Zeuzem S, Asselah T, Angus PW, Zarski JP, Larrey D, Mullhaupt B, et al. Strong antiviral activity and safety of IFN-sparing treatment with the protease inhibitor BI 201335, the HCV polymerase inhibitor BI 207127 and ribavirin in patients with chronic hepatitis C. Hepatology 52, AASLD 2010; Abstract LB-7.Google Scholar]. In conclusion, clinical proof-of-concept has recently been achieved with NS5A inhibitors, indicating that small molecules targeting a non-traditional viral protein without any known enzymatic activity can also have profound antiviral effects in HCV-infected subjects. Achieving high potency and selectivity against a non-mammalian target, the traditional goal of antiviral medicinal chemistry, has in the past translated into a wider therapeutic index in the clinic. Although preliminary, these data indicate that inhibitors of HCV NS5A offer considerable promise for the treatment of HCV infection. Once new DAAs become available, treatment strategies that combine several drugs with different mechanisms of action could hopefully result in IFN- and/or RBV-sparing regimens. Ongoing studies are directed towards demonstrating that such combinations of DAAs have synergistic antiviral potency, a low risk of resistance with a good safety profile. Conflict of interestThe authors who have taken part in this study have declared a relationship with the manufacturers of the drugs involved. Tarik Asselah has been an investigator or speaker for BMS, Gilead, Merck, Roche, and Tibotec. The authors who have taken part in this study have declared a relationship with the manufacturers of the drugs involved. Tarik Asselah has been an investigator or speaker for BMS, Gilead, Merck, Roche, and Tibotec.
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