APPROVED AND EXPERIMENTAL THERAPIES FOR TREATMENT OF HEPATITIS B AND C, AND MUTATIONS 13.ASSOCIATED WITH DRUG RESISTANCE • Luis Menéndez-Arias HEPATITIS B

Table 13.1. CURRENT DRUGS FOR TREATMENT OF HEPATITIS B

Drug name Drug class Manufacturer Status Pegasys (peginterferon α-2a) Interferon Genentech FDA-approved Intron A (interferon a-2b) Interferon Merck FDA-approved Hepsera (adefovir Nucleotide analogue Gilead Sciences FDA-approved dipivoxil)a Viread (tenofovir Nucleotide analogue Gilead Sciences FDA-approved b disoproxil fumarate) a Epivir-HBV, Zeffix and Nucleoside analogue Glaxo SmithKline FDA-approved Heptodin (lamivudine) a Baraclude (entecavir) Nucleoside analogue Bristol-Myers Squibb FDA-approved Tyzeka, Sebivo Nucleoside analogue Novartis FDA-approved (telbivudine) Vemlidy (TAF, tenofovir Tenofovir prodrug Gilead Sciences FDA-approved alafenamide, GS-7340)

Emtriva (emtricitabine; Nucleoside analogue Gilead Sciences FDA-approved b FTC) a

Levovir, Revovir Nucleoside analogue Bukwang Studies cancelled c (clevudine, L-FMAU) pharmaceuticals, Eisai (Japan) Besivo (LB80380, Nucleoside analogue IIDong Pharmaceutical Approved in S ANA380) Co. Ltd. Korea Zadaxin (thymosin alpha) Immune enhancer SciClone Approved outside U.S. ABX 203 Therapeutic vaccine ABIVAX Phase IIb/III ARC-520 RNAi gene silencer Arrowhead Research Phase II/III

641 Drug name Drug class Manufacturer Status Myrcludex B Entry inhibitor Hepatera (Russia), Phase IIa Myr-Gmbh (Germany) (Russia) NVR-1221 (NVR 3-778) Capsid inhibitor Novira Therapeutics Phase IIa AGX-1009 Tenofovir prodrug SHRG/YSY (China) Phase II, China TXL (CMX157) Tenofovir prodrug ContraVir Phase II Pharmaceuticals Pradefovir mesylate Adefovir prodrug Valeant Phase II (DB05478) Pharmaceuticals Morphothiadine mesilate Capsid inhibitor Sunshine Lake Phase II (GLS4) Pharma of HEC, China ISIS-HBV Rx RNA-based antisense ISIS Pharma with Phase II Glaxo SmithKline SB 9200 HBV Small nucleic acid Spring Bank Pharma Phase II hybrid ARB-1467 & ARB-1740 RNAi gene silencer Arbutus Biopharma Phase II Rep 2139-Ca HBsAg release REPLICor Inc Phase II inhibitor Rep 2165-Ca HBsAg release REPLICor Inc Phase II inhibitor GS-4774 Therapeutic vaccine Gilead Sciences Phase II with Globe Immune GS-9620 TLR7 agonist Gilead Sciences Phase II RO6864018 (RG7795, TLR7 agonist Roche Phase II ANA773) Nasvac Therapeutic vaccines CGEB, Cuba Phase II CYT107 (Interleukin-7) Immunomodulator Cytheris Phase I/IIa INO-1800 Therapeutic vaccine Inovio Phase I AIC 649 Capsid Inhibitor AiCuris Phase I HB-110 Therapeutic vaccine Ichor Medical Systems Phase I (with Janssen) TG 1050 Therapeutic vaccine Transgen Phase I

642 FDA, United States Food and Drug Administration. a This is an anti-HIV drug. For information on HIV drug-resistance, see Tables in Chapter 4. b This drug has been approved only for treatment of HIV infection, but is also an inhibitor of the hepatitis B virus polymerase. c Approved in South Korea and the Philippines for treatment of chronic hepatitis B. d Orphan drug approved in the U.S. for cancer treatment. Clinical studies on the efficacy of the drug in combination with nucleoside analogues are available [Wu et al. Expert Opin Biol Ther 2015; 15 (suppl 1): S129-S132]. References: Hepatitis B Foundation Drug Watch (http://www.hepb.org); Hadziyannis et al. Adv Pharmacol 2013; 67: 247-291; Liu et al. Int J Biochem Cell Biol 2013; 45: 1987- 1996; Menéndez-Arias et al. Curr Opin Virol 2014; 8: 1-9; Trépo et al. Lancet 2014; 384: 2053-2063; Block et al. Antiviral Res 2015; 121: 69-81; Isorce et al. Antiviral Res 2015; 122: 69-81; Jia et al. Future Med Chem 2015; 7: 587-607; Liang et al. Hepatology 2015; 62: 1893-1908; Wang et al. Virus Res 2016; 213: 205-213; Zoulim et al. Curr Opin Virol 2016; 18: 109-116; Testoni et al. Liver Int 2017; 37 (suppl. 1): 33-39; Scott & Chan. Drugs 2017; 77: 1017-1028.

Resistance to interferon

To date, ten hepatitis B virus (HBV) genotypes (A-J) have been identified. HBV genotypes are classified by more than 8% divergence of the full nucleotide sequence. Their geographic distribution varies significantly. HBV genotype A is common in Northern Europe, North America and Central Africa, whereas genotype D is found mainly in the Mediterranean area, Middle East and India. Genotypes B and C are prevalent in Asia, genotype E in West Africa, and genotype F in Central and South America and Polynesia. Genotype G was identified in Europe and North America. Genotype H has been recently identified in Central America. HBV genotypes can be further classified into sub-genotypes if a divergence of 4% is observed. Major sub-genotypes are A1 to A7 (genotype A), B1 to B9 (genotype B), C1 to C16 (genotype C), D1 to D8 (genotype D), and F1 to F4 (genotype F). Available evidence indicates a better sustained response to conventional interferon in patients with genotype B than those with C, and in patients with genotype A than those with D. In contrast, there are conflicting data regarding the response to pegylated interferon. The molecular basis of the observed differences are not known, although the lower response of genotype C patients has been related to the higher frequency of the basal core promoter mutations (A1762T/G1764A). Genotypes C and D also carry a higher risk of cirrhosis and development of hepatocellular carcinoma than genotypes A and B. A specialized database (HBVdb; http://hbvdb.ibcp.fr) dealing with the genetic variability of hepatitis B virus and its resistance to treatment has been established (Hayer et al. Nucleic Acids Res 2013; 41: D566-D570). The database allows sequence analysis and queries about drug resistance profiles.

643 A recent study has described an association between hepatitis B virus spliced variants and impaired response to interferon therapy (Chen et al. Sci Rep 2015; 5: 16459).

References: Coffin & Lee. Liver Int 2009; 29: 116-124; Shamliyan et al. Ann Intern Med 2009; 150: 111-124; Cooksley. J Viral Hepat 2010; 17: 601-610; Lin & Kao. J Gastroenterol Hepatol 2011; 26 (suppl. 1): 123-130; Liu et al. Curr Opin Immunol 2011; 23: 57-64; Wong & Sung. Curr Opin Infect Dis 2012; 25: 570-577; You et al. World J Gastroenterol 2014; 20: 13293-13305; Lin & Kao. Cold Spring Harb Perspect Med 2015; 5: a021436; Zhang et al. World J Gastroenterol 2016; 22: 126-144; Tian & Jia. Hepatol Int 2016; 10: 854-860.

Resistance to nucleoside analogue inhibitors

As in the case of HIV-1 and HIV-2, resistance to nucleoside analogue inhibitors maps within the viral polymerase. Hepatitis B virus (HBV) polymerase and HIV-1 reverse transcriptase share homology regions designated as motifs A, B, C, D and E (Fig. 16.1). Motif C contains the YMDD sequence which mutates in response to treatment with lamivudine and other HBV polymerase inhibitors.

Figure 13.1. Alignments of conserved motifs within hepatitis B virus (HBV) polymerase and HIV-1 reverse transcriptase. Conserved residues are boxed. Sequences were taken from EMBL/GenBank accession files J02205 (HBV subtype adw2) and M15654 (isolate BH10), and the alignments were based on those given by Poch et al. EMBO J 1989; 8: 3867-3874. Numbering of motifs in the HBV polymerase is given according to the standardized nomenclature of Stuyver et al. Hepatology 2001; 33: 751-757.

From a clinical point of view, the most relevant mutation is rtM204V which appears during treatment with lamivudine, and maps in the YMDD region. In many patients, it is accompanied by a second mutation (rtL180M), particularly in those previously treated with famciclovir. The emergence of these mutations is clinically associated with elevated

644 levels of alanine aminotransferase and viral DNA titers in serum. The YMDD mutation has a high prevalence of 56% among patients treated with lamivudine for 256 weeks and 70% in patients treated for more than 4 years (Leung et al. Hepatology 2001; 33: 1527- 1532). Studies focusing on the dynamics of HBV quasispecies variant populations have revealed that virological breakthrough was preceded by 2-4 months by the emergence of quasispecies variants bearing amino acid substitutions at RT position 204, i.e. within the YMDD catalytic motif (rtM204V/I) (Pallier et al. J Virol 2006; 80: 643-653). Compared with hepatitis B genotype C, genotype B appears to develop an earlier biochemical resistance to lamivudine (Hsieh et al. Antivir Ther 2009; 14: 1157-1163).

Table 13.2. HEPATITIS B VIRUS (HBV) POLYMERASE MUTATIONS ASSOCIATED WITH RESISTANCE TO NUCLEOSIDE INHIBITORS

Mutations a Comments rtS78T In a study involving >350 chronically HBV-infected patients in Europe, this (S426T) mutation showed more than ten-fold increased prevalence in adefovir-failing patients (Cento et al. J Infect 2013; 67: 303-312). The prevalence of this mutation was lower in lamivudine-treated patients (2.7%) in comparison with naïve individuals (7.9%). It can generate a truncated surface protein due to a stop codon at position 69 (sC69*) (Ding et al. Antiviral Res 2014; 102: 29-34). rtL80V/I Amino acid substitution associated with lamivudine resistance and enhanced (L428V/I) viral replication in vitro. Usually, rtL80V/I occur in virus containing the rtM204V mutation (Warner et al. Antimicrob Agents Chemother 2007; 51: 2285-2292). Associated in patients with poor response to adefovir (Lee et al. Liver Int 2009; 29: 552-556; Mirandola et al. Antiviral Res 2012; 96: 422- 429) and found as a minority variant in patients treated with telbivudine (Yin et al. J Gen Virol 2015; 96: 3302-3312). The rtL80I and rtL80V substitutions confer moderate resistance to telbivudine (Yin et al. J Gen Virol 2015; 96: 3302-3312). rtS117F Compensatory mutation for rtM204I appearing during lamivudine therapy (S465F) (Lin et al. J Antimicrob Chemother 2012; 67: 39-48). rtH124N Polymorphism associated with a reduced response to entecavir, in a study H472N carried out in Hong Kong (Wong et al. J Infect Dis 2014; 210: 701-707). Appears with higher frequency in the viral quasispecies obtained from patients failing therapy with lamivudine, as compared with those naïve for antiviral treatment. This substitution conferred 6.5-fold reduced susceptibility to tenofovir in vitro (Banerjee et al. Sci Rep 2017; 7: 44742). rtI163V Detected together with rtA186T and lamivudine resistance mutations rtL180M (I511V) and rtM204V in a patient with viral breakthrough and refractory to treatment with entecavir. The rtI163V had a minor impact on resistance but was important to sustain viral fitness (Hayashi et al. J Hepatol 2015; 63: 546-553).

645 Mutations a Comments rtI169T Emerged together with rtM250V during entecavir/lamivudine combination (I517T) therapy (Tenney et al. Antimicrob Agents Chemother 2004; 48: 3498-3507). rtV173L Appears after treatment with penciclovir (the prodrug of famciclovir) (Aye et (V521L) al. J Hepatol 1997; 26: 1148-1153). Associated with lamivudine therapy failure (Rhee et al. Antiviral Res 2010; 88: 269-275; Ma et al. PLoS One 2013; 8: e67606). It has also been observed at baseline with 9 – 22% of patients entering clinical trials of adefovir dipivoxil, due to lamivudine-resistance. In these cases, rtV173L was a third mutation present together with rtL180M/M204V that did not alter the viral resistance profile to lamivudine, penciclovir, adefovir or tenofovir, but increased viral replication capacity (Delaney IV et al. J Virol 2003; 77: 11833-11841; Lada et al. Antivir Ther 2004; 9: 353-363). Facilitates the emergence of the surface protein mutation sE164D (Ding et al. Antiviral Res 2014; 102: 29-34). rtP177G Confers reduced susceptibility to tenofovir in vitro (Qin et al. Antiviral Res (P525G) 2013; 97: 93-100). rtL180C Detected in one patient after 2 years of therapy with lamivudine, together (L528C) with mutation rtM204I. The double mutant showed high-level resistance to the inhibitor in vitro (Pai et al. Antimicrob Agents Chemother 2005; 49: 2618-2624). rtL180M This mutation has been shown to arise in HBV from patients treated with (L528M) penciclovir [Aye et al. J Hepatol 1997; 26: 1148-1153; Tillman et al. J Hepatol 1997; 26 (S1): 153; Seignères et al. J Infect Dis 2000; 181: 1221- 1233], and is always found in vivo associated with rtM204V in patients failing treatment with lamivudine (Ma et al. PLoS One 2013; 8: e67606). Associated with virological failure during adefovir monotherapy in patients with lamivudine-resistant chronic hepatitis B (Lee et al. J Gastroenterol Hepatol 2012; 27: 300-305). rtA181S Mutation appearing in combination with rtM204I in lamivudine-treated (A529S) patients. In phenotypic assays, this mutation pattern conferred more than 1,000-fold resistance to lamivudine and emtricitabine, 6-fold resistance to clevudine, and 28.2- and 5.6-fold resistance to adefovir and tenofovir, respectively (Karatayli et al. Antivir Ther 2007; 12: 761-768). Adefovir- related mutation detected in many patients infected with genotype C HBV and experiencing virologic breakthrough. Patient-derived virus showed moderate resistance to adefovir in phenotypic assays (Liu et al. J Viral Hepat 2015; 22: 328-334).

646 Mutations a Comments rtA181T This amino acid substitution appeared in a lamivudine-resistant strain of HBV, (A529T) which retained an intact YMDD motif. This mutation induced a unique amino acid change (W172L) in the overlapping hepatitis B surface protein. In vitro, rtA181T conferred a 3-fold decrease in lamivudine susceptibility (Yatsuji et al. Antimicrob Agents Chemother 2006; 50: 3867-3874). Appeared in one HIV- HBV co-infected patient under adefovir therapy (Lacombe et al. AIDS 2006; 20: 2229-2231). It conferred 2- to 4.5-fold resistance to adefovir and 2- to 3-fold resistance to tenofovir, as proved by in-vitro phenotypic analysis (Villet et al. J Hepatol 2008; 48: 747-755). Depending on the mutation causing the rtA181T amino acid change, expression of the overlapping surface antigen can be affected due to the introduction of a stop codon substituting Trp172 in the S protein (i.e. rtA181T/sW172*). The mutant virus shows a dominant negative secretory defect (Warner & Locarnini. Hepatology 2008; 48: 88-98). Depending on the codon usage, the rtA181T mutation could appear in combination with sW172S or sW172L. These mutants do not show the HBsAg secretion defect characteristic of rtA181T/sW172* (Ahn et al. J Virol 2014; 88: 6805-6818; Zhou et al. Sci Rep 2016; 6: 39260). The rtA181T mutation appears to be related to an increased risk of hepatoma occurrence in patients with lamivudine-resistant chronic hepatitis B (Yeh et al. BMC Cancer 2011; 11: 398). rtA181V Identified as an adefovir-resistance mutation in treated patients, which is (A529V) often associated with rtN236T (Fung et al. J Hepatol 2005; 43: 937-943; Borroto-Esoda et al. J Hepatol 2007; 47: 492-498; Villet et al. J Hepatol 2008; 48: 747-755). HBV variants carrying this mutation also showed decreased susceptibility to lamivudine. Although it is unusually found in patients under lamivudine monotherapy (Gerolami et al. Antivir Ther 2006; 11: 1103-1106; Laoi et al. J Antimicrob Chemother 2007; 59: 807-809), its presence at a low frequency in HBV selected under lamivudine therapy facilitates its emergence as a major adefovir resistance mutation (Cho et al. Antiviral Res 2014; 112: 8-17), particularly in combination with rtF/Y221L (Vincenti et al. Antiviral Res 2017; 143: 62-68). rtT184F Emerged in patients treated with entecavir usually in combination with (T532F) rtL180M (Wang et al. Exp Ther Med 2016; 11: 117-123). rtT184G Emerged together with rtS202I and lamivudine-resistance mutations (T532G) (rtV173L, rtL180M and rtM204V) during entecavir/lamivudine combination therapy, conferring reduced susceptibility to entecavir in vitro (Tenney et al. Antimicrob Agents Chemother 2004; 48: 3498-3507; Tenney et al. Antimicrob Agents Chemother 2007; 51: 902-911). Recombinant hepatitis B virus containing RT substitutions rtT184A, rtT184F, rtT184G, rtT184L, rtT184M or rtT184S in a background of rtL180M/rtM204V showed a higher level of phenotypic resistance to entecavir in cell culture (Baldick et al. Hepatology 2008; 47: 1473-1482).

647 Mutations a Comments rtT184L Associated with entecavir resistance in patients, but usually in combination (T532L) with rtL180M or rtM204V (Mukaide et al. Antimicrob Agents Chemother 2010; 54: 882-889). rtT184S Associated with lamivudine therapy (Rhee et al. Antiviral Res 2010; 88: (T532S) 269-275). rtA186T Associated with rtI163V, rtL180M and rtM204V in a patient failing treatment (A534T) with entecavir. This amino acid substitution had a significant impact on entecavir resistance (Hayashi et al. J Hepatol 2015; 63: 546-553). rtI187V Mutation found with high prevalence in Chinese patients infected with (I535V) genotype B. In vitro phenotypic assays show that it impairs viral replication without affecting lamivudine susceptibility in the presence or absence of rtM204I or rtL180M/rtM204V (Fan et al. J Gen Virol 2014; 95: 2523-2530). rtV191I Emerged in one HIV-hepatitis B co-infected patient during treatment with (V539I) tenofovir. Impairs replication capacity while conferring phenotypic resistance to lamivudine (Amini-Bavil-Olyaee et al. AIDS 2009; 23: 268-272). This mutation can generate a defective surface protein due to the introduction of a stop codon at position 182 (sW182*) (Ding et al. Antiviral Res 2014; 102: 29-34). rtA194T Appeared in combination with lamivudine-specific mutations rtL180M and (A542T) rtM204V in 2 out of 43 HIV-coinfected patients treated with tenofovir (Sheldon et al. Antivir Ther 2005; 10: 727-734; Pastor et al. World J Gastroenterol 2009; 15: 753-755; Zhu et al. Antivir Chem Chemother 2011; 22: 13-22). This mutation reduced the replication capacity, while conferring partial resistance to tenofovir in vitro. However, it does not affect telbivudine and entecavir susceptibility (Amini-Bavil-Olyaee et al. Hepatology 2009; 49: 1158-1165). rtA200V Low-prevalent mutation coexisting with rtM204V and rtM204I in a patient (A548V) treated with sequential monotherapies including lamivudine, adefovir and entecavir (Sede et al. Antiviral Res 2012; 94: 184-187). rtS202G Associated with entecavir resistance in patients, usually in combination with (S550G) lamivudine resistance mutations such as rtM204V or rtL180M (Kobashi et al. Hepatol Int 2009; 3: 403-410; Kurashige et al. Antivir Ther 2009; 14: 873- 877; Choe et al. Antivir Ther 2009; 14: 985-993; Mukaide et al. Antimicrob Agents Chemother 2010; 54: 882-889). Found during virological breakthrough together with rtL180M and rtM204V in a patient treated with tenofovir and emtricitabine (Suzuki et al. Drug Des Devel Ther 2014; 8: 869-873). rtS202I This substitution which appeared as a secondary mutation in lamivudine- (S550I) resistant hepatitis B virus becomes predominant in vivo during the acquisition of entecavir resistance (Tenney et al. Antimicrob Agents Chemother 2007; 51: 902-911). In addition, other mutations at this position (e.g. rtS202A, rtS202C or rtS202G) increased phenotypic resistance to entecavir when the combination rtL180M/rtM204V was present (Baldick et al. Hepatology 2008; 47: 1473-1482).

648 Mutations a Comments rtM204I Appears in lamivudine-resistant clinical isolates (Ling et al. Hepatology (M552I) 1996; 24: 711-713; Allen et al. Hepatology 1998; 27: 1670-1677; Benhamou et al. Hepatology 1999; 30: 1302-1306; Gutfreund et al. J Hepatol 2000; 33: 469-475; Ben-Ari et al. Amer J Gastroenterol 2003; 98: 151-159; Ma et al. PLoS One 2013; 8: e67606). Isolates carrying this mutation show higher resistance to lamivudine than those having rtM204V (Allen et al. Hepatology 1998; 27: 1670-1677). Decreases replication efficacy as evidenced by the reduced amounts of pregenomic RNA, encapsidated progeny DNA, polymerase activity and virion release. Compensatory mutations in the precore or basal core promoter regions restore replication efficacy (Tacke et al. J Virol 2004; 78: 8524-8535). Confers high-level resistance to telbivudine (reviewed in Han. Expert Opin Investig Drugs 2005; 14: 511-519; Seifer et al. Antiviral Res 2009; 81: 147-155) and to clevudine (Kwon et al. J Virol 2010; 84: 4494-4503). Recent studies indicate that this mutation as well as rtM204V is the most prevalent in European patients (Hermans et al. J Infect Dis 2016; 213: 39-48). This mutation can be associated with defective HBsAg secretion due to the introduction of a stop codon at position 196 (sW196*). The rtM204I/sW196* variant was found to induce apoptosis in infected cells and was highly cytopathic (Colledge et al. Virology 2017; 501: 70-78). rtM204Q Mutation associated with lamivudine resistance, identified in a cohort of (M552Q) Chinese patients. Phenotypic studies indicate that it confers 89.9% replication

capacity compared with the wild-type virus, and increases the EC50 for lamivudine 76 times (Liu et al. PLoS One 2014; 9: e89015). Compared with viruses having the rtM204I mutation, those carrying rtM204Q show lower levels of lamivudine resistance but retain higher replication capacity. rtM204S Identified in one patient under lamivudine treatment, associated with (M552S) rtL180M. The combination L180M/M204S conferred resistance to lamivudine in vitro (Bozdayi et al. J Viral Hepat 2003; 10: 256-265). The patient infected with that virus was successfully treated with adefovir dipivoxil (Bozdayi et al. J Clin Virol 2004; 31: 76-77). rtM204V b Appears in lamivudine-resistant clinical isolates (Ling et al. Hepatology (M552V) 1996; 24: 711-713; Allen et al. Hepatology 1998; 27: 1670-1677; Benhamou et al. Hepatology 1999; 30: 1302-1306; Thibault et al. J Clin Microbiol 1999; 37: 3013-3016; Gutfreund et al. J Hepatol 2000; 33: 469-475; Ma et al. PLoS One 2013; 8: e67606). Decreases viral fitness (Ladner et al. Antimicrob Agents Chemother 1998; 42: 2128-2131; Melegari et al. Hepatology 1998; 27: 628-633; Lau et al. Hepatology 2000; 32: 828-834; Ono et al. J Clin Invest 2001; 107: 449-455; Tacke et al. J Virol 2004; 78: 8524-8535; Brunelle et al. Hepatology 2005; 41: 1391-1398). The fitness loss is caused by the low affinity for the incoming dNTPs shown by the mutant polymerase, an effect that can be enhanced in the presence of low dNTP levels (Gaillard et al. Antimicrob Agents Chemother 2002; 46: 1005-1013). Two mutations in the fingers subdomain of the polymerase (rtT128N and rtW153Q) were able to restore the replication phenotype when introduced in a replication-competent

649 Mutations a Comments HBV vector containing mutations rtL180M/M204V (Torresi et al. Virology 2002; 299: 88-99). Mutations in the envelope (Bock et al. Gastroenterology 2002; 122: 264-273), or in the precore or basal core promoter regions of the viral genome (Tacke et al. J Virol 2004; 78: 8524-8535) have also been shown to increase the replication capacity of lamivudine-resistant HBV. In patients infected with HIV and HBV and treated with lamivudine, this mutation is selected both in HBV polymerase and in HIV-1 reverse transcriptase (Cooley et al. AIDS 2003; 17: 1649-1657). However, there is no simultaneous appearance of resistance in both viruses (Pillay et al. AIDS 2000; 14: 1111-1116). The rtM204V substitution facilitates the emergence of the surface protein mutation sI195M (Ding et al. Antiviral Res 2014; 102: 29-34). It shows a high prevalence in Europe in chronically infected patients (Hermans et al. J Infect Dis 2016; 213: 39-48). rtV207I Mutation related to penciclovir resistance, and conferring a 10-fold (V555I) decreased replication capacity (Zoulim et al. Hepatology 1997; 26: A1200). Also associated with resistance to lamivudine in vitro (Zöllner et al. J Clin Microbiol 2005; 43: 2503-2505) and in patients (Sede et al. Antiviral Res 2012; 94: 184-187; Rodríguez-Frías et al. PLoS One 2012; 7: e37874). rtV214A Mutation detected in patients who failed adefovir therapy (Bartholomeusz (V562A) et al. Antiviral Res 2005; 65: A32), or previously treated with nucleos(t)ide analogues (Pastor et al. World J Gastroenterol 2009; 15: 753-755). rtQ215S Although this mutation has been detected in patients failing therapy with (Q563S) adefovir (Bartholomeusz et al. Antiviral Res 2005; 65: A32), phenotypic analysis in vitro show that rtQ215 substitutions neither impair the viral replication efficiency nor the susceptibility to lamivudine or adefovir (Amini-Bavil-Olyaee et al. J Hepatol 2009; 51: 647-654). rtL217R This mutation appeared in virus from patients failing previous adefovir and (L565R) lamivudine treatment, but responding successfully to tenofovir (Schildgen et al. AIDS 2004; 18: 2325-2341; Bottecchia et al. J Antimicrob Chemother 2008; 62: 626-640). rtE218G Amino acid substitution emerging under treatment with adefovir. Phenotypic (E566G) analyses demonstrated that it could independently confer resistance to adefovir in vitro, with an IC50 5.5-fold higher than wild-type hepatitis B virus [Liu et al. J Viral Hepat 2010; 17 (suppl. 1): 66-72]. rtS219A Appeared together with rtL80[L/V], rtL91I, rtM204I, rtN238D and rtY245H (S567A) in virus obtained from one patient after successive failures of lamivudine, adefovir and entecavir monotherapies. The rtS219A mutation appears to contribute to the emergence of entecavir resistance (Karatayli et al. J Clin Virol 2012; 53: 130-134). rtF221L Associated with the use of adefovir in lamivudine experienced patients, rtY221L infected with HBV A or D genotypes, usually in combination with the (F/Y569L) substitution rtA181V (Vincenti et al. Antiviral Res 2017; 143: 62-68).

650 Mutations a Comments rtL229F Found in patients treated with lamivudine, before the selection of rtM204I. It (L577F) had no impact on drug susceptibility but restored the replication capacity of strains bearing rtM204I (Ji et al. J Clin Virol 2012; 54: 66-72). rtL229V Appearing during clevudine therapy, rtL229V was identified as a compensatory (L577V) mutation for impaired replication of mutant virus having rtM204I (Kwon et al. J Virol 2010; 84: 4494-4503). rtI233V It has been reported that this amino acid substitution causes adefovir (I581V) treatment failure (Schildgen et al. New Engl J Med 2006; 354: 1807-1812; Schildgen et al. J Clin Microbiol 2010; 48: 631-634), but these findings have been challenged by others (Curtis et al. J Infect Dis 2007; 196: 1483-1486; Geipel et al. N Engl J Med 2014; 370: 1667-1668), and the discussion is still active (Sirma & Schildgen. N Engl J Med 2014; 371: 482-483; Geipel et al. N Engl J Med 2014; 371: 483). Others have shown that the mutation (usually associated with rtA181T) may have a negative effect on viral replication (Ahn et al. J Virol 2014; 88: 6805-6818). However, rtI233V was found to restore the replication capacity of mutant HBV bearing the rtN236T substitution, functioning as a compensatory mutation associated with adefovir resistance (Liu et al. Antivir Ther 2016; 21: 9-16). rtN236T Appeared in patients treated with adefovir dipivoxil, and conferred resistance to (N584T) the drug when tested in vitro (Angus et al. Gastroenterology 2003; 125: 292-297; Hadziyannis et al. N Engl J Med 2005; 352: 2673-2681; Fung et al. J Hepatol 2005; 43: 937-943). Virus carrying this mutation together with L180M and M204V showed decreased susceptibility to adefovir, tenofovir, lamivudine and entecavir, and impaired replication in the absence of drugs (Brunelle et al. Hepatology 2005; 41: 1391-1398). However, in patients with rtN236T, wild- type and mutant virus showed similar rates of HBV DNA decline in adefovir- refractory individuals who switched to tenofovir or tenofovir/emtricitabine (Svarovskaia et al. J Viral Hepat 2013; 20: 131-140). See also rtA181V. rtN236V Infrequent amino acid substitution detected at virological breakthrough in (N584V) adefovir-treated patients. It confers moderate resistance to the drug and reduces the viral replication capacity in replicon-based assays (Liu et al. Antivir Ther 2014; 19: 551-558). rtY245H Contributes to the emergence of entecavir resistance (see above, rtS219A). (Y593H) rtF249A Confers reduced susceptibility to tenofovir in vitro (Qin et al. Antiviral Res (F597A) 2013; 97: 93-100).

651 Mutations a Comments rtM250V Confers reduced entecavir susceptibility in vitro and in vivo, in the presence (M598V) of lamivudine-resistance mutations (rtV173L, rtL180M and rtM204V) (Tenney et al. Antimicrob Agents Chemother 2004; 48: 3498-3507; Tenney et al. Antimicrob Agents Chemother 2007; 51: 902-911). Similar effects were observed for mutations rtM250C, rtM250I and rtM250L using recombinant hepatitis B virus clones (Baldick et al. Hepatology 2008; 47: 1473-1482). rtQ267H Often found in patients receiving lamivudine. It increases viral fitness but (Q615H) produces a modest reduction in the susceptibility to the drug in a wild-type sequence context and in the presence of rtL180M/rtM204V (Qin et al. Hepat Mon 2013; 13: e12160). The substitution rtH/L267Q appeared with higher prevalence in the viral quasispecies pool from patients failing lamivudine treatment as compared with untreated individuals (Banerjee et al. Sci Rep 2017; 7: 44742). rtL269I Compensatory mutation that appears in heavily mutated multidrug-resistant (L617I) HBV. Alone, it does not confer resistance to lamivudine, entecavir, adefovir or tenofovir, but when combined with a YMDD mutation, the replication capacity of the virus was enhanced by several folds in the presence of lamivudine or entecavir (Ahn et al. PLoS One 2015; 10: e0136728). a Mutations are designated according to the nomenclature proposed by Stuyver et al. Hepatology 2001; 33: 751-757. The notation indicated within parenthesis corresponds to the classical numbering for HBV subtype adw2. b This is a mutation of the YMDD sequence within motif C.

Other mutations probably with minor clinical significance are rtN53D, rtL91I, rtI121L, rtF/H122L, rtQ130P, rtN131D, rtF166L, rtT184S, rtA200V, rtS213T, rtH248N, rtR266I and rtV278I (for lamivudine resistance), rtN238H (for adefovir and lamivudine resistance), rtP177L and rtT184S (for penciclovir resistance), rtS246T (for entecavir resistance), and rtA181V (for adefovir dipivoxil resistance) (Angus et al. Gastroenterology 2003; 125: 292-297; Halfon et al. Revue Medec Intern 2003; 24: 786-793; Hu et al. J Med Virol 2012; 84: 34-43; Zhong et al. Antiviral Res 2012; 93: 185-190; Banerjee et al. Sci Rep 2017; 7: 44742). Sequence analysis using a large database including HBV-infected patients treated with nucleoside/nucleotide analogues showed a higher frequency of mutations rtL82M, rtS85A, rtA200V and rtQ215S in comparison with untreated individuals (Rhee et al. Antiviral Res. 2010; 88: 269-275). For reviews, see Locarnini & Warner. Antivir Ther 2007; 12 (suppl. 3): H15-H23; Iser & Sasadeusz. J Gastroenterol Hepatol 2008; 23: 699- 706; Yuen et al. Lancet Infect Dis 2009; 9: 256-264.

In another study involving patients mostly infected by HBV-genotypes A and D (Mirandola et al. Antiviral Res 2012; 96: 422-429), authors found that mutations rtS85F,

652 rtL91I and rtC256G were more prevalent in HBV genotype D of patients treated with lamivudine, while rtI53V, rtW153R and rtF221Y show increased prevalence in adefovir- treated patients infected with HBV genotype A.

There are several examples of nucleoside/nucleotide analogue resistance mutations (e.g. rtS78T, rtA181T and rtV191I) that produce an alteration of antigenicity due to changes in the HBV surface antigen (HBsAg). For a review, see Locarnini and Yuen. Antivir Ther 2010; 15: 451-461.

Deletions affecting the sequence of pre S2 domain of the surface antigen (HBsAg) are frequently observed in patients treated with nucleoside analogues, often associated with rtL190M or rtM204I (Zhang et al. BMC Microbiol 2012; 12: 307).

Figure 13.2. Resistance levels to nucleos(t)ide inhibitors conferred by mutations frequently found in treated HBV-infected patients. High-level (>20-fold increase of the

IC50 for the inhibitor), moderate (5- to 20-fold increase) and low-level (2.5- to 5-fold increase) resistance is indicated in solid, hatched and grey boxes, respectively. Open boxes are used to indicated amino acid substitutions or combinations of amino acid

653 changes that retain susceptibility to the drug. Double representations are used in those cases were we found significant discrepancies in the literature. Data were taken from the following references: L80I/M204I [1-3]; V173L/L180M/M204V [4-6]; L180M [5,7-10]; L180M/M204I [1,3,6-9,11]; L180M/M204V [1,3,5-10,12-15]; L180M/S202G/M204V [15]; L180M/M204V/N236T [14,16]; A181S [17,18]; A181S/M204I [18]; A181S/N236T [17]; A181T [15,18], A181T/N236T [15,19,20]; A181V/N236T [19]; A181V [1,3,17,19]; A194T [1]; M204I [1-3,6-10,12,13]; M204V [1,5,7,9-13,21]; I233V [22]; I233V/N236T [22]; N236T [1,14-17,19, 22]; and N236V [23].

1. Seifer et al. Antiviral Res 2009; 81: 147-155. 2. Yin et al. J Gen Virol 2015; 96: 3302-3312. 3. Geipel et al. Antivir Ther 2015; 20: 779-787. 4. Tenney et al. Antimicrob Agents Chemother 2004; 48: 3498-3507. 5. Delaney IV et al. J Virol 2003; 77: 11833-11841. 6. Yang et al. Antivir Ther 2005; 10: 625-633. 7. Das et al. J Virol 2001; 75: 4771-4779. 8. Delaney IV et al. Antimicrob Agents Chemother 2001; 45: 1705-1713. 9. Ono et al. J Clin Invest 2001; 107: 449-455. 10. Chin et al. Antimicrob Agents Chemother 2001; 45: 2495-2501. 11. Langley et al. J Virol 2007; 81: 3992-4001. 12. Xiong et al. Hepatology 1998; 28: 1669-1673. 13. Ono-Nita et al. J Clin Invest 1999; 103: 1635-1640. 14. Brunelle et al. Hepatology 2005; 41: 1391-1398. 15. Murakami et al. J Infect 2016; 72: 91-102. 16. Jacquard et al. Antimicrob Agents Chemother 2006; 50: 955-961. 17. Liu et al. J Viral Hepat 2015; 22: 328-334. 18. Karatayli et al. Antivir Ther 2007; 12: 761-768. 19. Qi et al. Antivir Ther 2007; 12: 355-362. 20. Takamatsu et al. Hepatology 2015; 62: 1024-1036. 21. Allen et al. Hepatology 1998; 27: 1670-1677. 22. Liu et al. Antivir Ther 2016; 21: 9-16. 23. Liu et al. Antivir Ther 2014; 19: 551-558.

Additional data can be obtained from reviews on the molecular biology of HBV (Ganem & Prince. N Engl J Med 2004; 350: 1118-1129; Lucifora & Zoulim. Future Virol 2011; 6: 599-614; Glebe & Bremer. Semin Liver Dis 2013; 33: 103-112; Delaney IV. Antiviral Res 2013; 99: 34-48), resistance to nucleoside and nucleotide analogues in chronic hepatitis B infection (Michailidis et al. Int J Biochem Cell Biol 2012; 44: 1060-1071; Menéndez- Arias et al. Curr Opin Virol 2014; 8: 1-9; Caligiuri et al. World J Gastroenterol 2016; 22: 145-154), lamivudine therapy (Jarvis & Faulds. Drugs 1999; 58: 101-141; Lau et al. Hepatology 2000; 32: 828-834), entecavir therapy (Rivkin. Curr Med Res Opin 2005; 21: 1845-1856; Tenney. Antivir Ther 2010; 15: 529-535), detection of HBV resistance (Neumann-Fraune et al. Intervirology 2014; 57: 232-236), and fitness of drug-resistant HBV mutants (Durantel. Antivir Ther 2010; 15: 521-527).

654 Table 13.3. HEPATITIS B VIRUS MUTATIONS OR COMBINATIONS OF MUTATIONS ASSOCIATED WITH RESISTANCE TO NUCLEOS(T)IDE RT INHIBITORS

Drug Major mutations Accessory mutations Lamivudine rtL80I/V, rtV173L, rtL180M, rtS117F, rtL180C, rtA181V, rtA181S, rtA181T, rtT184S, rtV191I, rtS202G, rtM204S, rtM204I/V, rtM204Q and rtV207I rtL217R, rtL229F, rtQ267H and rtL269I Adefovir rtL80I/V, rtA181S, rtA181T, rtS78T, rtL180M, rtV214A, rtA181V, rtE218G and rtN236T rtQ215S, rtL217R, rtI233V and rtN236V Entecavir rtI169T, rtT184A/F/G/L/M/S, rtI163V, rtA186T, rtS219A, rtS202C/G/I, rtM204I/V and rtY245H, rtS246T and rtL269I rtM250I/V/L Telbivudine rtL80I/V and rtM204I/V Tenofovir rtP177G, rtA181S, rtA181T, rtA194T and rtF249A Emtricitabine rtA181S and rtM204I/V Clevudine rtA181S and rtM204I/V rtL229V

Underlined bold amino acid substitutions are major mutations and by themselves confer high-level resistance to the drug. Important resistance mutations are indicated in bold, and other mutations associated with resistance to the corresponding inhibitor are shown in plain text. Accessory mutations shown on the right column are those that appeared sporadically, have limited effect on resistance, or may contribute to increase viral fitness of resistant mutants (Menéndez-Arias et al. Curr Opin Virol 2014; 8: 1-9). Additional mutations associated with resistance to lamivudine (rtL269I), entecavir (rtI163V, rtA186T and rtL269I) and telvibudine (rtL80I/V) have been recently described (Ahn et al. PLoS One 2015; 10: e0136728; Hayashi et al. J Hepatol 2015; 63: 546-553; Yin et al. J Gen Virol 2015; 96: 3302-3312).

655 HEPATITIS C

Table 13.4. CURRENT DRUGS APPROVED FOR TREATMENT OF HEPATITIS C

Regimen Drug name Drug class and comments Manufacturer Monotherapy Intron A Recombinant interferon α-2b Schering-Plough Roferon A Recombinant interferon α-2a Roche Infergen Consensus interferon α Cm1 Three Rivers Pharma PEG-Intron Pegylated interferon α-2b Schering-Plough Pegasys Pegylated interferon α-2a Roche Combination Roferon A + Recombinant interferon α-2a + Roche ribavirina ribavirin Rebetron Recombinant interferon α-2b + Schering-Plough (Intron A + ribavirin Rebetol) PEG-Intron Pegylated interferon α-2b + ribavirin Schering-Plough + Rebetol Pegasys + Pegylated interferon α-2a + ribavirin Roche Copegus Boceprevir HCV protease inhibitor (to be used in Merck (MSD) (Victrelis) combination with pegylated interferon and ribavirin for treatment of infections caused by HCV genotype 1) Telaprevir HCV protease inhibitor (to be used in Vertex (Incivek) combination with pegylated interferon Pharmaceuticals, and ribavirinfor treatment of infections Janssen Labs., caused by HCV genotype 1) Mitsubishi Simeprevir HCV protease inhibitor (to be used in Janssen (Olysio) combination with pegylated interferon Laboratories and ribavirin for treatment of infections caused by HCV genotypes 1a and 1b) Sofosbuvir Viral RNA polymerase inhibitor Gilead Sciences (Sovaldi) This drug should be used in combination with ribavirin for treating patients infected with HCV genotypes 2 and 3. When infected with genotypes 1 and 4, they should receive triple therapy including pegylated interferon 656 Regimen Drug name Drug class and comments Manufacturer Sofosbuvir Ledipasvir is an inhibitor of NS5A. The Gilead Sciences + Ledipasvir combination pill has been approved to (Harvoni) treat chronic HCV genotype 1 infection Daclatasvir HCV NS4A inhibitor indicated for use Bristol Myers (Daklinza) b with sofosbuvir for the treatment of Squibb patients with chronic HCV genotype 3 infection Paritaprevir/ Indicated in combination with ribavirin AbbVie ritonavir + for the treatment of patients with Dasabuvir + genotype 4 chronic HCV infection Ombitasvir without cirrhosis. As given, it is (Technivie) recommended for naïve patients that cannot tolerate ribavirin. Paritaprevir/ Indicated (with or without ribavirin) AbbVie ritonavir + for treatment of patients infected Ombitasvir with genotype 1 chronic hepatitis C tablets co- virus (HCV), including those with packaged with compensated cirrhosis Dasabuvir tablets (Viekira Pak) Elbasvir + Indicated for treatment of patients Merck Grazoprevir infected with HCV genotypes 1 and 4. (Zepatier) Sofosbuvir + Indicated for the treatment of patients Gilead Sciences Velpatasvir infected with HCV genotypes 1, 2, 3, 4, (Epclusa) 5, or 6, and having chronic hepatitis C. a Ribavirin is commercialized as Ribasphere, RibaPak, Copegus, Rebetol and RibaTab. b Daclatasvir in combination with asunaprevir (BMS-650032) has been approved for treatment of HCV-infected patients in Japan, but Bristol Myers Squibb stopped pursuing approval of the combination in the U.S. (as of October 2014). References: Nyalakonda & Utay. Curr Opin Infect Dis 2015; 28: 471-478; Feld & Foster. J Hepatol 2016; 65: S130-S142; Zopf et al. World J Hepatol 2016; 8: 139-147; Carter et al. J Clin Pharmacol 2017; 57: 287-296; Li & De Clercq. Antiviral Res 2017; 142: 83-122; Behara & Reau. Curr Opin Gastroenterol 2017; 33: 115-119; http://www.fda.gov/forpatients/ illness/hepatitisbc/ucm408658.htm HCV, hepatitis C virus

657 Table 13.5. EXPERIMENTAL DRUGS AND TREATMENTS FOR HEPATITIS C

Drug type Type of molecule Drug name and company Status Interferons Inteferon ß-1a Rebif (EMD Serono) Phase III Fusion protein Zalbin (albuferon α-2b) (Human Phase III interferon α-albumin Genome Sciences) Pegylated (Bristol Myers Squibb) Phase II interferon λ Purified multi- Multiferon (Viragen) Phase II subtype human interferon α Interferon Ω Omega interferon (Intarcia Phase II Therapeutics) Medusa interferon Medusa interferon (Flamel Technologies) Phase II Oral interferon α Oral interferon α Phase II (Amarillo Biosciences/CytoPharm) Controlled release of Locteron (Biolex/OctoPlus) Phase II interferon Pegylated alfacon-1 Peg-alfacon (InterMune) Phase I Ribavirin Amidine prodrug of Viramidine (Valeant Pharmaceuticals) Phase III alternatives ribavirin Antiviral drug Amantadine (Hoffmann-La Roche) Phase III Guanosine analogue Taribavirin (Valeant Pharmaceuticals) Phase II Immunomo- Stimulator of T cell Thymalfasin (Thymosin α-1, zadaxin) Phase III dulators function (Sci-Clone) Anti- Bavituximab (Peregrine Phase II phosphatidylserine Pharmaceuticals) monoclonal antibody Hepatoprotective NOV-205 (Novelos) Phase II agent STAT3 signalling SCV-07 (SciClone Pharma) Phase II inhibitor

658 Drug type Type of molecule Drug name and company Status Inhibitors NS3 serine protease Faldaprevir (BI-201335) (Boehringer Phase III of viral inhibitors Ingelheim) enzymes Danoprevir (RG7227, ITMN-191) Phase III (InterMune/Roche) Glecaprevir (ABT-493) (AbbVie) Phase III Voxilaprevir (GS-9857) (Gilead) Phase III GS-9256 (Gilead) Phase II Vedroprevir (GS-9451) (Gilead) Phase II BMS-791325 (Bristol Myers Squibb) Phase II IDX320 (Idenix) Phase II Sovaprevir (ACH-1625) (Achillion Phase II Pharmaceuticals) Deldeprevir (Neceprevir, ACH-2684) Phase II (Achillion Pharmaceuticals) VX-985 (Vertex Pharmaceuticals) Phase I/II Ciluprevir (BILN-2061) (Boehringer Studies Ingelheim) cancelled Narlaprevir (SCH 900518) (Schering- Studies Plough) cancelled Vaniprevir (MK-7009) (Merck) Discontinued VX-135 (Vertex) On hold RNA polymerase Deleobuvir (BI 207127) (Boehringer Phase III inhibitors Ingelheim) Beclabuvir (BMS-791325) (Bristol- Phase III Myers Squibb) Mericitabine (RG-7128, RO5024048) Phase III (Roche/Genentech) Tegobuvir (GS-9190) (Gilead) Phase II Setrobuvir (ANA-598, RG7790) Phase II (Roche/Genentech) R1626 (Roche) Phase II

659 Drug type Type of molecule Drug name and company Status ACH-3422 (Achillion) Phase II IDX184 (Idenix Pharmaceuticals) Phase II TMC-055 (Janssen) Phase II MK-8876 (Merck) Phase II GS-9669 (Gilead) Phase II Lomibuvir (VX-222, VCH-222) Phase II (Vertex Pharmaceuticals) TMC647055 (Janssen) Phase II VCH-916 (Vertex) Phase II VX-135 (Vertex) Phase II INX-08189 (Inhibitex Inc.) Phase I/II PSI-352938 (Pharmasset) Phase I/II

VX-759 (VCH 759) (Vertex) Phase I GS-9851 (PSI-7851) (Gilead) Phase I GSK625433 (Glaxo Smith Kline) Phase I MK-0608 (Merck) Phase I MK-3281 (Merck) Phase I TMC649128 (Janssen) Phase I RG7109 (Roche) Phase I BMS094 (INX-189) (Bristol-Myers Studies Squibb) cancelled Nesbuvir (HCV-796) Studies (ViroPharma/Wyeth) cancelled Valopicitabine (NM-283) Studies (Idenix Pharmaceuticals) cancelled

Filibuvir (PF-00868554) (Pfizer) Discontinued ABT-072 (AbbVie) Discontinued

660 Drug type Type of molecule Drug name and company Status NS5A inhibitors Pibrentasvir (ABT-530) (AbbVie) Phase III Ravidasvir (PPI-668) (Presidio) Phase II/III Samatasvir (IDX719) (Idenix Phase II Pharmaceuticals) BMS-824393 (Bristol-Myers Squibb) Phase II PPI-461 (Presidio) Phase II GSK2336805 (Glaxo Smith Kline) Phase II Odalasvir (ACH-3102) (Achillion Phase II Pharmaceuticals) EDP-239 (Enanta) Phase II Ruzasvir (MK-8408) (Merck) Phase II ACH-2928 (Achillion Pharmaceuticals) Phase II BMS-824393 (Bristol-Myers Squibb) Phase II AZD7295 (Astra Zeneca) Phase II Helicase activity BTN10 and BTN11 Phase I of NS3 Trixsalen Phase I Therapeutic Civacir (immunoglobulin) (Biotest Phase III vaccines Pharmaceutical Corporation) Chronvac-C [Chron Tech Pharma Phase II (Triprep) / Inovio] GI5005 (Globeimmune) Phase II IC41 (Intercell/Novartis) Phase II TG4040 (MVA-HCV) (Transgene) Phase II Others Oral phospholipic IP-501 (Indevus Pharmaceuticals) Phase III antifibrotics Antifibrotic G1262570 (Glaxo Smith Kline) Phase II Cyclophilin Alisporivir (Debio-025) (Novartis) a Phase III inhibitors NIM811 (Novartis) Phase II SCY-635 (Scynexis) Phase II

661 Drug type Type of molecule Drug name and company Status Others Entry inhibitors ITX-5061 (iTherX) Phase II (continuation) EI-1 (Bristol-Myers Squibb) Phase I PRO0371155 (Progenics Phase I Pharmaceuticals, Inc.) b NS5B inhibitors GSK4809 c Phase I IRES analogue VGX-410 (VGX Pharmaceuticals) Phase II Interferon enhancers EMZ702 (Transition Therapeutics) Phase II Triterpenoids: toosendanin Phase I Caspase inhibitors ID-6556 (Idun Pharmaceuticals) Phase III Salicylamide Alinia (nitazosanide) (Romark Phase II (antiprotozoal agent) Laboratories) Antihistamine Chlorcyclizine HCl d Anti-CD20 Rituximab (Genentech) Phase I/II monoclonal antibody Monoclonal XLT-6865 (XTL) Phase I/II antibodies MBL-HCV (Univ. Mass. Med. School) Phase I AR5A (Novo Nordisk, Scripps) Preclinical HMG-CoA Fluvastatin Phase I/II reductase inhibitor Hepatoprotective Silibinin (silybin) e Phase II agent microRNA-targeted Miravirsen (anti-miR-122, SPC3649) Phase II drug (Santaris Pharma) miRNA antagonists RG-101 (Regulus Therapeutics) Phase II (miR-122) a Alisporivir (Debio-025) inhibits HIV-1 by interfering with an early event in the replication cycle (Daelemans et al. Antiviral Res 2010; 85: 418-421; Flisiak et al. Expert Opin Investig Drugs 2012; 21: 375-382; Gallay & Lin. Drug Des Devel Ther 2013; 7: 105-115). Currently is on clinical hold because of pancreatitis when combined with peg- interferon α and ribavirin (Scheel & Rice. Nat Med 2013; 19: 837-849).

662 b PRO0371155 is a 1,3,5-triazine derivative that inhibits HCV genotype 1a/2a entry. Resistance to this compound is mediated by a single amino acid substitution (V719G) in the transmembrane domain of E2 (Coburn et al. PLoS One 2012; 7: e35351). c Shows less than 100 nM activity against the HCV replicon in tissue culture, but reduced efficiency against HCV genotype 2 strains due to the presence of a single amino acid change at position 98 (F98L) (Arico-Muendel et al. Antimicrob Agents Chemother 2015; 59: 3450-3459). d Chlorcyclizine HCl is a first-generation antihistamine drug approved in the 1940s that showed high antiviral activity and synergistic with several approved anti-HCV drugs (He et al. Sci Transl Med 2015; 7: 282ra49). e The mechanism of action of silibinin is not completely clear. A direct inhibition of the viral polymerase has been reported in vitro (Ahmed-Belkacem et al. Gastroenterology 2010; 138: 1112-1122). However, recently reported evidence suggests that silibinin might target an interaction between NS4B and NS3/4A. NS4B is a key element that induces alterations in the membrane HCV replication sites. An NS4B mutation selected in vitro in the presence of silibinin confers partial resistance to the drug (Esser-Nobis et al. Hepatology 2013; 57: 953-963). Live-cell imaging studies show that silibinin inhibits HCV entry into human hepatocytes by hindering clathrin-dependent trafficking, and therefore interfering with endocytosis (Blaising et al. Cell Microbiol 2013; 15: 1866-1882).

References: General (Ahmed & Felmlee. Viruses 2015; 7: 6716-6729; Del Bello et al. Curr Opin HIV AIDS 2015; 10: 337-347; Lontok et al. Hepatology 2015; 62: 1623-1632; Chen et al. Sci Rep 2016; 6: 20310; Sarrazin. J Hepatol 2016; 64: 486-504; Pawlotsky. Gastroenterology 2016; 151; 70-86; Li & De Clercq. Antiviral Res 2017; 142: 83-122); inhibitors of the viral protease (Wu et al. World J Gastroenterol 2013, 19: 8940-8948; De Luca et al. Curr Opin Pharmacol 2014; 18: 9-17; Kieffer & George. Curr Opin Virol 2014; 8. 16-21; McCauley & Rudd. Curr Opin Pharmacol 2016; 30: 84-92); NS4B inhibitors (Arico-Muendel et al. Antimicrob Agents Chemother 2015; 59: 3450-3459); NS5A inhibitors (Pawlotsky. J Hepatol 2013; 59: 375-382; Gao. Curr Opin Virol 2013; 3: 514-520; Belema et al. J Med Chem 2014; 57: 1643-1672; Nakamoto et al. World J Gastroenterol 2014; 20: 2902-2912; Issur & Götte. Viruses 2014; 6: 4227-4241; Lim & Gallay. Curr Opin Virol 2014; 8: 30-37; Ivanenkov et al. Expert Opin Ther Pat 2017; 27: 401-414); NS5B polymerase inhibitors (Götte. Curr Opin Virol 2014; 8: 104-108; Eltahla et al. Antimicrob Agents Chemother 2014; 58: 7215-7224; Eltahla et al. Viruses 2015; 7: 5206-5224); cyclophilin inhibitors (Tang. Viruses 2010; 2: 1621-1634); and entry inhibitors (Fofana et al. Antiviral Res 2014; 104: 136-142; Colpitts & Baumert. Hepatol Int 2016; 10: 741-748).

663 HCV serine protease 1 APITAYAQQT RGLLGCIITS LTGRDKNQVE GEVQIVSTAA QTFLATCING 51 VCWTVYHGAG TRTIASPKGP VIQMYTNVDQ DLVGWPAPQG SRSLTPCTCG 101 SSDLYLVTRH ADVIPVRRRG DSRGSLLSPR PISYLKGSSG GPLLCPAGHA 151 VGIFRAAVCT RGVAKAVDFI PVENLETTMR S

Figure 13.3. Amino acid sequence of the hepatitis C virus protease genotype 1a (GenBank accession number M62321).

NS5A 1 SGSWLRDIWD WICEVLSDFK TWLKAKLMPQ LPGIPFVSCQ RGYKGVWRVD 51 GIMHTRCHCG AEITGHVKNG TMRIVGPRTC RNMWSGTFPI NAYTTGPCTP 101 LPAPNYTFAL WRVSAEEYVE IRQVGDFHYV TGMTTDNLKC PCQVPSPEFF 151 TELDGVRLHR FAPPCKPLLR EEVSFRVGLH EYPVGSQLPC EPEPDVAVLT 201 SMLTDPSHIT AEAAGRRLAR GSPPSVASSS ASQLSAPSLK ATCTANHDSP 251 DAELIEANLL WRQEMGGNIT RVESENKVVI LDSFDPLVAE EDEREISVPA 301 EILRKSRRFA QALPVWARPD YNPPLVETWK KPDYEPPVVH GCPLPPPKSP 351 PVPPPRKKRT VVLTESTLST ALAELATRSF GSSSTSGITG DNTTTSSEPA 401 PSGCPPDSDA ESYSSMPPLE GEPGDPDLSD GSWSTVSSEA NAEDVVCC

Figure 13.4. Amino acid sequence of the hepatitis C virus NS5A protein genotype 1a (GenBank accession number M62321).

664 HCV RNA-dependent RNA polymerase (NS5B) 1 SMSYSWTGAL VTPCAAEEQK LPINALSNSL LRHHNLVYST TSRSACQRQK 51 KVTFDRLQVL DSHYQDVLKE VKAAASKVKA NLLSVEEACS LTPPHSAKSK 101 FGYGAKDVRC HARKAVTHIN SVWKDLLEDN VTPIDTTIMA KNEVFCVQPE 151 KGGRKPARLI VFPDLGVRVC EKMALYDVVT KLPLAVMGSS YGFQYSPGQR 201 VEFLVQAWKS KKTPMGFSYD TRCFDSTVTE SDIRTEEAIY QCCDLDPQAR 251 VAIKSLTERL YVGGPLTNSR GENCGYRRCR ASGVLTTSCG NTLTCYIKAR 301 AACRAAGLQD CTMLVCGDDL VVICESAGVQ EDAASLRAFT EAMTRYSAPP 351 GDPPQPEYDL ELITSCSSNV SVAHDGAGKR VYYLTRDPTT PLARAAWETA 401 RHTPVNSWLG NIIMFAPTLW ARMILMTHFF SVLIARDQLE QALDCEIYGA 451 CYSIEPLDLP PIIQRLHGLS AFSLHSYSPG EINRVAACLR KLGVPPLRAW 501 RHRARSVRAR LLARGGRAAI CGKYLFNWAV RTKLKLTPIA AAGQLDLSGW 551 FTAGYSGGDI YHSVSHARPR WIWFCLLLLA AGVGIYLLPN R

Figure 13.5. Amino acid sequence of the RNA-dependent RNA polymerase of hepatitis C virus genotype 1a (GenBank accession number M62321).

665 Table 13.6. DATA ON RESISTANCE TO DRUGS TARGETING HEPATITIS C VIRUS (HCV)

1. Interferon

Comments Over 50% of patients infected with HCV genotype 1 are resistant to current therapies. Resistance to interferon has been associated with nucleotide-sequence patterns within the NS5A gene of HCV genotype 1b, at the interferon-sensitivity-determining region (ISDR; amino acids 2209 to 2248 according to numbering of HCV-J) (Enomoto et al. J Clin Invest 1995; 96: 224-230; Witherell & Beineke. J Med Virol 2001; 63: 8-16; reviewed in Hofmann et al. J Clin Virol 2005; 32: 86-91; Enomoto & Nishiguchi. World J Hepatol 2015; 7: 2681-2687). NS5A binds and modulates PKR, a cellular protein kinase induced by interferon that phosphorylates eukaryotic initiation factor 2 (eIF-2) shutting off protein synthesis (Gale et al. Mol Cell Biol 1998; 18: 5208-5218). ISG56, an antagonist of HCV RNA translation that acts on the polyribosome complex, has been identified as a host factor that contributes to selection of interferon-resistant viral strains (Sumpter et al. J Virol 2004; 78: 11591-11604). In addition, the number of amino acid substitutions in the viral RNA polymerase (NS5B) correlate with the number of mutations in the ISDR, suggesting an effect on interferon response (Watanabe et al. J Med Virol 2005; 75: 504-512). Interferon-resistance patterns are rather complex and algorithms are being developed to predict resistance from the nucleotide sequence (Sarrazin et al. J Virol 2002; 76: 11079-11090). Multiple serial passages in the presence of different doses of interferon α led to the selection of virus with enhanced progeny production. These viruses contained multiple, nonsynonymous mutations scattered throughout the genome. A common trait of these viruses was an increased shutoff of host cell protein synthesis (Perales et al. J Virol 2013; 87: 7593-7607). Apart from mutations in the ISDR, amino acid substitutions in the HCV core protein such as R70Q (Akuta et al. J Med Virol 2006; 78: 83-90; Okanoue et al. J Gastroenterol 2009; 44: 952- 963; Kurbanov et al. J Infect Dis 2010; 201: 1663-1671; Hayashi et al. J Viral Hepat 2011; 18: 280-286), T75A (Alhamlan et al. J Med Virol 2014; 86: 224-234), L91M (Akuta et al. J Med Virol 2006; 78: 83-90; Kitamura et al. Antivir Ther 2010; 15: 1087-1097) and T110N (Di Lello et al. Arch Virol 2014; 159: 3345-3351) correlate with an increase risk of failure to treatment with pegylated interferon and ribavirin. This association is stronger with HCV genotype 1b. Gln70 in the HCV core protein and additional mutations in NS3 and NS5A are significantly associated with the development of hepatocellular carcinoma (El-Shamy et al. Hepatology 2013; 58: 555- 563). The association of L91M with treatment failure has not been found in all studies, and the association of T110N was observed in a cohort of 100 Caucasian patients infected with HCV genotype 1b. The expression level of an interferon signal attenuator, SOCS3, was significantly higher for the R70Q, R70H and L91M mutants of the HCV core protein than for the wild type, supporting interferon resistance controlled by interleukin 6 (Funaoka et al. J Virol 2011; 85: 5986-5984). Interestingly, the substitution Y93H that confers high-level resistance to daclastavir and other NS5A inhibitors renders HCV more susceptible to interferon-based therapy (Itakura et al. PLoS One 2015; 10: e0138060), and seems to be associated to the IL28B TT genotype (Itakura et al. Hepatol Res 2015; 45: E115-E121). Other mutations in the NS5A protein seem to have additional influence on treatment response to chronic HCV-1b infection (Maekawa et al.

666 Comments Hepatology 2012; 56: 1611-1621). In vitro studies have shown the selection of escape mutations in the E1 protein (I348T) in HCV genotype 1a, as well as F345V/V414A (in the E1/E2 proteins) in HCV genotype 3a replicons. These mutations increase viral fitness by facilitating virus entry into target cells (Serre et al. J Virol 2013; 87: 12776-12793).

In general, HCV genotypes 1 and 4 are less sensitive to treatment than genotypes 2 and 3 (reviewed in Taylor et al. Microbes & Infection 2000; 2: 1743-1756; Pawlotsky. Antiviral Res 2003; 59: 1-11; Pawlotsky. Curr Opin Infect Dis 2003; 16: 587-592). Patients with chronic hepatitis C genotype 6 respond better to pegylated interferon and ribavirin combination therapy than patients with genotype 1 (Tsang et al. J Gastroenterol Hepatol 2010; 25: 766-771). A genetic polymorphism near the IL28B gene, encoding interferon-λ-3, is associated with a better response to treatment with peginterferon and ribavirin, particularly in European patients (Ge et al. Nature 2009; 461: 399-401; Thomas et al. Nature 2009; 461: 798-801; Thompson et al. Gastroenterology 2010; 139: 120-129; Fischer et al. Hepatology 2012; 1700-1710; for recent reviews, see Clark & Thompson. J Gastroenterol Hepatol 2012; 27: 212-222; Chayama et al. Hepatol Res 2012; 42: 841-853; Stättermayer & Ferenci. Curr Opin Virol 2015; 14: 50-55). The genetic polymorphism does not seem to have an impact on treatment responses when patients are infected with HCV genotypes 2 or 3 (Jia et al. PLoS One 2012; 7: e45698; Jeng et al. PLoS One 2012; 7: e48217; Hayashi et al. J Gastroenterol Hepatol 2015; 30: 178-183), or to sustained virological response to telaprevir-based therapies (Pol et al. J Hepatol 2013; 58: 883-889). Currently, IL28B genotyping is not recommended before initiation of interferon-free therapies. Other polymorphisms associated with sustained virological response that improve the predictive value of IL-28 appear in the following genes: low-density lipoprotein receptor (LDLR), transforming growth factor β (TGF-β), aquaporine 2 (AQP-2), very-low-density lipoprotein receptor, Sp110 nuclear body protein, interferon a/β receptor 1, 2’-5’-oligoadenylate synthase 1 and apolipoprotein B (Neukam et al. AIDS 2013; 17: 2715-2724), histone deacetylases (López-Rodríguez et al. Genes Immun 2013; 14: 317-324), HLA-Bw4 (Nozawa et al. PLoS One 2013; 8: e83381) and KIR2DS (Keane et al. PLoS One 2013; 8: e66831; Nozawa et al. PLoS One 2013; 8: e83381). Expression of genes in the interferon response pathway can be predictive of a positive response to interferon therapy (e.g. STAT1 and XAF1) or associated with poorer response to therapy (e.g. RSAD2, IFI6, IFI16 and CCL5) (Pfeffer et al. PLoS One 2014; 9: e104202). In HIV-1/HCV-coinfected patients, interferon therapy response has been associated with the expression of the gene encoding the interferon-λ 4 protein (IFNL4) (Franco et al. AIDS 2014; 28: 133-136), but this correlation has not been observed in patients infected only with HCV genotypes 1 and 4 (Real et al. PLoS One 2014; 9: e95515). HCV replicon cell populations selected after passage in the presence of unpegylated recombinant interferon lambda (λ) (currently in clinical development) also showed a defect in the activation of the interferon-dependent JAK-STAT signaling pathway (Friborg et al. Virology 2013; 444: 384-393).

667 2. HCV protease (NS3/4A) inhibitors

Drug Comments Asunaprevir HCV protease inhibitor in clinical development. Resistance to (BMS-650032) asunaprevir appears to be mediated by R155K and D168A/E/T/V/Y, as determined in clinical assays where patients were given the drug in combination with daclastavir (Lok et al. N Engl J Med 2012; 366: 216-224; McPhee et al. Hepatology 2013; 58: 902-911; for a review see Akamatsu et al. Expert Rev Anti Infect Ther 2015; 13: 1307-1317) or in combination with pegylated interferon and ribavirin (Bronowicki et al. J Hepatol 2014; 61: 1220-1227). Amino acid substitutions R155K, D168G and I170T were selected in vitro with HCV genotype 1a replicons and conferred low to moderate level resistance to asunaprevir. For genotype 1b, those mutations conferred a higher level of resistance (170- to 400- fold relative to the wild-type replicon). Predominant mutations were found at position 168 and were associated with high-level asunaprevir resistance and impaired replication capacity (McPhee et al. Antimicrob Agents Chemother 2012; 56: 3670-3681; Jensen et al. Antimicrob Agents Chemother 2015; 59: 7426-7436).

Boceprevir Peptidic ketoamide inhibitor of the HCV protease (Njoroge et al. Acc (SCH 503034, Chem Res 2008; 41: 50-59). T54A, V170A and A156S conferred low Victrelis) to moderate levels of resistance (<20-fold), but longer exposure to the drug led to the selection of a more resistant variant, A156T (>100-fold) (Lin et al. J Biol Chem 2004; 279: 17508-17514). A156T and A156S reduced viral fitness in a colony formation efficiency assay, while V170A did not affect replicon fitness (Tong et al. Antiviral Res 2006; 70: 28-38). Two additional mutations in the HCV protease (V55A and V158I) were identified in patients after automated analysis of sequence polymorphisms (Qiu et al. Nucleic Acids Res 2009; 37: e74). V158I conferred 3.3-fold increased resistance to the inhibitor as determined with the replicon system. Resistant replicons bearing the M175L change in the protease were selected after exposure to a combination treatment of boceprevir and a viral polymerase inhibitor (Chase et al. Antiviral Res 2009; 84: 178-184). In HCV genotype 6, resistance to boceprevir is mostly associated with mutations at codon 122, instead of A156T or A156V (Aloia et al. Antivir Ther 2015; 20: 271-280). Unlike telaprevir, boceprevir might not be effective on genotype 4 isolates with currently used dosages (Khattab et al. J Hepatol 2011; 54: 1250-1262).

668 Drug Comments Ciluprevir (BILN HCV NS3/4A protease inhibitor. Resistant replicons were selected 2061) in vitro and contained mutations A156V, D168A or D168V. In all cases these amino acid substitutions conferred >100-fold resistance to the inhibitor (Lin et al. J Biol Chem 2004; 279: 17508-17514; Mo et al. Antimicrob Agents Chemother 2005; 49: 4305-4314). Those ciluprevir-resistant replicons were susceptible to telaprevir (VX- 950). However, mutations A156V and A156T confer cross resistance to telaprevir, while diminishing replication capacity in a transient replicon cell system (Lin et al. J Biol Chem 2005; 280: 36784-36791). Mechanistic details involved in resistance mediated by mutations R155Q, A156T, D168A and D168V have been discussed based on a theoretical model of NS3/4A complexed with ciluprevir (Courcambeck et al. Antivir Ther 2006; 11: 847-855). Studies with recombinant NS3/4A and NS3 proteins have demonstrated that ciluprevir shows decreased affinity for proteases of genotypes 2 and 3 compared with those of genotype 1 (Thibeault et al. J Virol 2004; 78: 7352-7359; Tong et al. Biochemistry 2006; 45: 1353-1361). Residues at positions 78, 79, 80, 122, 132 and 168 appear to be responsible for the observed differences (Thibeault et al. J Virol 2004; 78: 7352-7359). Enzymatic assays revealed that the substitutions found at genotype 2 and conferring resistance were D79E, Q80G and S122K, while D168Q was largely responsible for the differences between genotypes 3 and 1b (Tong et al. Biochemistry 2006; 45: 1353-1361). Amino acid substitutions found in the resistant HCV genotype 2a strain J6/JFH-1 were A156G, D168A and D168V. A156G was not found using the genotype 2a replicon, although A156T and A156V were selected in this case (Cheng et al. Antimicrob Agents Chemother 2011; 55: 2197-2205). Compound 1 Tripeptide inhibitor of the HCV protease, NS3/4A. Resistance mutations to this drug were identified using a subgenomic HCV RNA (replicon). The combination of mutations D168A and E176K in NS3, plus an insertion of three nucleotides (encoding Lys) in position 67 of NS5A, and the combination of the insertion with mutations D168Y or D168V and E176G in NS3 were found in resistant replicons (Trozzi et al. J Virol 2003; 77: 3669-3679). As in the case of ciluprevir (BILN 2061), compound 1 was less effective on genotypes 2 and 3 than on genotype 1b, although the D168Q mutation had a small influence in the increase of the inhibition constant (Tong et al. Biochemistry 2006; 45: 1353-1361).

669 Drug Comments

Danoprevir Noncovalent macrocyclic protesase inhibitor (Seiwert et al. Antimicrob (ITMN-191/ Agents Chemother 2008; 52: 4432-4441) effective on genotype 1 chronic RG7227) HCV. Danoprevir susceptibility decreases >50-fold in the presence of R155K, R155Q, D168T or D168V. These mutations were also found in patients treated with danoprevir, although R155K was the most frequent among all of them (Lim et al. Antimicrob Agents Chemother 2012; 56: 271-279; Gane et al. Antimicrob Agents Chemother 2014; 58: 1136-1145). In phase II clinical trials, R155K was found at viral breakthrough in all patients infected with genotype 1a. D168V was predominant in patients infected with genotype 1b, although R155K was also identified in some of them (Tong et al. Antimicrob Agents Chemother 2014; 58: 3105-3114).

Deldeprevir Pan-genotypic HCV protease inhibitor that showed higher potency (neceprevir, ACH- in vitro against genotype 1a and 4a HCV strains (Jensen et al. 2864) Antimicrob Agents Chemother 2015; 59: 7426-7436). High-level resistance to this drug was observed in HCV variants carrying R155K (all genotypes except 3a), D/Q168A (genotypes 1a, 2a and 5a), D/Q168G/H (genotype 6a), and D/Q268V (genotypes 2a and 5a) (Jensen et al. Antimicrob Agents Chemother 2015; 59: 7426-7436).

Faldaprevir (BI Linear tripeptide inhibitor containing a C-terminal carboxylic acid 201335) (Llinás-Brunet et al. J Med Chem 2010; 53: 6466-6476) active against HCV genotype 1. When administered in combination with pegylated interferon and ribavirin, it selected for mutations R155K and D168V in the viral NS3 protease. By themselves, these mutations decreased viral susceptibility to the inhibitor >100 times. Similar effects were also observed in vitro with single mutations: A156V, A156T and D168A (Lagacé et al. Antimicrob Agents Chemother 2012; 56: 569-572). R155K also confers high-level resistance to the drug in genotypes 2a, 4a, 5a and 6a, while D168G confers >70-fold increased resistance in strains of genotypes 2a and 6a (Jensen et al. Antimicrob Agents Chemother 2015; 59: 7426-7436). During faldaprevir treatment (in phase I clinical studies) virologic breakthrough was common and was associated to the emergence of mutations R155K in genotype 1a and D168V in genotype 1b. D168V conferred a greater reduction in faldaprevir susceptibility, but was generally less fit than R155K. A rare NS3 (V/I)170T polymorphism produced a small reduction in drug susceptibility in vitro (Berger et al. Antimicrob Agents Chemother 2013; 57: 4928-4936).

Glecaprevir HCV protease inhibitor identified by AbbVie and Enanta that showed (ABT-493) potent activity against all major HCV genotypes. Currently in clinical trials in combination with the NS5A inhibitor pibrentasvir (ABT-530). The presence of NS3 baseline variants T54S, V55A/I, Q80K, S122G/T, and I170V did not affect viral load declines during ABT-493 monotherapy (Lawitz et al. Antimicrob Agents Chemother 2016; 60: 1546-1555).

670 Drug Comments Grazoprevir Novel P2-P4 quinoxaline macrocyclic inhibitor of the HCV protease, (MK-5172) NS3/4A, and therefore structurally related to vaniprevir (MK-7009). Active against genotypes 1a, 1b, 2a, 2b, 3a, 4a and 6a (EC50<4 nM with the replicon assay) (Lahser et al. Antimicrob Agents Chemother 2016; 60: 2954-2964). Selected 3a strains from untreated patients may show some resistance (Lahser et al. Antimicrob Agents Chemother 2016; 60: 2954-2964; Guo et al. J Biol Chem 2017; 292: 6202-6212). A156T and A156V confer high-level resistance to the inhibitor. Mutations selected by grazoprevir (MK-5172) include Q41H, F43S, A156T and D168A/V/G (Summa et al. Antimicrob Agents Chemother 2012; 56: 4161-4167). A156T is considered as the major resistance mutation for this drug. D168A confers >100-fold increased resistance to grazoprevir in replicon assays with HCV genotype 1a (Lahser et al. Antimicrob Agents Chemother 2016; 60: 2954-2964). However, its combination with R155K nullified this effect (Guo et al. J Biol Chem 2017; 292: 6202-6212). In comparison with first-generation protease inhibitors such as boceprevir or telaprevir, grazoprevir (MK-5172) has a better pharmacokinetic profile, higher barrier to resistance, is active against HCV genotype 3 and against most of the variants that confer resistance to boceprevir or telaprevir (for a review, see Gentile et al. Expert Opin Investig Drugs 2014; 23: 719-728). Narlaprevir Ketoamide inhibitor related to boceprevir, and active against proteases of (SCH 900518) genotypes 1 to 3. Selection of replicon cells with narlaprevir resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V substitutions) (Tong et al. Antimicrob Agents Chemother 2010; 54: 2365- 2370). Emergence of mutations at positions 36, 54, 155 and 156 have been observed in patients treated with the inhibitor in combination with pegylated interferon/ribavirin in phase I clinical trials. All mutations except R155K disappeared within six months following treatment with the drug (de Bruijne et al. J Viral Hepat 2013; 20: 779-789).

Paritaprevir Protease inhibitor used with a minimal dose of ritonavir to improve its (ABT-450) bioavailability. It is used in combination with ombitasvir and desabuvir in interferon-free therapies (for a review, see Cheng et al. Expert Opin Pharmacother 2015; 16: 2835-2848). In these trials, D168V has been selected in patients infected with HCV genotypes 1a and 1b (Poordad et al. N Engl J Med 2014; 370: 1973-1982), as well as in patients infected with genotypes 4a and 4d (Schnell et al. Antimicrob Agents Chemother 2015; 59: 6807-6815). In vitro, the largest effects on ABT-450 resistance were observed with the substitution D168Y, although other mutations at positions 155, 156 and 168 may contribute to confer reduced susceptibility to HCV genotype 1 (Pilot-Marias et al. Antimicrob Agents Chemother 2015; 59: 988-997). R155K confers high-level resistance to the drug in HCV genotypes 1a, 4a and 5a, R155Q in genotype 1a and R155T, as well as D168G in genotypes 2a and 6a (Jensen et al. Antimicrob Agents Chemother 2015; 59: 7426-7436).

671 Drug Comments SCH6 Peptidic ketoamide inhibitor of the HCV protease. Resistant genotype (SCH446211) 1b replicons, selected in culture under SCH6 pressure contained mutations A156T, A156V and R109K. R109K conferred moderate resistance to the drug, while having a minimal effect on the enzymatic activity. A156T had a large effect on replicon fitness, but mutations Q86R, P89L, and G162R were capable of partially reversing A156T- associated defects (Yi et al. J Biol Chem 2006; 281: 8205-8215).

Simeprevir Simeprevir is a macrocyclic inhibitor active against HCV genotype 1 (TMC435, strains that shows antiviral activity against genotypes 4, 5 and 6, but not TMC435350) against genotype 3 (probably due to the mutation D168Q) (reviewed in Tanwar et al. Expert Opin Investig Drugs 2012; 21: 1193-1209; Flanagan et al. Expert Rev Clin Pharmacol 2014; 7: 691-704). In genotype 1b, high-level resistance is conferred by mutations D168I or D168V, and moderate resistance by the amino acid substitutions D168G and D168N, and several mutations at codon 80 of the protease-coding region (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887). The presence of Q80K in genotype 1a resulted in a median 11-fold reduction in sime- previr activity (Verbinnen et al. Antimicrob Agents Chemother 2015; 59: 7548-7557). Resistance pathways of simeprevir were similar in genotype 1 and in other genotypes. For example, mutations V36L, Q80G and S122R were selected in genotype 2, and S122T or S122N in genotype 6. In patients, viral breakthrough in genotypes 4, 5 and 6 was associated with emerging mutations including Q80R, R155K and/or D168E/V (Lenz et al. J Hepatol 2013; 58: 445-451). In a phase IIb trial involving simeprevir treatment of drug-experienced patients infected with HCV genotype 1, emerging mutations identified at viral breakthrough were R155K (predominant in genotype 1a isolates), D168V (predominant in genotype 1b) and a combination of Q80K or R, R155K and/or D168E (Zeuzem et al. Gastroenterology 2014; 146: 430-441).

Telaprevir Substrate-analogue HCV protease inhibitor (reviewed in Matthews (VX-950, & Lancaster. Clin Ther 2012; 34: 1857-1882). Telaprevir-resistant Incivek) replicons were obtained after serial passages in the presence of the drug. These constructs contained the mutation A156S in the viral protease-coding region, which conferred 10-fold resistance to the inhibitor (Lin et al. J Biol Chem 2004; 279: 17508-17514). Cross- resistance with ciluprevir is conferred by mutations A156V or A156T (Lin et al. J Biol Chem 2005; 280: 36784-36791). Telaprevir showed similar potency on the proteases derived from genotypes 1b, 2 or 3 (Tong et al. Biochemistry 2006; 45: 1353-1361).

672 Drug Comments Mutations conferring low-level resistance (V36A/M, T54A, R155K/T and A156S) and high-level resistance (A156T/V, and V36A/M + R155K/T or V36A/M + A156S/T/V) were selected in patients treated with the inhibitor (Sarrazin et al. Gastroenterology 2007; 132: 1767-1777; Macartney et al. Antiviral Res 2014; 105: 112-117; Talal et al. Hepatology 2014; 60: 1826- 1837). V36M, R155K and R155T predominate in patients infected with HCV genotype 1a, while V36A, T54A and A156S are more frequently selected in patients infected with genotype 1b (Dierynck et al. J Viral Hepat 2014; 21: 835-842). Telaprevir-resistant strains in naïve patients are more common in genotype 1a than in genotype 1b HCV isolates (Sullivan et al. Clin Infect Dis 2013; 57: 221-229).

V36C produces a 4-fold increase in the IC50 for telaprevir in an enzyme assay, and a 9.5-fold increase in the replicon model. V36C has a minor effect on the replication capacity of the HCV replicon (Barbotte et al. Antimicrob Agents Chemother 2010; 54: 2681-2683). The amino acid substitutions T54A and A156S were selected with the HCV genotype 2a strain J6/JFH-1 and were also found in genotype 2 patients (Cheng et al. Antimicrob Agents Chemother 2011; 55: 2197-2205). Vaniprevir Competitive macrocyclic inhibitor of the HCV NS3/4A protease. (MK-7009) Resistance-associated amino acid variants were identified at positions 155 and 168 in patients infected with genotype 1a virus. The largest increases

in the EC50 conferred by single mutations were observed with D168V, R155G, A156V, D168A and R155S (>70-fold increases relative to the wild-type in all cases) (Lawitz et al. Antiviral Res 2013; 99: 214-220).

Vedroprevir Macrocyclic inhibitor highly active in patients infected with HCV (GS-9451) genotypes 1a and 1b, but less active against genotype 2a replicons. Stable in hepatic microsomes and hepatocytes. It shows good availability in combination with pegylated interferon, ribavirin and the RNA polymerase inhibitors GS-6620 and tegobuvir (Yang et al. Antimicrob Agents Chemother 2014; 58: 647-653). Mutations D168E/G/V and R155K conferring high-level resistance to the drug were detected in patients infected with genotype 1a and 1b virus (Dvory-Sobol et al. Antimicrob Agents Chemother 2012; 56: 5289-5295). Voxilaprevir NS3/4A inhibitor with potent in vitro activity against genotypes 1-6 (GS-9857) and improved resistance profile against common genotype 1 NS3 resistance-associated variants (Taylor et al. J Hepatol 2015; 62: S681). Phase Ib clinical studies supported further development of this drug for its administration in once-daily dosing (Rodriguez-Torres et al. J Viral Hepat 2016; 23: 614-622). Voxilaprevir looks a promising candidate for treating cirrhotic patients infected with HCV genotype 3, when coformulated with sofosbuvir and velpatasvir (Johnson et al. Curr Infect Dis Rep 2017; 19: 22).

673 3. NS4B inhibitors

Drug Comments 2-(4-sulfonamido- Compounds synthesized and optimized against HCV genotype 1b phenyl)-indole replicons, but also active against genotypes 1a, 2a and 3a. Amino acid 3-carboxamides substitutions F98C and V105M were selected in the presence of the drug and individually conferred high-level resistance to the inhibitor (Zhang et al. Bioorg Med Chem Lett 2016; 26: 594-601). AP80978 Tetrahydropyrazolo-pyrimidine that inhibits HCV genotypes 1a and 1b, but not genotype 2a. It inhibits the HCV genotype 1b with an

EC50 of 630 nM. NS4B mutations F98V and F98L confer >20-fold increased resistance to the drug (Dufner-Beattie et al. Antimicrob Agents Chemother 2014; 58: 3399-3410). PTC725 Pyridine sulfonamide that inhibits HCV genotype 1b and 3 replicons at nanomolar concentration. Replicons (1b) selected for resistance to PTC725 harbored amino acid substitutions F98C/L and V105M in NS4B (Gu et al. Antimicrob Agents Chemother 2013; 57: 3250-3261). Single amino acid substitutions S59C, F98C, V105M and L109R decreased replicon susceptibility to the inhibitor by >100-fold, while other changes

(e.g. S59G, H94R, F98L, V105L and L109I) increased the IC50 to the drug by 6-80 fold (Graci et al. Antimicrob Agents Chemother 2016; 60: 7060-7066). Selected mutations were essentially the same in genotypes 1b and 3, but F97H/Y was more prevalent than F98C/L in HCV genotype 3, while other minor substitutions (e.g. L64I, S85A, and S88C) were also common in this sequence context (Graci et al. Antimicrob Agents Chemother 2016; 60: 7060-7066). Spirocyclic Inhibit HCV genotype 1a and 1b replicons at nanomolar concentration. compounds 6j, Genotype 2a replicons are resistant to these compounds. Mutations 6p, 22b and 27c H94N and V105M in genotype 1b confer different degrees of resistance to those drugs (Tai et al. Bioorg Med Chem Lett 2014; 24: 2288-2294).

4. NS5A inhibitors (selecting for mutations in the N-terminal domain of NS5A)

Drug Comments Biphenyl It shows potent activity against both genotype 1a and 1b replicons, with

derivative EC50 values of 23.1 and 9.8 pM, respectively. Its resistance profile is compound 1 similar to that of daclatasvir, with M28T, Q30H/K/R, L31M/V and Y93C as major resistance mutations in genotype 1a, and Y93H/N in genotype 1b (Bhattacharya et al. J Gen Virol 2014; 95: 363-372).

674 Drug Comments BP008 Thiazole analogue with improved potency against HCV genotype 1b and 2a replicons, as well as against 2a infectious virus. The BP008 resistance profile suggests that mutations Q24L, P58S/T/L, Y93H, F149L and V153M in HCV NS5A are the important determinants for mediating BP008 sensitivity. Y93H was the mutation with the largest impact on resistance as determined with an HCV genotype 1b replicon assay (Lin et al. Antimicrob Agents Chemother 2012; 56: 44-53). Daclatasvir Small molecule inhibitor of the HCV NS5A protein (Lemm et al. (BMS-790052) Antimicrob Agents Chemother 2011; 55: 3795-3802; Herbst & Reddy. Expert Opin Investig Drugs 2013; 22: 1337-1346; for reviews, see Lee. Drug Des Devel Ther 2013; 7: 1223-1233; Bunchorntavakul & Reddy. Aliment Pharmacol Ther 2015; 42: 258-272; Degasperi et al. Expert Opin Pharmacother 2015; 16: 2679-2688). It is quite effective in patients infected with genotype 1b, but has a lower barrier of resistance with other genotypes. The rank order of resistance barriers to daclatasvir is 1b > 4a > 5a > 6a > 1a > 2a(JFH) > 3a > 2a(M31) (Wang et al. Antimicrob Agents Chemother 2014; 58: 5155-5163). Resistance has been detected using an in vitro replicon system (reviewed in Lemm et al. J Virol 2010; 84: 482-491; Asselah. J Hepatol 2011; 54: 1069-1072). Single amino acid changes such as M28T, Q30H, Q30R, L31M, L31V and Y93C in NS5A conferred >2000- fold decreased susceptibility to the inhibitor. Some of those mutations (i.e. M28T, Q30H/R and L31M/V for genotype 1a, and L31V and Y93H for genotype 1b) were found in patients treated with the inhibitor during clinical trials (Gao et al. Nature 2010; 465: 96-100; Zeuzem et al. J Hepatol 2016; 64: 292-300; for a recent review see Pawlotsky. J Hepatol 2013; 59: 375-382). Response to daclatasvir varied a lot depending on the HCV genotype and strains, with isolate J6 (genotype 2a) and SG2 (genotype 3a)

showing the highest IC50 values (Scheel et al. Gastroenterology 2011; 140: 1032-1042). However, the lower susceptibility of genotype 3a does not seem to be related to the NS5A structure, since hybrid replicons containing the protein of genotype 3a were efficiently inhibited by the drug (Wang et al. Antimicrob Agents Chemother 2013; 57: 611-613). For genotype 1b, L31V and Y93H were identified as primary resistance mutations, while L23F, R30Q, P32L, P58S and Y93N acted as secondary resistance mutations. For genotype 1a, substitutions M28T, Q30E/H, L31M and Y93H were the ones most frequently selected, while Q30K and Y93N appeared in a lower proportion (Fridell et al. Antimicrob Agents Chemother 2010; 54: 3641-3650; Wang et al. Antimicrob Agents Chemother 2012; 56: 1350-1358). Amino acid substitutions A30K, L31F, L31V, L31M and particularly Y93H conferred high-level resistance to daclastavir in HCV genotype 3a NS5A (Wang et al. Antimicrob Agents Chemother 2013; 57: 611-613; Hernandez et al. J Clin Virol 2013; 57: 13-18). All of those mutations reduced viral replication capacity.

675 Drug Comments All NS5A sequences of genotype 4 harbor the mutation L31M, while the combination L31M/Y93H was found in 7% of isolates of HCV genotype 1b and in 13% of those of genotype 4 (Plaza et al. Antivir Ther 2012; 17: 921-926; Suzuki et al. J Clin Virol 2012; 54: 352-354). L28S together with M31I and Y93H have been associated with daclatasvir resistance in patients infected with HCV genotype 4 variants (Bartolini et al. J Clin Virol 2015; 66: 38-43). Despite the diversity of circulating genotype 4 subtypes, polymorphisms conferring high-level resistance to daclatasvir are uncommon before therapy. In general, baseline polymorphisms seem to have a minor impact to daclatasvir-containing regimens (Zhou et al. J Infect Dis 2016; 213: 206-215). In vitro studies showed that single amino acid substitutions L31V, L31W, Y93H and Y93N conferred high-level resistance (>20-fold increase of

the IC50) when introduced in 1b replicon variants, although only the first two retained wild-type replication capacity (Wang et al. Antimicrob Agents Chemother 2012; 56: 1350-1358). In the case of genotype 1a, the largest effects on drug susceptibility were observed with mutations: M28A/T, Q30D/E/G/H/K/R, L31M/V, P32L, H58D and Y93C/H/N (Fridell et al. Hepatology 2011; 54: 1924-1935; Wang et al. Antimicrob Agents Chemother 2012; 56: 1350-1358). Studies carried out with genotype 2a JFH1 replicon cells identified major resistance mutations: F28S, L31M, C92R and Y93H. C92R and Y93H had a negative impact on viral fitness, and second-site replacements (e.g. K30E/Q) restored efficient replication of the C92R variant (Fridell et al. J Virol 2011; 85: 7312-7320). Daclastavir is also effective against HCV genotype 4a strains, although natural variability at positions 28, 30, 32, 58 and 93 of NS5A may facilitate the emergence of drug resistant strains. In the context of genotype 4a strains, R30G, R30H, R30S, L30H, Y93H, Y30R and the double mutants M28L/L30H and L30I/Y93R show more than 60- fold decreased daclastavir susceptibility in comparison with the wild-type strain (Wang et al. Antimicrob Agents Chemother 2012; 56: 1588-1590). HCV passage in hepatoma cells carried out in the presence of daclatasvir and interferon α2b led to the selection of Y93H in all tested genotypes (wild-type isolate QC69 of genotype 7a contains His at position 93 of NS5A) (Scheel et al. Gastroenterology 2011; 140: 1032-1042). The Y93H mutation pre-existed as a minor population (representing 0.1-0.5% of the total) in HCV genotype 1b-infected patients, as observed in clinical trials of daclatasvir combined with pegylated interferon and ribavirin (Murakami et al. Antimicrob Agents Chemother 2014; 58: 2105-2112).

676 Drug Comments In clinical assays involving patients infected with genotype 1a, NS5A mutations Q30E/R, L31I/M/V and Y93C/N have been detected at failure (McPhee et al. Hepatology 2013; 58: 902-911; McPhee et al. Antivir Ther 2014; 19: 479-490). Unusual combinations of resistance mutations such as L31F/ΔP32 and L28M/R30Q/A92K have been detected in resistant variants emerging in prior non-responders (McPhee et al. Antivir Ther 2014; 19: 479-490). In combination with sofosbuvir, daclatasvir has shown efficacy against genotypes 1, 2 and 3 in patients failing treatment with telaprevir or boceprevir (Sulkowski et al. N Engl J Med 2014; 370: 211-221). Response was better with patients infected with HCV genotype 1a in comparison with 1b, and the presence of polymorphism A30K in patients infected with genotype 2 could produce a loss of daclatasvir susceptibility. Resensitization of daclatasvir-resistant HCV variants by using analogues of NS5A inhibitors (e.g. Syn-395) has been demonstrated in vitro (Sun et al. Nature 2015; 527: 245-248). Daclatasvir and NS5A synergists such as as Syn-690 were highly efficient at clearing cells of viruses. Although S38F and S38T have been selected in response to Syn-690 and Syn-535, respectively, these compounds seem to increase the genetic barrier for daclatasvir resistance (O’Boyle II et al. Antimicrob Agents Chemother 2016; 60: 1573-1583). DBPR110 Dithiazol analogue derivative related to BP008. Very effective on HCV genotype 2a. Amino acid substitutions in the N-terminal region (domain I) of NS5A were associated with decreased inhibitor susceptibility: P58L or T and Y93H or N in genotype 1b and T24A, P58L and Y93H in genotype 2a replicons were critical for resistance selection. V153M, M202L and M265V played a compensatory role in replication and drug resistance (Lin et al. Antimicrob Agents Chemother 2013; 57: 723-733).

EDP-239 HCV inhibitor with in vitro 50% effective concentrations (EC50s) of 34 pM and 4 pM against genotype 1a (GT1a) and GT1b replicons, respectively. Its potency is apparently reduced by amino acid substitutions L31M, L31V or Y93H (Owens et al. Antimicrob Agents Chemother 2016; 60: 6207-6215).

677 Drug Comments Elbasvir (MK- Highly potent HCV inhibitor used in combination with grazoprevir 8742) (an NS3 inhibitor). HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a and 6 are susceptible to the inhibitor in replicon assays (Lahser et al. Antimicrob Agents Chemother 2016; 60: 2954-2964; reviewed in Vallet-Pichard & Pol. Therap Adv Gastroenterol 2017; 10: 155-167). In clinical trials, resistance- associated variants of NS5A contained the amino acid substitutions M28T, Q30H/L/R, L31M and Y93H/N (Lawitz et al. Lancet 2015; 385; 1075- 1086; Sulkowski et al. Lancet 2015; 385: 1087-1097). By themselves Q30E, L31V, Y93H and Y93N confer high-level resistance to the inhibitor (Lahser et al. Antimicrob Agents Chemother 2016; 60: 2954-2964). GSK2336805 Inhibitor with picomolar activity against standard HCV genotypes 1a, 1b and 2a, as well as genotype 1b L31V and Y93H mutants. Resistance mutations selected in genotype 1a are Q30H and L31M. Resistant HCV genotype 1b strains have a mixture of L31G/M/V. Single amino acid substitutions L31F/V/W and Y93N in HCV genotype 1b, and M28T, Q30K/R, L31V and Y93C in genotype 1a confer decreased susceptibility to the drug (Kazmierski et al. J Med Chem 2014; 57: 2058-2073; Walker et al. Antimicrob Agents Chemother 2014; 58: 38-47). Ledipasvir (GS- Inhibitor of HCV replication with EC50 values around 4-34 pM against 5885) genotype 1a and 1b replicons (for reviews, see Gentile et al. Expert Opin Investig Drugs 2014; 23: 561-571; Keating. Drugs 2015; 75: 675-685). As described for daclatasvir, it showed less potency against clinical variants of HCV genotype 1a carrying mutations at positions 28, 30, 31 and 93 (Hernandez et al. J Clin Virol 2013; 57: 13-18; Wong et al. Antimicrob Agents Chemother 2014; 57: 6333-6340). When introduced in HCV genotype 1a replicons single amino acid substitutions M28T, Q30E/H/R, L31M and Y93C/H/N produced >90-fold reductions in ledipasvir susceptibility (Wong et al. Antimicrob Agents Chemother 2014; 57: 6333-6340). L31M in genotype 1a and Y93H in genotype 1b have been selected in patients treated with the drug in combination with sofosbuvir, in interferon-free therapies (Afdhal et al. N Engl J Med 2014; 370: 1889-1898; Wilson et al. Clin Infect Dis 2016; 62: 280-288). Ledipasvir is also active against HCV genotypes 4a, 4d, 5a and 6a

with EC50 values of 0.11 to 1.1 nM, but it has reduced activity against genotypes 2a, 2b, 3a and 6e. Y93H and Q30E are major resistance mutations both in vitro and in vivo (Cheng et al. Antimicrob Agents Chemother 2016; 60: 1847-1853).

678 Drug Comments Ombitasvir NS5A inhibitor used in combination with ABT-450/r (NS3 inhibitor) and (ABT-267) desabuvir (NS5B inhibitor) in interferon-free regimens (Poordad et al. N Engl J Med 2014; 370: 1973-1982; for a review, see Stirnimann. Expert Opin Pharmacother 2014; 15: 2609-2622). Q30R in genotype 1a and Y93H in genotype 1b have been selected in these patients. Ombitasvir- resistant HCV genotype 1a variants carrying mutations M28T, Q30R or Y93C/H/N showed >800-fold decreased susceptibility to the drug. High- level resistance mutations in genotype 1b are L28T (661-fold increase

in the EC50), and the double-mutants L31M/Y93H and L31F/Y93H (DeGoey et al. J Med Chem 2014; 57: 2047-2057). L28V is the most relevant resistance mutation in HCV genotypes 4a and 4d (Schnell et al. Antimicrob Agents Chemother 2015; 59: 6807-6815). Pibrentasvir NS5A inhibitor whose antiviral activity and safety is being tested in (ABT-530) combination with the protease inhibitor glecaprevir (ABT-493). It is active

against HCV genotypes 1 to 6 with EC50 values of 1.4 to 5.0 nM. Amino acid substitutions at positions 28, 30, 31 and 93 found in several HCV genotypes and responsible for resistance to other NS5A inhibitors seem to have no effect on pibrentasvir resistance. In selection experiments, the major resistance-associated mutation found in HCV genotypes 1a and 3a was Y93H. Other changes conferring increased resistance to the drug using several HCV replicons were Q30D, Y93N and H58D/Y93H (genotype 1a), and F28S/M31I and P29S/K30G (genotype 2a) (Ng et al. Antimicrob Agents Chemother 2017; 61: e02558-16). Pibrentasvir treatment produced less robust virologial responses in clinical studies, but further studies are needed to confirm this observation (Lawitz et al. Antimicrob Agents Chemother 2016; 60: 1546-1555). On the other hand, clinical studies show some promise for treating patients infected with HCV genotype 3 (Poordad et al. Liver Int 2016; 36: 1125-1132). Samatasvir NS5A inhibitor with EC50s in the range of 2 to 24 pM for HCV genotype (IDX719) 1 to 5 replicons. Resistance selection experiments with genotype 1a led to the identification of mutations at codons 28, 30, 31, 32 and 93 associated with reduced susceptibility to the drug (Bilello et al. Antimicrob Agents Chemother 2014; 58: 4431-4442).

679 Drug Comments Velpatasvir NS5A inhibitor with demonstrated pan-genotypic activity in vitro with

(GS-5816) EC50 values ranging from 6 to 130 pM against genotype 1-6 replicons [Cheng et al. J Hepatol 2013; 58 (suppl.): S484]. Deep sequence analysis of the clinical resistance to velpatasvir revealed that in HCV genotype 1a, resistance-associated mutations emerge at positions M28, Q30, L31, P32, H58, E92 and Y93, although the most prevalent are L31M, E92R and Y93H. In genotypes 1b and 2, resistance is associated with substitutions at positions 31 and 93, while in genotype 3 this occurs at residues 31, 92 and 93, and in genotype 4 at positions 28, 31 and 93, together with the substitution P32L (Lawitz et al. Antimicrob Agents Chemother 2016; 60: 5368-5378). In phase II/III clinical trials, amino acid substitutions Q30R, L31M, and Y93H/N were frequently observed after treatment with the inhibitor in combination with sofosbuvir (Feld et al. N Engl J Med 2015; 373: 2599-2607; Foster et al. N Engl J Med 2015; 373: 2608-2617).

5. NS5A inhibitors (selecting for mutations in the protein domain II)

Drug Comments Alisporivir Cyclic endecapeptide that binds cyclophilin A, a protein with peptidyl- (DEB025, Debio- prolyl isomerase activity (for a review, see Flisiak et al. Expert Opin 025) Investig Drugs 2012; 21: 375-382). It is active against many different HCV genotypes and shows a high barrier to viral resistance. Alisporivir plus ribavirin has been proposed as an effective interferon-free therapy for some patients infected with genotype 2 or 3 strains (Pawlotsky et al. Hepatology 2015; 62: 1013-1023). Increasing evidence indicates that cyclophilin A binds NS5A, and the only mutation found in alisporivir- resistant replicons was D320E, located in the NS5A domain II (Coelmont et al. PLoS One 2010; 5: e13687). D320E decreases susceptibility to alisporivir by 3-fold. Prolines at positions 310 and 341, and a conserved Trp in the spacer region are required for cyclophilin A binding and HCV replication (Grisé et al. J Virol 2012; 86: 4811-4822). Cyclosporine A HCV replication is suppressed by cyclosporine A through a mechanism that it is still poorly understood. D320E in the NS5A domain II is a major mutation associated with resistance to the drug (Arai et al. Biochem Biophys Res Commun 2014; 448: 56-62). HCV genotype 2a (but not 1b) shows increased susceptibility to cyclosporine. Mutations E199A, G200A, H217V and H250A that impair viral replication confer resistance to the drug (Madan et al. Gastroenterology 2014; 146: 1361-1372).

680 Drug Comments SCY-635 Nonimmunosuppressive cyclophilin inhibitor derived from cyclosporine (Hopkins et al. Antimicrob Agents Chemother 2010; 54: 660-672). It targets the interaction between NS5A and cyclophilin A. Both mutations D320E and Y321N render HCV (genotype 1b) more resistant to SCY- 635 than the wild-type virus. Those mutations had no effect on viral fitness in the absence of the drug (Hopkins et al. Antimicrob Agents Chemother 2012; 56: 3888-3897). STG-175 Cyclosporine A derivate with a high (EC50 11.5 – 38.9 nM) multi- genotypic (genotypes 1a to 4a) anti-HCV activity (Gallay et al. PLoS One 2016; 11: e0152036). As in the case of SCY-635, resistance was associated with the emergence of D320E and Y312N, although both mutations conferred only partial resistance to the inhibitor (2-3-fold

increase in the EC50).

6. RNA-dependent RNA polymerase (NS5B) inhibitors (nucleoside analogues)

Drug Comments 2´C-methyl- Nucleoside analogue (Carroll et al. J Biol Chem 2003; 278: 11979-11984; adenosine Draffan et al. ACS Med Chem Lett 2014; 5: 679-684). HCV replicons passaged in cell culture developed resistance by selecting mutation S282T in NS5B. This mutation was also selected in an HCV-infected chimpanzee (Ludmerer et al. Antimicrob Agents Chemother 2005; 49: 2059-2069). However, the S282T substitution impaired replication. GS-6620 Prodrug of adenosine analogue monophosphate, active against HCV

replicons 1 to 6 and against HCV genotype 2a with EC50 values in the range 48-680 nM. The mutation S282T is the major resistance mutation selected in vitro and confers >30-fold increased resistance to the drug (Feng et al. Antimicrob Agents Chemother 2014; 58: 1930-1942). INX-08189 Prodrug of 6-O-methyl-2´-C-methyl guanosine, with an IC50 of 10 nM against HCV genotype 1b. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred approximately 10-fold decreased sensitivity to the inhibitor (Vernachio et al. Antimicrob Agents Chemother 2011; 55: 1843-1851).

681 Drug Comments Mericitabine Mericitabine is a triester prodrug of PSI-6130 (β-D-2’-deoxy-2’-fluoro- 2’-C-methylcytidine). The major resistance mutation found in vitro has been detected in 2 out of 30 patients treated with the drug in combination with danoprevir in clinical trials. Other mutations found in NS5B at viral breakthrough were L159F and S189N. Another mutation (Q47H) associated with sustained virological response was more prevalent in patients infected with genotype 1a and reduced the viral replication capacity (Tong et al. Antimicrob Agents Chemother 2014; 58: 3105- 3114). L159F in combination with L320F confers low-level resistance to mericitabine (Tong et al. J Infect Dis 2014; 209: 668-675). NITD008 Adenosine analogue that was previously identified as an inhibitor of dengue virus replication. It showed efficacy in inhibiting the replication of HCV genotypes 1a, 1b and 2a, but selected for mutation S282T that produced a 76.5-fold decrease in viral susceptibility (Qing et al. Antiviral Res 2016; 126: 46-54). ODE-S-HPMPA ODE-S-HPMPA and ODE-S-MPMPA are alkoxyalkyl esters of acyclic and ODE-S- nucleoside phosphonates. Selection experiments with the replicon MPMPA allowed the identification of Q49L, K50N and Q58L as related to drug

resistance. K50N confers a modest increase of the IC50, but also increases the replication capacity of the replicon. Cross-resistance with the previously described active site mutant S282T was also observed [Wyles et al. Antivir Ther 2010; 15 (suppl. 2): A26]. PSI-352938 and Prodrugs of β-D-2´-deoxy-2´-α-fluoro-2´-β-C-methylguanosine PSI-353661 monophosphate. PSI-352938 is a cyclic phosphate nucleotide and PSI- 353661 is a phosphoramidate. Cross-resistance studies showed that both PSI-352938 and PSI-353661 were fully active against replicons containing the S282T and the S96T/N142T mutations (Lam et al. Antimicrob Agents Chemother 2011; 55: 2566-2575). HCV genotype 2a replicon cells with 19.2-fold reduced susceptibility to PSI-352938 were selected in cell culture. Resistance-associated mutations found were S15G, R222Q, C223Y/H, L320I and V321I. Mutations C223Y and C223H were lethal for the genotype 1b replicon. Variants of the genotype 2a replicon containing mutations S15G/C223H/- V321I and S15G/R222Q/C223H/V321I showed 10- to 20-fold increased resistance to both PSI-352938 and PSI-353661. C223H alone conferred 3.7-fold increased resistance to PSI-352938, but the replication capacity of this variant was very low (Lam et al. J Virol 2011; 85: 12334-12342).

682 Drug Comments R1626 This is the prodrug of R1479 (4´-azidocytidine), a drug related to valopicitabine. S96T and N142T could be involved in phenotypic resistance but results are still preliminary. The mutation S282T does not confer resistance to R1626 (Roberts et al. J Hepatol 2006; 44: S269; Le Pogam et al. Virology 2006; 351: 349-359). Ribavirin Several mechanisms of action have been proposed for this guanosine analogue, including the enhancement of cell-mediated host immunity, depletion of intracellular dGTP pool, inhibition of the viral polymerase, and a mutagenic effect on viral replication. A ribavirin-resistance mutation (F415Y) in NS5B (RNA-dependent RNA polymerase) has been identified in ribavirin-treated patients infected with HCV genotype 1a (Young et al. Hepatology 2003; 38: 869-878). Its relevance to resistance was confirmed using HCV replicons, and discontinuation of treatment led to replacement of Tyr by wild-type Phe at position 415 of NS5B. It has also been proposed that an uptake defect specific for ribavirin could mediate resistance in peripheral blood mononuclear cells (Ibarra et al. J Virol 2011; 85: 7273- 7283). For a recent review on the usage of ribavirin in therapy of hepatitis C, see Thomas et al. Antivir Chem Chemother 2012; 23: 1-12. Sofosbuvir (GS- This compound is a purified diastereroisomer of PSI-7851. This drug 7977, PSI-7977) showed efficacy against genotypes 1, 2, 3, 4 and 6 (Gane et al. N Engl J Med 2013; 368: 34-44; Jacobson et al. N Engl J Med 2013; 368: 1867- 1877; Lawitz et al. N Engl J Med 2013; 368: 1878-1887; reviewed in Abraham & Spooner. Clin Infect Dis 2014; 59: 411-415; Keating. Drugs 2014; 74: 1127-1146). Cells expressing the S96T/N142T mutations remained fully susceptible to PSI-7851, and this compound was less active against the S282T replicon mutant (Lam et al. Antimicrob Agents Chemother 2010; 54: 3187-3196). PSI-7977, a prodrug of β-D-2’-deoxy-2’-fluoro-2’- C-methyluridine monophosphate (Sofia et al. J Med Chem 2010; 53: 7202-7218) was active against replicons of genotypes 1a, 1b and 2a (strain JFG-1). HCV replicons with genotypes 1a and 1b carrying the S282T substitution were resistant to sofosbuvir, but the mutation had a reduced effect on resistance in the context of genotype 2a. Sequence analysis of the JFH-1 NS5B region indicated that additional amino acid changes (T179A, M289L, I293L, M434T and H479P) were selected prior to and after the emergence of S282T (Lam et al. Antimicrob Agents Chemother 2012; 56: 3359-3368).

683 Drug Comments In patients, the selection of sofosbuvir-resistant HCV is rare for genotypes 1-6 and is associated with a significant reduction of viral fitness (Svarovskaia et al. Clin Infect Dis 2014; 54: 1666-1674). It has been estimated that NS5B substitutions, L159F (sometimes in combination with L320F or C316N) and V321A, emerge in 2.2%- 4.4% of subjects who fail sofosbuvir treatment in clinical trials, and polymorphisms at position 316 were potentially associated with reduced response rates in subjects infected with HCV genotype 1b (Sulkowski et al. JAMA 2014; 312: 353-361; Zeuzem et al. N Engl J Med 2014; 370: 1993-2001; Donaldson et al. Hepatology 2015; 61: 56-65). IFNL4-DG genotype is associated with slower viral clearance in patients infected with HCV genotype 1 and treated with sofosbuvir and ribavirin (Meissner et al. J Infect Dis 2014; 209: 1700-1704). TMC647078 Nucleoside inhibitor (2´-deoxy-2´-spirocyclopropylcytidine) effective on HCV genotype 1, with reduced inhibitory activity on strains containing the S282T mutation. However, it maintained full activity against mutants resistant to R1479 (4´-azidocytidine) such as those containing S96T, N142T or S96T/N142T (Berke et al. Antimicrob Agents Chemother 2011; 55: 3812-3820). In vitro resistance selection experiments showed that mutations at position 495 (e.g. P495S, P495L and P495T) were responsible for the largest effects of drug susceptibility, while L392I had a relatively minor influence (Devogelaere et al. Antimicrob Agents Chemother 2012; 56: 4676-4684).

Valopicitabine NM-283 is an oral prodrug of 2´-C-methyl-cytidine. S282T confers resistance (NM-283) to 2’-modified nucleotide analogues (see above, 2´-C-methyl-adenosine), while decreasing viral fitness. Natural nucleotides become 5- to 20-fold less efficiently incorporated into RNA by the mutant NS5B polymerase (Dutrarte et al. Antimicrob Agents Chemother 2006; 50: 4161-4169).

7. RNA-dependent RNA polymerase (NS5B) inhibitors (nonnucleosides)

Drug Comments 4,5-dihydroxy- In assays using recombinant HCV RNA polymerase, mutations G152E pyrimidine and P156L have been shown to confer resistance to this compound carboxylate (Powdrill et al. Antimicrob Agents Chemother 2010; 54: 977-983). A-848837 Specific inhibitor of the viral RNA polymerase. Resistant replicons selected in cell culture contained 1 to 3 mutations of: G46A, S368T, Y392F, M414T, Y448H, Q514R, G554D, D559G and Y586C. Each mutation by itself conferred 4- to 255-fold reduced susceptibility to the inhibitor. All mutants except Y392F exhibited reduced replication capacity (Molla et al. Antivir Ther 2006; 11: S6).

684 Drug Comments Beclabuvir (BMS- Allosteric inhibitor of the NS5B polymerase with low-nanomolar 791325) potency against genotypes 1a and 1b (EC50 values of 3 nM and 7 nM, respectively) (for a review, see Gentile et al. Expert Opin Investig Drugs 2015; 24: 1111-1121). Selected mutations in HCV genotype 1 replicons map at position 495 (e.g. P495A/S/L/T). P495L/S has been transiently observed in one patient treated with the drug (Lemm et al. Antimicrob Agents Chemother 2014; 58: 3485-3495; Sims et al. Antimicrob Agents Chemother 2014; 58: 3496-3503). Genotype 1a has the highest resistance barrier versus the inhibitor while genotype 6a has the lowest. The EC50 of HCV genotype 6a isolates is around 30 nM. Reduced potency against some genotype 6a isolates is due to the presence of the polymorphism Ala494, found in approximately 21% of the sequences in the European HCV database (Liu et al. Antimicrob Agents Chemother 2014; 58: 7416-7423). Benzimidazole Non-nucleoside inhibitors of the HCV NS5B polymerase. Resistance 5-carboxamide mutations in the replicon cell system emerged at positions 495 (P495A/S/-­ compounds L/T; high-level resistance), 496 (P496A/S; moderate resistance) and 499 (V499A; low-level resistance) (Kukolj et al. J Biol Chem 2005; 280: 39260-39267). Mutations T389S, C445F and P459A were selected in HCV genotype 1b replicons cultured in the presence of the compound JT- 16. Replicons having the substitutions T389A and T389S have moderate levels of resistance to JT-16 (7- and 13-fold, respectively), while P495A is associated with high-level (44-fold) resistance (Delang et al. Antiviral Res 2012; 93: 30-38). Benzothiadiazines These drugs target the HCV replicase complex (reviewed in Sarisky. J Antimicrob Chemother 2004; 54: 14-16). Mutations were selected upon passage of HCV replicon cells in the presence of a benzo- 1,2-thiadiazine designated as compound 4. Further analysis showed that the amino acid substitution M414T in NS5B (RNA-dependent RNA polymerase) was sufficient to confer significant resistance to compound 4 (Nguyen et al. Antimicrob Agents Chemother 2003; 47: 3525-3530). In addition, M414T as well as other mutations in NS5B such as H95R, C451R and G558R, were found to confer resistance to benzothiadiazines (Tomei et al. J Virol 2004; 78: 938-946). Replicons selected in the presence of compound A-782759 (an N-1- aza-4-hydroxyquinolone benzothiadiazine) contained single amino acid substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H), with the largest effects being observed with mutations at position 414 (Mo et al. Antimicrob Agents Chemother 2005; 49: 4305-4314). Mutations M414L/V499A (and M414I alone) were shown to confer resistance to benzothiadiazine NNI-2 in phenotypic assays (Le Pougam et al. J Antimicrob Chemother 2008; 61: 1205-1216). C316Y and M414T were shown to confer high-level resistance to A-782759 in phenotypic assays (Delang et al. Antimicrob Agents Chemother 2011; 55: 4103-4113).

685 Drug Comments Dasabuvir Non-nucleoside polymerase inhibitor that shows high rates of sustained (ABT-333) virological response in combination with paritaprevir and ombitasvir (for reviews, see Trivella et al. Expert Opin Pharmacother 2015; 16: 617-624; Mantry & Pathak. Expert Rev Antiinfect Ther 2016; 14: 157- 165). In vitro studies carried out with replicon systems showed that C316Y, M414T, Y448C/H and S556G were the most frequent mutations selected by the drug (Koev et al. J Hepatol 2009; 50: S346-S347). Major mutations found in virus from patients treated with an interferon-free drug combination including desabuvir (ABT-333) were C316Y and M414T (Poordad et al. N Engl J Med 2014; 370: 1973-1982). Deleobuvir Non-nucleoside inhibitor of HCV RNA polymerase with 50% effective (BI 207127) concentration values in the replicon system of 23 nM for HCV genotype 1a and 11 nM for genotype 1b [Beaulieu et al. J Hepatol 2012; 56 (suppl. 2): S231]. Amino acid substitutions affecting Pro495 and Pro496 and V499A were associated with decreased susceptibility to the drug. P495L has been found in patients treated with the inhibitor and reduced drug susceptibility by 120- to 310-fold (Larrey et al. Antimicrob Agents Chemother 2013; 57: 4727-4735). Filibuvir Non-nucleoside inhibitor of HCV RNA polymerase, effective in vitro (PF-00868554) on genotypes 1a and 1b. M423T is the primary mutation associated with filibuvir resistance and no cross-resistance to other polymerase inhibitors was identified in replicons having that amino acid change (Shi et al. Antimicrob Agents Chemother 2009; 53: 2544-2552). Studies carried out with HCV genotype 1b replicons showed that amino acid substitutions M423I, M423T, M423V, R422K and I483S conferred >80-fold increased resistance to the inhibitor, while M423A, M426A, V494A and I482T produced moderate increases of the IC50 for the inhibitor (Troke et al. Antimicrob Agents Chemother 2012; 56: 1331- 1341). Selection in patients of an NS5B mutation at position 423 was associated with reduced replicative capacity in vitro (relative to the pretherapy sequence) (Wagner et al. Hepatology 2011; 54: 50-59; Troke et al. Antimicrob Agents Chemother 2012; 56: 1331-1341). Filibuvir efficacy is reduced by 40-59-fold in HCV replicons of genotypes 2a and 3a (Eltahla et al. Antimicrob Agents Chemother 2014; 58: 7215-7224). In a phase II clinical study of patients treated with pegylated interferon, ribavirin and filibuvir, the presence of resistance mutations R422K, M423I, M426A/T, V494A and the double-mutant R422K/M426T was not associated to poor response to filibuvir therapy (Rodríguez-Torres et al. Ann Hepatol 2014; 13: 464-475).

686 Drug Comments GS-9669 Inhibitor structurally related to lomibuvir and filibuvir. HCV replicons passaged in vitro in the presence of low concentrations of the drug led to the selection of M423T with genotypes 1a and 1b. M423I was found in genotype 1a and M423V in genotype 1b. At higher drug concentrations other selected mutations were L419M/W, R422K and I482L (Dvory-Sobol et al. Antimicrob Agents Chemother 2014; 58: 6599-6606). By themselves, amino acid substitutions L419M, R422K, I482L and A486V confer high-level resistance to the drug, although the effects of mutations at position 423 (i.e. M423I, M423T and M423V)

are less pronounced with 3.4- to 12-fold increases in the EC50 for the drug (compared with the wild-type HCV 1b replicon) (Fenaux et al. Antimicrob Agents Chemother 2013; 57: 804-810; Dvory-Sobol et al. Antimicrob Agents Chemother 2014; 58: 6599-6606). JTK-109 Benzimidazole derivative that inhibits replication of the HCV genotype

1b replicon with an EC50 of 257.1 nM. It shows 6-fold reduced efficacy against genotype 3a and 41-fold against genotype 2a (Eltahla et al. Antimicrob Agents Chemother 2014; 58: 7215-7224). Major mutations conferring resistance to this drug affect position 495 (P495A/L/S/T). JTK-853 Piperazine derivative with inhibitory activity against HCV polymerases of genotypes 1a and 1b (Ando et al. Intervirology 2013; 56: 302-309). Mutations C316Y, M414T, Y452H and L466V were detected in JTK- 853 replicon cells. Each of them produced at least a 20-fold increase

in the IC50 for the inhibitor when introduced in HCV replicons (Ando et al. Antimicrob Agents Chemother 2012; 56: 4250-4256). In patients, treatment with JTK-853 led to the selection of M414T, C445R, Y448C/H and L466F. M414T and L466V conferred high-level resistance to the inhibitor, while C445F and Y448H produce a 5- to 6-fold increase in resistance (Ogura et al. Antimicrob Agents Chemother 2013; 57: 436-444). Lomibuvir (VX- Thumb II allosteric inhibitor that shows an EC50 of 5.9 nM against the 222, VCX-222) HCV genotype 1b replicon, and reduced efficacy against genotypes 2a and 3a (Eltahla et al. Antimicrob Agents Chemother 2014; 58: 7215- 7224). Single amino acid substitutions L419M, R422K, M423I, M423T, M423V and I482L confer >24-fold increased resistance to the drug in phenotypic assays using HCV genotype 1b replicons (Fenaux et al. Antimicrob Agents Chemother 2013; 57: 804-810). Mutations selected in vitro after increasing the doses of lomibuvir were L419C/I/M/P/S/V, R422K, M423I/T/V, I482L/N/T, A486S/T/V and V494A. The highest levels of resistance were obtained with the amino acid substitutions L419C, L419S and R422K that by themselves conferred >200-fold decreased susceptibility to the drug (Jiang et al. Antimicrob Agents Chemother 2014; 58: 5456-5465).

687 Drug Comments Nesbuvir Allosteric inhibitor with EC50s in the range of 16.6-43.3 nM for HCV (HCV-796) genotypes 1b, 2a and 3a (Eltahla et al. Antimicrob Agents Chemother 2014; 58: 7215-7224). Known resistance mutations to this drug map to positions 316 and 365 of the NS5B polymerase. Pyranoindoles Nonnucleoside RNA-dependent RNA polymerase inhibitor. Mutations (HCV-371 and L419M or M423V confer 8- to 10-fold increased resistance to HCV- HCV-570) 371, while this value goes up to 17-fold with replicons containing the combination T19P/M71V/A338V/M423V/A442T. These studies confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb subdomain (Howe et al. Antimicrob Agents Chemother 2006; 50: 4103-4113). Setrobuvir Allosteric inhibitor of the NS5B polymerase that shows an EC50 of 8.1 nM (ANA-598, against the HCV genotype 1b replicon, but >1000-fold reduced efficacy RG7790) against genotypes 2a and 3a (Eltahla et al. Antimicrob Agents Chemother 2014; 58: 7215-7224). Mutations M414L/T, G554D, S556G and D559G could impact resistance to this drug (Thompson et al. Hepatology 2008; 48: 1164A). SPBS inhibitors Inhibitors that bind the thumb site II in the HCV polymerase. Resistance (N-phenylbenzene- to these drugs is mediated by substitutions at positions 419 (e.g. L419M), sulphonamides) 423 (e.g. M423L, M423V or M423K), 476 (e.g. S476A) or 482 (e.g. I482S or I482T) (May et al. Antiviral Res 2012; 95: 182-191). Tegobuvir Imidazopyridine inhibitor of HCV replication with potent antiviral (GS-9190) activity against genotype 1, but with reduced efficiency against genotypes 2a and 3a (Shih et al. Antimicrob Agents Chemother 2011; 55: 4196-4203; Eltahla et al. Antimicrob Agents Chemother 2014; 58: 7215-7224). Resistance selection experiments in HCV genotype 1b replicon cells revealed several mutations that contributed to the drug resistance phenotype: C316Y, Y448H, Y452H and C445F. The strongest effects on resistance were observed with Y448H (36-fold increase in the

IC50), followed by C316Y (8.8-fold increase) (Shih et al. Antimicrob Agents Chemother 2011; 55: 4196-4203). Thiophene-2- Inhibitor of the RNA-dependent RNA polymerase. Resistant HCV carboxilic acid replicon variants contained mutations at positions 419, 423 and 482 in the (NNI-1). polymerase thumb subdomain. Selected double mutants (M414L/M423T) showed reduced replication capacity compared to wild-type replicons (Le Pogam et al. Antivir Ther 2006; 11: S5). TMC647055 Macrocyclic indole currently evaluated with simeprevir in phase II clinical trials. The amino acid substitution P495L produces a 371-fold reduction in drug susceptibility. L392I and V494A mutants have 9- and 3-fold decreased susceptibility to the drug (Devogelaere et al. Antimicrob Agents Chemother 2012; 56: 4676-4684; Cummings et al. J Med Chem 2014; 57: 1880-1892).

688 8. Entry inhibitors

Drug Comments EI-1 Triazine inhibitor that blocks cell-free entry and cell-to-cell transmission of virus. Sequential passage in the presence of EI-1 led to selection of changes in the C-terminal transmembrane anchor region of E2 (e.g. V719F, V719G), conferring >50-fold increased resistance to the inhibitor (Baldick et al. PLoS Pathog 2010; 6: e1001086). Ferroquine Chloroquine analogue that inhibits HCV infection by impairing the fusion process. Resistance to ferroquine was conferred by a single mutation in the E1 glycoprotein (S327A) (Vausselin et al. Hepatology 2013; 58: 86-97). GS-563253 The HCV infectivity inhibitor 1(HCV II-1) is a tetrahydroquinolinone (HCV II-1) that selectively inhibits genotype 1 and 2 HCV in the low nanomolar

range (EC50). Resistance mutations map to the HCV E2 protein. Amino acid substitutions conferring resistance to this inhibitor are P687S that is selected in genotypes 1b and 2a, and R643G/K in HCV genotype 2a (Bush et al. Antimicrob Agents Chemother 2014; 58: 386-396).

Table 13.7. HEPATITIS C VIRUS (HCV) SERINE PROTEASE MUTATIONS ASSOCIATED WITH RESISTANCE TO ANTIVIRAL DRUGS a

Amino acid In vivo / Comments and references substitution In vitro V36A In vivo Selected in patients with boceprevir (Susser et al. Hepatology 2009; 50: 1709-1718) and telaprevir (Sarrazin et al. Gastroenterology 2007; 132: 1767-1777; Talal et al. Hepatology 2014; 60: 1826-1837). In vitro It produces a 7.4-fold increase in the IC50 for telaprevir in replicon-based assays. It has a minor impact on viral fitness (Barbotte et al. Antimicrob Agents Chemother 2010; 54: 2681-2683). Classified as a low-level telaprevir resistance mutation. Confers low-level resistance to boceprevir and danoprevir (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887).

689 Amino acid In vivo / Comments and references substitution In vitro V36C In vitro It produces a 9.5-fold increase in the IC50 for telaprevir in replicon-based assays. It has a minor impact on viral fitness (Barbotte et al. Antimicrob Agents Chemother 2010; 54: 2681-2683). V36L In vivo Found in the HCV protease of patients treated with boceprevir and telaprevir (Hoffmann et al. Virol J 2013; 10: 57). Frequent in genotype 6 isolates (Chen et al. Sci Rep 2016; 6: 20310). In vitro Present in HCV genotypes 2, 3, 4 and 5, this substitution contributes to simeprevir (TMC435) resistance [Lenz et al. Antivir Ther 2011; 16 (suppl. 1): A24]. V36M In vivo Selected in patients treated with telaprevir (Sarrazin et al. Gastroenterology 2007; 132: 1767-1777; Macartney et al. Antiviral Res 2014; 105: 112-117; Talal et al. Hepatology 2014; 60: 1826-1837). Identified in patients treated with boceprevir [Susser et al. Hepatology 2009; 50: 1709-1718; Qiu et al. Nucleic Acids Res 2009; 37: e74; Barnard et al. Antivir Ther 2011; 16 (suppl. 1): A26]. In vitro It produces a 7-fold increase in the IC50 for telaprevir in replicon-based assays. It has a minor impact on viral fitness (Barbotte et al. Antimicrob Agents Chemother 2010; 54: 2681-2683). Classified as a low-level telaprevir resistance mutation, except when appearing in combination with R155K. Q41H In vitro Confers low-level resistance to telaprevir in phenotypic assays (Welsch et al. Gastroenterology 2012; 142: 654-663). Q41R In vitro Confers 3.6-fold increased resistance to grazoprevir (MK- 5172) in HCV genotype 1b (Summa et al. Antimicrob Agents Chemother 2012; 56: 4161-4167). F43I In vitro Decrease simeprevir (TMC435) susceptibility >12-fold, F43S and F43S confers low-level resistance to boceprevir and F43V telaprevir (Lenz et al. Antimicrob Agents Chemother 2010;

54: 1878-1887). F43S increased the IC50 for grazoprevir (MK-5172) by 2.6-fold (Summa et al. Antimicrob Agents Chemother 2012; 56: 4161-4167).

690 Amino acid In vivo / Comments and references substitution In vitro T54A In vivo Selected in patients infected with HCV genotype 1 and treated with telaprevir (Sarrazin et al. Gastroenterology 2007; 132: 1767-1777; Talal et al. Hepatology 2014; 60: 1826-1837) and with boceprevir (Susser et al. Hepatology 2009; 50: 1709-1718; Qiu et al. Nucleic Acids Res 2009; 37: e74; Macartney et al. Antiviral Res 2014; 105: 112-117). Also selected in patients infected with genotype 4 and treated with telaprevir (De Meyer et al. Virol J 2014; 11: 93). In vitro Confers low-level resistance to boceprevir and moderate

resistance (7.5-fold change of the IC50) to telaprevir (Lin et al. J Biol Chem 2004; 279: 17508-17514; Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887). Selected in the presence of telaprevir with the HCV genotype 2a strain J6/JFH-1 (Cheng et al. Antimicrob Agents Chemother 2011; 55: 2197-2205). T54S In vivo Identified in patients treated with boceprevir during phase II and phase III clinical trials [Susser et al. Hepatology 2009; 50: 1709-1718; Qiu et al. Nucleic Acids Res 2009; 37: e74; Barnard et al. Antivir Ther 2011; 16 (suppl. 1): A26; Hoffmann et al. Virol J 2013; 10: 57]. In vitro Confers moderate resistance to boceprevir (8.5-fold

increase in the IC50) in replicon-based assays (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887). V55A In vivo Found in some patients treated with boceprevir during phase II and phase III clinical trials (Susser et al. Hepatology 2009; 50: 1709-1718; Qiu et al. Nucleic Acids Res 2009; 37: e74; Susser et al. J Clin Virol 2011; 52: 321-327; Hoffmann et al. Virol J 2013; 10: 57). In vitro It confers low-level resistance to boceprevir in phenotypic assays (Welsch et al. Antimicrob Agents Chemother 2012; 56: 1907-1915).

691 Amino acid In vivo / Comments and references substitution In vitro Y56H In vivo Found in patients failing therapy with grazoprevir (Lawitz et al. Lancet 2015; 385; 1075-1086; Sulkowski et al. Lancet 2015; 385: 1087-1097) and in individuals infected with HCV genotype 4a and treated with paritaprevir (Schnell et al. Antimicrob Agents Chemother 2015; 59: 6807-6815). Also selected under treatment with simeprevir or deldeprevir (Serre et al. Antimicrob Agents Chemother 2016; 60: 3563-3578). In vitro Alone and in the context of HCV genotype 4a replicons, it confers 8-fold increased resistance to paritaprevir. It also enhances the effects of D168V when combined with this mutation (Schnell et al. Antimicrob Agents Chemother 2015; 59: 6807-6815). P67S In vivo Associated with predisposition to boceprevir or telaprevir treatment failure when present at baseline (Cuypers et al. Infect Genet Evol 2017; 53: 15-23). D79E In vitro Enzymatic assays suggest that this mutation could be responsible in part for the reduced susceptibility to ciluprevir of HCV genotype 2 (Tong et al. Biochemistry 2006; 45: 1353-1361). Q80G In vitro Responsible in part for the ciluprevir resistance observed with HCV genotype 2 (Tong et al. Biochemistry 2006; 45: 1353- 1361). Confers moderate resistance to danoprevir (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887). Q80H In vivo Q80K and Q80R were found in HCV genotype 1-infected Q80K drug-experienced patients treated with simeprevir (Zeuzem Q80R et al. Gastroenterology 2014; 146: 430-441; Jabara et al. Antimicrob Agents Chemother 2014; 58: 6079-6092) and grazoprevir (Lawitz et al. Lancet 2015; 385; 1075-1086). Q80K is a frequent polymorphism in genotype 1a (De Luca et al. Open Forum Infect Dis 2015; 2: ofv043), although it can also be found in genotypes 1b and 6 (Chen et al. Sci Rep 2016; 6: 20310), usually associated with reduced susceptibility to simeprevir. Q80R was also a frequent mutation in isolates of HCV genotypes 4, 5 or 6 from patients treated with simeprevir (Lenz et al. J Hepatol 2013; 58: 445-451). In contrast, clinical studies indicate that Q80K does not have a significant impact on the virological response to faldaprevir (Berger et al. Antimicrob Agents Chemother 2014; 58: 698-705) and asunaprevir (Bronowicki et al. J Hepatol 2014; 61: 1220-1227). 692 Amino acid In vivo / Comments and references substitution In vitro In vitro Decrease simeprevir (TMC435) susceptibility in HCV genotypes 1a and 1b (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887; Verbinnen et al. Antimicrob Agents Chemother 2015; 59: 7548-7557). The presence of Q80K at baseline could have an impact on initial virologic response rates to asunaprevir, but its long-term effects are uncertain (McPhee et al. Antimicrob Agents Chemother 2012; 56: 3670-3681). Confers a small reduction (5.3-fold) in susceptibility to GS-9256 (Mo et al. Antiviral Res 2017; 140: 151-157). Q86R In vitro This substitution (selected with SCH6) can partially revert the replicative defect of mutants carrying A156T (Yi et al. J Biol Chem 2006; 281: 8205-8215). P89L In vitro This substitution (selected with SCH6) can partially revert the replicative defect of mutants carrying A156T (Yi et al. J Biol Chem 2006; 281: 8205-8215). A98T In vitro This amino acid substitution produces an increase in viral fitness when combined with other resistance mutations (Serre et al. Antimicrob Agents Chemother 2016; 60: 3563- 3578). It has been identified in HCV genotype 3 patients either naïve or failing telaprevir therapy without apparent resistance (Paolucci et al. Virol J 2012; 9: 245; De Meyer et al. J Viral Hepat 2013; 20: 395-403). R109K In vitro Confers moderate (2-fold) resistance to SCH6 in the replicon assay, while having a minimal effect on protease activity (Yi et al. J Biol Chem 2006; 281: 8205-8215). Selected in vitro after passage of HCV genotype 1a in the presence of grazoprevir (Lahser et al. Antimicrob Agents Chemother 2016; 60: 2954-2964). R117H In vivo Deep sequencing methods have shown that this mutation together with S174F could pre-exist and persist during a long term in patients treated with telaprevir and boceprevir [Susser et al. Antivir Ther 2012; 17 (suppl. 1): A93]. N122D/S In vitro Associated with boceprevir resistance in the HCV genotype 6 (Aloia et al. Antivir Ther 2015; 20: 271-280).

693 Amino acid In vivo / Comments and references substitution In vitro S122G In vivo Found in HCV genotype 1a-infected patients failing treatment with danoprevir and mericitabine, that developed the R155K mutation. In vitro studies indicate that S122G has no effect on drug susceptibility but improves the viral replication capacity by 2- to 4-fold (Tong et al. Antimicrob Agents Chemother 2014; 58. 3105-3114). Also found in patients treated with grazoprevir (Lawitz et al. Lancet 2015; 385; 1075-1086). S122K In vitro Contributes to ciluprevir resistance observed with HCV genotype 2, as determined in enzymatic assays (Tong et al. Biochemistry 2006; 45: 1353-1361). S122R In vitro This mutation has been associated with resistance to simeprevir (TMC435) during phase III clinical trials. It also enhances resistance mediated by R155K [Fevery et al. Antivir Ther 2012; 17 (suppl. 1): A100]. I132L In vivo Polymorphism found in treatment-experienced HCV genotype 4-infected patients (Fevery et al. J Viral Hepat 2017; 24: 28-36). In vitro HCV genotype 4 clinical isolates bearing this amino acid substitution had 4-fold reduced susceptibility to simeprevir in phenotypic assays (Fevery et al. J Viral Hepat 2017; 24: 28-36). S138T In vitro Decrease ciluprevir, simeprevir and danoprevir susceptibility about 3- to 4.5-fold (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887). R155G In vitro Confers >100-fold increased resistance to vaniprevir (Lawitz et al. Antiviral Res 2013; 99: 214-220). R155K In vivo Selected in patients treated with telaprevir (Sarrazin et al. Gastroenterology 2007; 132: 1767-1777; Macartney et al. Antiviral Res 2014; 105: 112-117). This mutation was shown to persist for around two years in one patient, in the absence of protease-inhibitor based therapy (Colson & Gérolami. J Infect Dis 2011; 203: 1341-1342). Found in patients failing treatment with boceprevir [Susser et al. Hepatology 2009; 50: 1709-1718; Barnard et al. Antivir Ther 2011; 16 (suppl. 1): A26] and vaniprevir (Lawitz et al. Antiviral Res 2013; 99: 214- 220). Predominant mutation appearing in patients treated with asunaprevir (Bronowicki et al. J Hepatol 2014; 61: 1220-1227; Muir et al. JAMA 2015; 313: 1736-1744), danoprevir (Lim et

694 Amino acid In vivo / Comments and references substitution In vitro al. Antimicrob Agents Chemother 2012; 56: 271-279; Gane et al. Antimicrob Agents Chemother 2014; 58: 1136-1145; Tong et al. Antimicrob Agents Chemother 2014; 58: 3105-3114), GS-9451 (Dvory-Sobol et al. Antimicrob Agents Chemother 2012; 56: 5289-5295; Lawitz et al. Antivir Ther 2013; 18: 311- 319) and narlaprevir (de Bruijne et al. J Viral Hepat 2013; 20: 779-789). Also found in virus isolated from patients infected with HCV genotypes 4-6 and treated with simeprevir (Lenz et al. J Hepatol 2013; 58: 445-451). In vitro R155K, as well as R155I, R155M and R155T are con- sidered as low-level resistance mutations for telaprevir, boceprevir, narlaprevir and grazoprevir (MK-5172), and confer high-level resistance to asunaprevir, ciluprevir, danoprevir, faldaprevir, vaniprevir, GS-9256 and GS-9451 (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878- 1887; Lim et al. Antimicrob Agents Chemother 2012; 56: 271-279; Lagacé et al. Antimicrob Agents Chemother 2012; 56: 569-572; McPhee et al. Antimicrob Agents Chemother 2012; 56: 3670-3681; Summa et al. Antimicrob Agents Chemother 2012; 56: 4161-4167; Dvory-Sobol et al. Antimicrob Agents Chemother 2012; 56: 5289-5295; Lawitz et al. Antiviral Res 2013; 99: 214-220; Mo et al. Antiviral Res 2017; 140: 151-157). In phenotypic assays, R155K produces a 2.1- and 8.8-fold increase in resis- tance to boceprevir and telaprevir, respectively (Welsch et al. Gastroenterology 2012; 142: 654-663). Although this mutation has a mild effect on resistance to all protease inhibitors when present in HCV genotype 3a, it con- fers high-level resistance (>80-fold increase in the EC50) when tested against simeprevir (genotype 4a), vaniprevir (genotypes 1a, 4a and 5a), faldaprevir (genotypes 4a, 5a and 6a), paritaprevir (genotype 1a), and deldeprevir (1a, 2a, 4a, 5a and 6a) (Jensen et al. Antimicrob Agents Chemother 2015; 59: 7426-7436). R155M In vivo R155M has been identified in patients treated with R155Q telaprevir (Talal et al. Hepatology 2014; 60: 1826-1837). In vitro Associated with ciluprevir and danoprevir resistance (Courcambeck et al. Antivir Ther 2006; 11: 847-855; Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887).

695 Amino acid In vivo / Comments and references substitution In vitro R155S In vivo Selected in patients treated with boceprevir (Susser et al. R155T Hepatology 2009; 50: 1709-1718) or telaprevir (Sarrazin et al. Gastroenterology 2007; 132: 1767-1777; Talal et al. Hepatology 2014; 60: 1826-1837). In vitro R155T confers intermediate levels of resistance to boceprevir, telaprevir, simeprevir and vaniprevir, and high- level resistance to danoprevir, faldaprevir and deldeprevir (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878- 1887; Jensen et al. Antimicrob Agents Chemother 2015; 59: 7426-7436). Intermediate levels of resistance to vaniprevir were also observed with R155S (Lawitz et al. Antiviral Res 2013; 99: 214-220). A156G In vitro Amino acid substitution found in ciluprevir-resistant HCV genotype 2a strain J6/JFH-1, but not in the genotype 2a replicon (Cheng et al. Antimicrob Agents Chemother 2011; 55: 2197-2205). Confers resistance to simeprevir, vaniprevir and faldaprevir in different HCV genotypes (e.g. 1a, 2a, 4a, 5a and 6a) (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887; Jensen et al. Antimicrob Agents Chemother 2015; 59: 7426-7436). A156S In vivo Selected in patients during phase I clinical trials with telaprevir (Sarrazin et al. Gastroenterology 2007; 132: 1767- 1777). Identified in patients treated with boceprevir during phase II clinical trials (Susser et al. Hepatology 2009; 50: 1709-1718; Qiu et al. Nucleic Acids Res 2009; 37: e74). In vitro Selected in vitro in the presence of telaprevir. It confers 11-fold increased resistance to telaprevir, <20-fold increased resistance to boceprevir, and no effect on ciluprevir or simeprevir (TMC435) resistance (Lin et al. J Biol Chem 2004; 279: 17508-17514; Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887; Serre et al. Antimicrob Agents Chemother 2016; 60: 3563-3578). It has a negative effect on fitness, as determined in a colony formation assay (Tong et al. Antiviral Res 2006; 70: 28-38). Also selected in the presence of telaprevir with the HCV genotype 2a strain J6/JFH-1 (Cheng et al. Antimicrob Agents Chemother 2011; 55: 2197-2205). It also confers significant levels of resistance to telaprevir when found in HCV genotypes 1a and 5a (Jensen et al. Antimicrob Agents Chemother 2015; 59: 7426-7436). When introduced in HCV genotypes 4a and 5a, A156S increases asunaprevir resistance by more than 10-fold (Jensen et al. Antimicrob

696 Amino acid In vivo / Comments and references substitution In vitro Agents Chemother 2015; 59: 7426-7436). A156S mutants had decreased replication capacity and increased viral assembly (Serre et al. Antimicrob Agents Chemother 2016; 60: 3563- 3578). A156T In vivo Selected in patients treated with telaprevir (Sarrazin et al. Gastroenterology 2007; 132: 1767-1777; Talal et al. Hepatology 2014; 60: 1826-1837) and grazoprevir (Lawitz et al. Lancet 2015; 385; 1075-1086; Sulkowski et al. Lancet 2015; 385: 1087-1097). In vitro Confers high-level resistance (>70-fold) to boceprevir, telaprevir, ciluprevir, simeprevir, danoprevir, faldaprevir, grazoprevir (MK-5172) and GS-9256 (Lin et al. J Biol Chem 2004; 279: 17508-17514; Lin et al. J Biol Chem 2005; 280: 36784-36791; Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887; Lagacé et al. Antimicrob Agents Chemother 2012; 56: 569-572; Summa et al. Antimicrob Agents Chemother 2012; 56: 4161-4167; Mo et al. Antiviral Res 2017; 140: 151-157). Selected in vitro with the HCV replicon in the presence of SCH6 (SCH446211) (Yi et al. J Biol Chem 2006; 281: 8205-8215). It has a large negative effect on fitness (Tong et al. Antiviral Res 2006; 70: 28-38). A156V In vivo Selected in patients during phase I clinical trials with telaprevir (Sarrazin et al. Gastroenterology 2007; 132: 1767- 1777). Found in patients treated with vaniprevir (Lawitz et al. Antiviral Res 2013; 99: 214-220) and grazoprevir (Lawitz et al. Lancet 2015; 385; 1075-1086). In vitro Confers >70-fold increased resistance to boceprevir, telaprevir, ciluprevir, simeprevir, danoprevir, faldaprevir, vaniprevir and grazoprevir (MK-5172) (Lin et al. J Biol Chem 2005; 280: 36784-36791; Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887; Lagacé et al. Antimicrob Agents Chemother 2012; 56: 569-572; Summa et al. Antimicrob Agents Chemother 2012; 56: 4161-4167; Lawitz et al. Antiviral Res 2013; 99: 214-220). However, grazoprevir showed relatively high efficacy against HCV genotype 2a replicons containing this amino acid substitution (Serre et al. Antimicrob Agents Chemother 2016; 60: 3563- 3578). Selected in vitro with the HCV genotype 1 replicons in the presence of SCH6 (SCH446211) (Yi et al. J Biol Chem 2006; 281: 8205-8215).

697 Amino acid In vivo / Comments and references substitution In vitro V158I In vivo Identified in patients treated with boceprevir during phase II clinical trials (Qiu et al. Nucleic Acids Res 2009; 37: e74). In vitro Confers 3.3-fold increased resistance to boceprevir as determined with the replicon system (Qiu et al. Nucleic Acids Res 2009; 37: e74). G162R In vitro This substitution (selected with SCH6) can partially revert the replicative defect of mutants carrying A156T (Yi et al. J Biol Chem 2006; 281: 8205-8215).

D168A In vivo This amino acid substitution and other changes at this position (e.g. D168G/H/V/Y) are commonly found in patients treated with asunaprevir (McPhee et al. Antimicrob Agents Chemother 2012; 56: 3670-3681; Bronowicki et al. J Hepatol 2014; 61: 1220-1227), vaniprevir (Lawitz et al. Antiviral Res 2013; 99: 214-220), and in patients treated with grazoprevir (Lawitz et al. Lancet 2015; 385; 1075-1086; Sulkowski et al. Lancet 2015; 385: 1087-1097). D168A is associated with high-level resistance to the inhibitor and impaired replication capacity. In vitro High-level resistance mutation selected in vitro after expo- sure to ciluprevir (Lin et al. J Biol Chem 2004; 279: 17508- 17514; Mo et al. Antimicrob Agents Chemother 2005; 49: 4305-4314) or vaniprevir (Lawitz et al. Antiviral Res 2013; 99: 214-220). It has no effect on telaprevir or boceprevir re- sistance. Confers high-level resistance to faldaprevir (Lagacé et al. Antimicrob Agents Chemother 2012; 56: 569-572). In genotypes 1a, 5a and 6a, D168A confers high-level resis­ tance­ to simeprevir, asunaprevir, vaniprevir, paritaprevir, deldeprevir and grazoprevir (Jensen et al. Antimicrob Agents Chemother 2015; 59: 7426-7436). However, the combination D168A/R155K was found to be susceptible to grazoprevir, while showing high-level resistance to simeprevir (Guo et al. J Biol Chem 2017; 292: 6202-6212). D168E In vivo D168E has been detected in patients treated with GS-9451 D168H and infected with genotypes 1 and 2 (Dvory-Sobol et al. Antimicrob Agents Chemother 2012; 56: 5289-5295), in patients treated with simeprevir and infected with genotypes 4-6 (Lenz et al. J Hepatol 2013; 58: 445-451), in patients infected with HCV genotype 4 and receiving asunaprevir in combination with pegylated interferon and ribavirin (Bronowicki et al. J Hepatol 2014; 61: 1220-1227), and in patients receiving grazoprevir in combination with elbasvir (Lawitz et al. Lancet 2015; 385; 1075-1086).

698 Amino acid In vivo / Comments and references substitution In vitro In vitro Confer resistance to ciluprevir, simeprevir, danoprevir and GS-9451 (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887; Dvory-Sobol et al. Antimicrob Agents Chemother 2012; 56: 5289-5295). D168E confers resistance to asunaprevir [McPhee et al. Hepatology 2009; 50 (suppl. 4): 1048A] and grazoprevir (Guo et al. J Biol Chem 2017; 292: 6202-6212), and has been found occasionally in patients failing danoprevir treatment (Lim et al. Antimicrob Agents Chemother 2012; 56: 271-279), and in patients receiving GS-9451 (Dvory-Sobol et al. Antimicrob Agents Chemother 2012; 56: 5289-5295). D168G In vivo D168G has been detected in patients treated with GS-9451 D168N (Dvory-Sobol et al. Antimicrob Agents Chemother 2012; 56: 5289-5295) and vaniprevir (Lawitz et al. Antiviral Res 2013; 99: 214-220). In vitro These amino acid substitutions confer moderate resistance to simeprevir (TMC435) and faldaprevir (i.e. >10-fold

change in the IC50 relative to the wild-type replicon) (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887; Lagacé et al. Antimicrob Agents Chemother 2012; 56: 569-572). D168G confers high-level resistance to asunaprevir, vaniprevir, faldaprevir, deldeprevir and GS- 9451, particularly when appearing in HCV genotypes 1b and 6a (McPhee et al. Antimicrob Agents Chemother 2012; 56: 3670-3681; Dvory-Sobol et al. Antimicrob Agents Chemother 2012; 56: 5289-5295; Lawitz et al. Antiviral Res 2013; 99: 214-220; Jensen et al. Antimicrob Agents Chemother 2015; 59: 7426-7436), and 24-fold decreased susceptibility to grazoprevir in HCV genotype 1a replicons (Guo et al. J Biol Chem 2017; 292: 6202-6212). D168I In vitro Confers high-level (>2000-fold change in the IC50) resistance to simeprevir (TMC435) (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887). D168N In vivo Found in patients experiencing virological failure to grazoprevir (Sulkowski et al. Lancet 2015; 385: 1087-1097). D168Q In vitro This mutation, present in HCV genotype 3 appears to be responsible for resistance to the macrocyclic inhibitor simeprevir (TMC435) [Lenz et al. Antivir Ther 2011; 16 (suppl. 1): A24], and for differences in susceptibility to ciluprevir and other inhibitors between genotypes 3 and 1b (Tong et al. Biochemistry 2006; 45: 1353-1361). 699 Amino acid In vivo / Comments and references substitution In vitro D168T In vitro Confers high-level resistance (i.e. >200-fold increase in the IC50 relative to the wild-type virus) to ciluprevir, simeprevir and danoprevir (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887; Lim et al. Antimicrob Agents Chemother 2012; 56: 271-279). D168V In vivo Found in virus from patients treated with GS-9451 (Dvory- Sobol et al. Antimicrob Agents Chemother 2012; 56: 5289-5295), simeprevir (Lenz et al. J Hepatol 2013; 58: 445-451: Fevery et al. J Viral Hepat 2017; 24: 28-36), vaniprevir (Lawitz et al. Antiviral Res 2013; 99: 214-220) and ritonavir-boosted paritaprevir (ABT-450) (Poordad et al. N Engl J Med 2014; 370: 1973-1982; Schnell et al. Antimicrob Agents Chemother 2015; 59: 6807-6815). Predominant mutation in HCV genotype 1b isolates of patients failing treatment with danoprevir (in combination with the RNA polymerase inhibitor mericitabine) (Tong et al. Antimicrob Agents Chemother 2014; 58: 3105-3114). It confers >300-fold increased resistance to paritaprevir in genotype 4a and 4d HCV replicons (Schnell et al. Antimicrob Agents Chemother 2015; 59: 6807-6815). In vitro High-level resistance mutation selected in vitro after exposure to ciluprevir (Lin et al. J Biol Chem 2004; 279: 17508-17514; Delang et al. Antimicrob Agents Chemother 2011; 55: 4103-4113), simeprevir (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887), vaniprevir (Lawitz et al. Antiviral Res 2013; 99: 214-220) and GS- 9451 (Dvory-Sobol et al. Antimicrob Agents Chemother 2012; 56: 5289-5295). It has no effect on telaprevir or boceprevir resistance, but confers high-level resistance to faldaprevir (Lagacé et al. Antimicrob Agents Chemother 2012; 56: 569-572) and GS-9256 (Mo et al. Antiviral Res 2017; 140: 151-157), and has a significant impact on resistance to grazoprevir (MK-5172) (Summa et al. Antimicrob Agents Chemother 2012; 56: 4161-4167; Guo et al. J Biol Chem 2017; 292: 6202-6212). D168V confers high-level resistance to asunaprevir, vaniprevir, paritaprevir and deldeprevir when present in HCV genotype 5a (Jensen et al. Antimicrob Agents Chemother 2015; 59: 7426-7436). D168Y In vivo Found in virus from patients treated with grazoprevir (Lawitz et al. Lancet 2015; 385; 1075-1086).

700 Amino acid In vivo / Comments and references substitution In vitro In vitro Mutation selected in vitro in HCV replicons grown in the presence of a tripeptide inhibitor of the HCV protease (compound 1). Appears together with E176G (Trozzi et al. J Virol 2003; 77: 3669-3679). Confers resistance to grazoprevir (MK-5172) (Summa et al. Antimicrob Agents Chemother 2012; 56: 4161-4167; Guo et al. J Biol Chem 2017; 292: 6202-6212) and paritaprevir (ABT-450) (Pilot-Marias et al. Antimicrob Agents Chemother 2015; 59: 988-997). I170T In vitro Rare polymorphism that confers resistance to asunaprevir and faldaprevir in HCV replicons 1a and 1b (McPhee et al. Antimicrob Agents Chemother 2012; 56: 3670-3681; Berger et al. Antimicrob Agents Chemother 2013; 57: 4928-4936). V170A In vivo Identified in patients treated with boceprevir during phase II clinical trials (Susser et al. Hepatology 2009; 50: 1709- 1718; Qiu et al. Nucleic Acids Res 2009; 37: e74). In vitro Confers some resistance to boceprevir (Lin et al. J Biol Chem 2004; 279: 17508-17514), but does not affect ciluprevir, simeprevir or danoprevir susceptibility (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887). V170T In vitro Rare polymorphism that produces a 5- to 7-fold decrease in ciluprevir, simeprevir (TMC435) and faldaprevir susceptibility (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887; Berger et al. Antimicrob Agents Chemother 2013; 57: 4928-4936). N174H In vivo Identified as potentially related to boceprevir resistance (S174H) after extensive bioinformatics analysis using HCV sequences from treated patients (Cuypers et al. Infect Genet Evol 2017; 53: 15-23). S174F In vivo See R117H. M175L In vitro Selected in cell culture with the HCV genotype 1b replicon in the presence of a combination of boceprevir and a viral polymerase inhibitor. This mutation confers 3-fold increased resistance to boceprevir (Chase et al. Antiviral Res 2009; 84: 178-184). E176G In vitro Selected in vitro in HCV replicons grown in the presence of a tripeptide inhibitor of the HCV protease (compound 1). Appears together with mutations at codon 168 in replicons showing high-level resistance to the inhibitor (Trozzi et al. J Virol 2003; 77: 3669-3679).

701 Amino acid In vivo / Comments and references substitution In vitro E176K In vitro HCV replicons displaying high-level resistance to compound 1 (a tripeptide HCV protease inhibitor) contained additional mutations: P67S, D168A and R512K (Trozzi et al. J Virol 2003; 77: 3669-3679). a Underlined substitutions appear in the protease of other genotypes different from 1a.

702 Figure 13.6. Effect of NS3 mutations on antiviral activities of several serine protease (NS3/4A) inhibitors. Assays were carried out with single or multiple-site-directed mutants in a genotype 1b replicon backbone (Lenz et al. Antimicrob Agents Chemother 2010; 54: 1878-1887; Jiang et al. Antimicrob Agents Chemother 2013; 57: 6236-6245; Kieffer & George. Curr Opin Virol 2014; 8: 16-21). Vanoprevir and faldaprevir data were taken from Lagacé et al. Antimicrob Agents Chemother 2012; 56: 569-572, and Berger et al. Antimicrob Agents Chemother 2013; 57: 4928-4936. Additional data were collected for asunaprevir (McPhee et al. Antimicrob Agents Chemother 2012; 56: 3670-3681), grazoprevir (MK-5172) (Summa et al. Antimicrob Agents Chemother 2012; 56: 4161- 4167; Lahser et al Antimicrob Agents Chemother 2016; 60: 2954-2964), paritaprevir (ABT-450) (Pilot-Matias et al. Antimicrob Agents Chemother 2015; 59: 988-997) and GS- 9256 (Mo et al. Antiviral Res 2017; 140: 151-157). When the replication capacity is >70% relative to the wild-type clone, this is indicated with black boxes, when the value is 20- 70% with hatched boxes, and for replication capacities below 20%, values are represented with white boxes. High-level (>50-fold increase of the IC50 for the inhibitor), moderate (10- to 50-fold increase) and low-level (2.5- to 10-fold increase) resistance are represented by solid, hatched and grey boxes, respectively. Open boxes indicate susceptible viruses

(<2.5-fold increase in the IC50 for the inhibitor).

703 Table 13.8. HEPATITIS C VIRUS (HCV) NS5A MUTATIONS ASSOCIATED WITH RESISTANCE INHIBITORS a

Amino acid In vivo / substitution In vitro Comments and references L23F In vitro Secondary mutation selected with daclatasvir (genotype 1b) (Fridell et al. Antimicrob Agents Chemother 2010; 54: 3641-3650; Wang et al. Antimicrob Agents Chemother 2012; 56: 1350-1358). K24G In vitro Confers 50-fold decreased susceptibility to EDP-239 in drug susceptibility assays carried out with HCV replicon 1a (Owens et al. Antimicrob Agents Chemother 2016; 60: 6216-6226). K24R In vitro It has no effect on EDP-239 resistance, but confers 32-fold decreased susceptibility to EDP-239 in combination with F36L (Owens et al. Antimicrob Agents Chemother 2016; 60: 6216-6226). F28S In vitro Major daclatasvir resistance mutation identified in genotype 2a JFH1 replicons (Fridell et al. J Virol 2011; 85: 7312- 7320; O’Boyle II et al. Antimicrob Agents Chemother 2016; 60: 1573-1583). Found associated with M31I in pibrentasvir-resistant HCV genotype 2a replicons (Ng et al. Antimicrob Agents Chemother 2017; 61: e02558-16). L28S In vivo L28S together with M31I and Y93H have been associated with daclatasvir resistance in patients infected with HCV genotype 4 variants (Bartolini et al. J Clin Virol 2015; 66: 38-43). L28V In vitro Major ombitasvir resistance mutation in HCV genotypes 4a and 4d (Schnell et al. Antimicrob Agents Chemother 2015; 59: 6807-6815). M28T In vivo Found in patients infected with HCV genotype 1a and (L28T) treated with daclatasvir (for a review see Pawlotsky. J Hepatol 2013; 59: 375-382) or elbasvir (Lawitz et al. Lancet 2015; 385; 1075-1086; Sulkowski et al. Lancet 2015; 385: 1087-1097).

704 Amino acid In vivo / substitution In vitro Comments and references In vitro See Figure 13.7 for effects in HCV genotype 1a replicons. M28T in HCV genotype 1a confers high-level resistance to ombitasvir and GSK2336805 (DeGoey et al. J Med Chem 2014; 57: 2047-2057; Kazmierski et al. J Med Chem 2014; 57: 2058-2073; Walker et al. Antimicrob Agents Chemother 2014; 58: 38-47), while L28T in genotype 1b confers high-level resistance to ombitasvir (DeGoey et al. J Med Chem 2014; 57: 2047-2057). M28V In vitro Selected in patients infected with HCV genotype 1a, treated with EDP-239 (Owens et al. Antimicrob Agents Chemother 2016; 60: 6216-6226). P29S In vitro The combination P29S/K30G was selected in genotype 2a replicons after exposure to pibrentasvir (Ng et al. Antimicrob Agents Chemother 2017; 61: e02558-16). A30K In vivo The presence of this mutation at baseline in HCV genotype 2 variants could lead to the loss of daclatasvir efficacy (Sulkowski et al. N Engl J Med 2014; 370: 211-221). Also found in patients treated with velpatasvir (Lee et al. Hepatol Int 2017; 11: 161-170). K30G In vitro See P29S. Q30E In vivo Major mutation selected in patients treated with daclatasvir (genotype 1a) (Fridell et al. Antimicrob Agents Chemother 2010; 54: 3641-3650; Wang et al. Antimicrob Agents Chemother 2012; 56: 1350-1358; McPhee et al. Hepatology 2013; 58: 902-911; McPhee et al. Antivir Ther 2014; 19: 479-490). In vitro See Figure 13.7 for effects in HCV genotype 1a replicons. Major ledipasvir resistance-associated mutation for all genotypes (Wong et al. Antimicrob Agents Chemother 2014; 57: 6333-6340; Cheng et al. Antimicrob Agents Chemother 2016; 60: 1847-1853). Q30H In vivo Selected in patients infected with HCV genotype 1a and (L30H, R30H) treated with daclatasvir (for a review see Pawlotsky. J Hepatol 2013; 59: 375-382), elbasvir (Lawitz et al. Lancet 2015; 385; 1075-1086; Sulkowski et al. Lancet 2015; 385: 1087-1097), ledipasvir (Wyles et al. J Hepatol 2017; 66: 703-710), velpatasvir (Lawitz et al. J Viral Hepat 2015; 22: 1011-1019), and EDP-239 (Owens et al. Antimicrob Agents Chemother 2016; 60: 6216-6226).

705 Amino acid In vivo / substitution In vitro Comments and references In vitro See Figure 13.7 for effects in HCV genotype 1a replicons. In genotype 4a strains, it produces >60-fold decreased daclastavir susceptibility in comparison with the wild-type strain (Wang et al. Antimicrob Agents Chemother 2012; 56: 1588-1590). Q30K In vivo Mutation selected in patients treated with daclatasvir (genotype 1a), although less frequent than Q30E or Q30H (Fridell et al. Antimicrob Agents Chemother 2010; 54: 3641-3650; Wang et al. Antimicrob Agents Chemother 2012; 56: 1350-1358). In vitro Confers high-level resistance to GSK2336805 in HCV genotype 1a (Kazmierski et al. J Med Chem 2014; 57: 2058-2073; Walker et al. Antimicrob Agents Chemother 2014; 58: 38-47). Q30L In vivo Found in patients treated with elbasvir (Lawitz et al. Lancet 2015; 385; 1075-1086; Sulkowski et al. Lancet 2015; 385: 1087-1097). Q30R In vivo Selected in patients infected with HCV genotype 1a and treated with daclatasvir (reviewed in Pawlotsky. J Hepatol 2013; 59: 375-382; McPhee et al. Hepatology 2013; 58: 902- 911; McPhee et al. Antivir Ther 2014; 19: 479-490), elbasvir (Lawitz et al. Lancet 2015; 385; 1075-1086; Sulkowski et al. Lancet 2015; 385: 1087-1097), ledipasvir (Wyles et al. J Hepatol 2017; 66: 703-710), velpatasvir (Feld et al. N Engl J Med 2015; 373: 2599-2607; Foster et al. N Engl J Med 2015; 373: 2608-2617) and EDP-239 (Owens et al. Antimicrob Agents Chemother 2016; 60: 6216-6226). Major ombitasvir resistance mutation in HCV genotype 1a-infected patients (DeGoey et al. J Med Chem 2014; 57: 2047-2057). In vitro See Figure 13.7 for effects in HCV genotype 1a replicons. Confers high-level resistance to ombitasvir and GSK2336805 in HCV genotype 1a replicons (DeGoey et al. J Med Chem 2014; 57: 2047-2057; Kazmierski et al. J Med Chem 2014; 57: 2058-2073; Walker et al. Antimicrob Agents Chemother 2014; 58: 38-47).

706 Amino acid In vivo / substitution In vitro Comments and references R30G In vitro Phenotypic assays showed that in HCV genotype 4a strains, R30S both changes produce >60-fold decreased daclastavir (Q30G) susceptibility relative to the wild-type strain (Wang et al. Antimicrob Agents Chemother 2012; 56: 1588-1590). The substitution Q30G in HCV 1a replicons confers high-level resistance to elbasvir (Lahser et al. Antimicrob Agents Chemother 2016; 60: 2954-2964). L31F In vitro Confers high-level resistance to daclatasvir in HCV genotype 3a replicons (Wang et al. Antimicrob Agents Chemother 2013; 57: 611-613; Hernandez et al. J Clin Virol 2013; 57: 13-18). L31I In vivo Detected at failure in patients treated with daclatasvir and infected with genotype 1a (McPhee et al. Hepatology 2013; 58: 902-911; McPhee et al. Antivir Ther 2014; 19: 479-490). Also found in patients infected with genotype 2a HCV, treated with ledipasvir and sofosbuvir (Gane et al. Gastroenterology 2017; 152: 1366-1371). L31M In vivo Selected in patients infected with HCV genotype 1a and treated with daclatasvir (for a review see Pawlotsky. J Hepatol 2013; 59: 375-382), elbasvir (Lawitz et al. Lancet 2015; 385; 1075-1086; Sulkowski et al. Lancet 2015; 385: 1087-1097), ledipasvir (Afdhal et al. N Engl J Med 2014; 370: 1889-1898; Wilson et al. Clin Infect Dis 2016; 62: 280-288; Wyles et al. J Hepatol 2017; 66: 703-710; Gane et al. Gastroenterology 2017; 152: 1366-1371) or velpatasvir (Feld et al. N Engl J Med 2015; 373: 2599-2607; Foster et al. N Engl J Med 2015; 373: 2608-2617; Lee et al. Hepatol Int 2017; 11: 161-170). In vitro See Figure 13.7 for effects in HCV genotype 1a replicons. Confers high-level resistance to daclatasvir in HCV genotype 3a replicons (Wang et al. Antimicrob Agents Chemother 2013; 57: 611-613; Hernandez et al. J Clin Virol 2013; 57: 13-18).

707 Amino acid In vivo / substitution In vitro Comments and references L31V In vivo Natural polymorphism in HCV genotype 4 isolates. Selected in patients infected with HCV genotypes 1a and 1b and treated with daclatasvir (reviewed in Pawlotsky. J Hepatol 2013; 59: 375-382) In vitro See Figure 13.7 for effects in HCV genotypes 1a and 1b replicons. Confers high-level resistance to daclatasvir in HCV genotype 3a replicons (Wang et al. Antimicrob Agents Chemother 2013; 57: 611-613; Hernandez et al. J Clin Virol 2013; 57: 13-18) and to GSK2336805 in HCV genotype 1a replicons (Kazmierski et al. J Med Chem 2014; 57: 2058-2073; Walker et al. Antimicrob Agents Chemother 2014; 58: 38-47). L31W In vitro Confers high-level resistnce to daclatasvir (genotype 1b) (Wang et al. Antimicrob Agents Chemother 2012; 56: 1350-1358). M31I In vivo Associated with daclatasvir resistance in genotype 4 variants (Bartolini et al. J Clin Virol 2015; 66: 38-43). In vitro Confers high-level resistance to pibrentasvir when combined with F28S in HCV genotype 2a replicons (Ng et al. Antimicrob Agents Chemother 2017; 61: e02558-16). P32L In vitro Associated with resistance to daclatasvir in HCV genotypes 1a and 1b (Fridell et al. Antimicrob Agents Chemother 2010; 54: 3641-3650; Fridell et al. Hepatology 2011; 54: 1924-1935; Wang et al. Antimicrob Agents Chemother 2012; 56: 1350-1358). F36L In vitro See K24R. S38F In vitro Identified in rebound virus obtained from treatment with S38T NS5A synergist inhibitors Syn-535 and Syn-690 (O’Boyle II et al. Antimicrob Agents Chemother 2016; 60: 1573-1583). H58D In vivo Selected in patients infected with HCV genotype 1a and treated with velpatasvir (Lawitz et al. J Viral Hepat 2015; 22: 1011-1019). In vitro See Figure 13.7 for effects in HCV genotype 1a replicons. It enhances pibrentasvir resistance when combined with Y93H, in HCV genotype 1a replicons (Ng et al. Antimicrob Agents Chemother 2017; 61: e02558-16).

708 Amino acid In vivo / substitution In vitro Comments and references P58S In vitro Secondary mutation selected with daclatasvir (genotype 1b) (Fridell et al. Antimicrob Agents Chemother 2010; 54: 3641-3650; Wang et al. Antimicrob Agents Chemother 2012; 56: 1350-1358). A92V In vitro Decreases HCV genotype 1b susceptibility to EDP-239 when the Y93H substitution is present (Owens et al. Antimicrob Agents Chemother 2016; 60: 6216-6226). C92R In vitro Selected in HCV genotype 2a replicons grown in the presence of daclatasvir. It has a negative impact on their replication capacity (Fridell et al. J Virol 2011; 85: 7312- 7320). Y93C In vivo Selected in patients infected with HCV genotype 1a and treated with daclatasvir (for a review see Pawlotsky. J Hepatol 2013; 59: 375-382) In vitro See Figure 13.7 for effects in HCV genotype 1a replicons. In HCV genotype 1a replicons it confers high-level resistance to ombitasvir (DeGoey et al. J Med Chem 2014; 57: 2047-2057) and GSK2336805 (Kazmierski et al. J Med Chem 2014; 57: 2058-2073; Walker et al. Antimicrob Agents Chemother 2014; 58: 38-47).

Y93F In vivo Selected in patients infected with HCV genotype 1a and treated with EDP-239 (Owens et al. Antimicrob Agents Chemother 2016; 60: 6216-6226). Y93H In vivo Associated with daclatasvir resistance in genotypes 1 and 4 (reviewed in Pawlotsky. J Hepatol 2013; 59: 375-382; Bartolini et al. J Clin Virol 2015; 66: 38-43). Found in virus from patients treated with elbasvir (Lawitz et al. Lancet 2015; 385; 1075-1086; Sulkowski et al. Lancet 2015; 385: 1087-1097), ledipasvir (Afdhal et al. N Engl J Med 2014; 370: 1889-1898; Wilson et al. Clin Infect Dis 2016; 62: 280-288; Cheng et al. Antimicrob Agents Chemother 2016; 60: 1847-1853; Gane et al. Gastroenterology 2017; 152: 1366-1371), velpatasvir (Feld et al. N Engl J Med 2015; 373: 2599-2607; Foster et al. N Engl J Med 2015; 373: 2608-2617) and EDP-239 (Owens et al. Antimicrob Agents Chemother 2016; 60: 6216-6226). Major ombitasvir resistance mutation in HCV genotype 1b-infected patients (DeGoey et al. J Med Chem 2014; 57: 2047-2057).

709 Amino acid In vivo / substitution In vitro Comments and references In vitro See Figure 13.7 for effects in HCV genotypes 1a and 1b replicons. Confers high-level resistance to daclatasvir in HCV genotype 3a replicons (Wang et al. Antimicrob Agents Chemother 2013; 57: 611-613; Hernandez et al. J Clin Virol 2013; 57: 13-18). Major ledipasvir resistance-associated mutation for all genotypes (Cheng et al. Antimicrob Agents Chemother 2016; 60: 1847-1853). Confers high-level resistance to ombitasvir (DeGoey et al. J Med Chem 2014; 57: 2047-2057) and BP008 (Lin et al. Antimicrob Agents Chemother 2012; 56: 44-53), and low-level resistance to pibrentasvir (Ng et al. Antimicrob Agents Chemother 2017; 61: e02558-16), in HCV genotype 1a replicon assays. In genotype 4a replicons, it produces >60-fold decreased daclatasvir susceptibility in comparison with the wild-type strain (Wang et al. Antimicrob Agents Chemother 2012; 56: 1588-1590). Y93N In vivo Mutation selected in patients treated with daclatasvir (genotype 1a), although less frequently than Y93H (Fridell et al. Antimicrob Agents Chemother 2010; 54: 3641-3650; Wang et al. Antimicrob Agents Chemother 2012; 56: 1350-1358) and in patients treated with elbasvir (Lawitz et al. Lancet 2015; 385; 1075-1086; Sulkowski et al. Lancet 2015; 385: 1087-1097), ledipasvir (Wyles et al. J Hepatol 2017; 66: 703-710) or velpatasvir (Feld et al. N Engl J Med 2015; 373: 2599-2607; Foster et al. N Engl J Med 2015; 373: 2608-2617). In vitro See Figure 13.7 for effects in HCV genotype 1a replicons. In these assays, Y93N was found to confer high-level resistance to ledipasvir (Wong et al. Antimicrob Agents Chemother 2014; 57: 6333-6340) and ombitasvir (DeGoey et al. J Med Chem 2014; 57: 2047-2057), and low-level resistance to pibrentasvir (Ng et al. Antimicrob Agents Chemother 2017; 61: e02558-16). Y93N is also a secondary mutation selected with daclatasvir in HCV genotype 1b replicons. It confers resistance to the inhibitor (Fridell et al. Antimicrob Agents Chemother 2010; 54: 3641-3650; Wang et al. Antimicrob Agents Chemother 2012; 56: 1350-1358). Confers high-level resistance to GSK2336805 in HCV genotype 1b replicons (Kazmierski et al. J Med Chem 2014; 57: 2058-2073; Walker et al. Antimicrob Agents Chemother 2014; 58: 38-47).

710 Amino acid In vivo / substitution In vitro Comments and references Y93R In vitro In genotype 4a replicons, it produces >60-fold decreased daclastavir susceptibility in comparison with the wild-type strain (Wang et al. Antimicrob Agents Chemother 2012; 56: 1588-1590). D320E In vitro Found in alisporivir-resistant replicons (Coelmont et al. PLoS One 2010; 5: e13687), it decreases viral susceptibility by 3-fold (Grisé et al. J Virol 2012; 86: 4811-4822). In HCV genotype 1b, it confers resistance to cyclosporine A (Arai et al. Biochem Biophys Res Commun 2014; 448: 56-62), and to SCY-635 when found in combination with Y321N (Hopkins et al. Antimicrob Agents Chemother 2012; 56: 3888-3897). Y321N In vitro See D320E.

711

Figure 13.7. Effect of NS5A mutations on antiviral activities of selected inhibitors. Assays were carried out with different replicons and with single- and double-mutants made in genotypes 1a and 1b replicon backbones. High-level (>400-fold increase of the EC50 for the inhibitor), moderate (50- to 400-fold increase) and low-level (5- to 50-fold increase) resistance are represented by solid, hatched and grey boxes, respectively. Open boxes indicate susceptible viruses (<5-fold increase in the EC50 for the inhibitor). For reviews see: Gao. Curr Opin Virol 2013; 3: 514-520; Issur & Götte. Viruses 2014; 6: 4227-4241. References for the original data shown were obtained for daclastavir (Gao et al. Nature 2010; 465: 96- 100; Fridell et al. Antimicrob Agents Chemother 2010; 54: 3641-3650; Fridell et al. J Virol 2011; 85: 7312-7320); BMS-766 (Gao et al. 46th EASL 2011, Berlin, Germany, Abstract and poster 787); ledipasvir (Lawitz et al. J Hepatol 2012; 57: 24-31; Wong et al. Antimicrob Agents Chemother 2013; 57: 6333-6340; Cheng et al. Antimicrob Agents Chemother 2016; 60: 1847-1853); velpatasvir (GS-5816) [Cheng et al. J Hepatol 2013; 58 (suppl.): S484; Lawitz et al. Antimicrob Agents Chemother 2016; 60: 5368-5378]; odalasvir (ACH-3102) (Zhao et al. 47th EASL 2012, Barcelona, Spain, Abstract 845); elbasvir (MK-8742) (Liu et al. 47th EASL 2012, Barcelona, Spain, Poster 858; Lahser et al. Antimicrob Agents Chemother 2016; 60: 2954-2964); GSK-805 (Bechtel et al. 46th EASL 2011, Berlin, Germany, Poster 764); ravidasvir (PPI-668) (Colonno et al. 46th EASL 2011, Berlin, Germany, Poster 1200); samatasvir (IDX-719) (Bilello et al. Antimicrob Agents Chemother 2014; 58: 4431-4442); pibrentasvir (Ng et al. Antimicrob Agents Chemother 2017; 61: e02558-16); and EDP-239 (Owens et al. Antimicrob Agents Chemother 2016; 60: 6216-6226).

712 Table 13.9. HEPATITIS C VIRUS (HCV) RNA POLYMERASE MUTATIONS ASSOCIATED WITH RESISTANCE TO NUCLEOSIDE AND NONNUCLEOSIDE INHIBITORS a

Amino acid In vivo / Comments and references substitution In vitro S15G In vitro See C223H and C223Y. T19P In vitro See M423V. L30S In vitro Selected in the replicon system in the presence of BMS- 791325. It decreases polymerase activity but it does not affect the inhibitor binding constant (Rigat et al. J Biol Chem 2014; 289: 33456-33468). G46A In vitro Selected in HCV genotype 1b replicons passaged in the presence of the inhibitor A-848837 (Molla et al. Antivir Ther 2006; 11: S6). Q47H In vivo This mutation was found to be more prevalent in HCV genotype 1a-infected patients in a clinical trial testing the efficacy of mericitabine combined with danoprevir (Tong et al. Antimicrob Agents Chemother 2014; 58: 3105-3114). It reduces viral replication capacity by 2- to 4-fold. Q49L In vitro Identified as accessory mutation in HCV genotype 1b replicons selected in the presence of ODE-S-HPMPA and ODE-S- MPMPA [Wyles et al. Antivir Ther 2010; 15 (suppl. 2): A26]. K50N In vitro Identified in HCV genotype 1b replicons selected in the presence of ODE-S-HPMPA and ODE-S-MPMPA, confers increased replicative capacity [Wyles et al. Antivir Ther 2010; 15 (suppl. 2): A26]. Q58L In vitro Identified as accessory mutation in HCV genotype 1b replicons selected in the presence of ODE-S-HPMPA and ODE-S-MPMPA [Wyles et al. Antivir Ther 2010; 15 (suppl. 2): A26]. M71V In vitro See M423V. H95Q In vitro Both mutations confer high-level resistance to various H95R benzothiadiazines (Tomei et al. J Virol 2004; 78: 938-946; Mo et al. Antimicrob Agents Chemother 2005; 49: 4305-4314).

713 Amino acid In vivo / Comments and references substitution In vitro S96T In vitro There is evidence that suggests that this mutation together with N142T could be involved in phenotypic resistance to 4´-azidocytidine (R1479 and its pro-drug R1626) (Roberts et al. J Hepatol 2006; 44: S269; Le Pogam et al. Virology 2006; 351: 349-359). N142T In vivo Found in patients treated with sofosbuvir (combined with velpatasvir) (Lee et al. Hepatol Int 2017; 11: 161- 170). See also S96T for additional data on resistance to 4´-azidocytidine. G152E In vitro Confers resistance to 4,5-dihydroxypyrimidine carboxylates in cell-based assays (Tomei et al. Antivir Chem Chemother 2005; 16: 225-245). P156L In vitro By itself, this mutation confers resistance to 4,5-dihydroxypyrimidine carboxylates in cell-based assays (Tomei et al. Antivir Chem Chemother 2005; 16: 225-245). These findings have been confirmed also in enzymatic assays (Powdrill et al. Antimicrob Agents Chemother 2010; 54: 977-983). L159F In vivo Found in clinical trials with sofosbuvir in patients infected with genotypes 1-6 (Sulkowski et al. JAMA 2014; 312: 353- 361; Zeuzem et al. N Engl J Med 2014; 370: 1993-2001; Svarovskaia et al. Clin Infect Dis 2014; 59: 1666-1674; Molina et al. Lancet 2015; 385: 1098-1106; Lee et al. Hepatol Int 2017; 11: 161-170), and in patients treated with mericitabine and danoprevir (Tong et al. Antimicrob Agents Chemother 2014; 58: 3105-3114). L159F alone has no effect on resistance but combined with L320F it confers low-level resistance to sofosbuvir and mericitabine (Tong et al. J Infect Dis 2014; 209: 668-675). The L159F/L320F combination has been also observed in one patient treated with mericitabine and ritonavir- boosted danoprevir (Gane et al. Liver Int 2015; 35: 79-89). L159F is a relatively frequent polymorphism in genotype 1b isolates (Chen et al. Sci Rep 2016; 6: 20310). T179A In vitro Appears in HCV genotype 2a after passage in the presence of sofosbuvir (Najera. Curr Opin Virol 2013; 3: 508-513). R222Q In vitro See C223H and C223Y.

714 Amino acid In vivo / Comments and references substitution In vitro C223H In vitro Resistance-associated mutations found in HCV genotype C223Y 2a replicons cultured in the presence of PSI-352938, a cyclic phosphate prodrug of β-D-2´-deoxy-2´-α-fluoro-2´- β-C-methylguanosine monophosphate. Appear together with S15G, R222Q, L320I and V321I in resistant strains. These mutations had a strong negative effect on the replication capacity of HCV genotype 1b and 2a replicons (Lam et al. Antimicrob Agents Chemother 2011; 55: 2566-2575; Lam et al. J Virol 2011; 85: 12334-12342). E237G In vivo Occassionally selected in patients treated with sofosbuvir (Lee et al. Hepatol Int 2017; 11: 161-170), but its relevance for resistance has not been demonstrated S282C In vivo Observed as minority variants detected using next generation S282G sequencing in HCV present in patients treated with S282N sofosbuvir (Ji et al. Virology 2015; 477: 1-9; Maimone et al. S282R Antivir Ther 2015; 20: 245-247; Molina et al. Lancet 2015; 385: 1098-1106). S282T In vivo Found in patients infected with genotype 1 HCV and treated with mericitabine (NS5B inhibitor) and ritonavir-boosted danoprevir (Gane et al. Liver Int 2015; 35: 79-89). Rarely found in patients infected with HCV genotype 1b variants and treated with sofosbuvir (Maimone et al. Antivir Ther 2015; 20: 245-247). In vitro This amino acid substitution confers resistance to 2´-modified nucleoside analogues (e.g. 2´-C-methyl adenosine, valopicitabine, INX-08189 and TMC647078) and to alkoxyalkyl esters of acyclic nucleoside phosphonates (e.g. ODE-S-HPMPA and ODE-S-MPMPA), while decreasing viral fitness, as determined with HCV replicons [Ludmerer et al. Antimicrob Agents Chemother 2005; 49: 2059-2069; Dutrarte et al. Antimicrob Agents Chemother 2006; 50: 4161-4169; Wyles et al. Antivir Ther 2010; 15 (suppl. 2): A26; Vernachio et al. Antimicrob Agents Chemother 2011; 55: 1843-1851; Berke et al. Antimicrob Agents Chemother 2011; 55: 3812-3820]. Selected under passage with sofosbuvir (genotypes 1a, 1b and 2a) and with mericitabine (Najera. Curr Opin Virol 2013; 3: 508--513; Ramirez et al. Gastroenterology 2016; 151: 973-985).

715 Amino acid In vivo / Comments and references substitution In vitro M289I In vivo Found during clinical trials in patients treated with sofosbuvir plus velpatasvir (Lee et al. Hepatol Int 2017; 11: 161-170), or ledipasvir (Gane et al. Gastroenterology 2017; 152: 1366-1371). M289L In vitro Selected in HCV genotype 2a after passage in the presence of sofosbuvir (Gallego et al. Antimicrob Agents Chemother 2016; 60: 3786-3793), sometimes in combination with T179A and I293L (Najera. Curr Opin Virol 2013; 3: 508- 513). It is a frequent polymorphism in genotype 6 isolates (Chen et al. Sci Rep 2016; 6: 20310). I293L In vitro Appears together with T179A and M289L in HCV genotype 2a after passage in the presence of sofosbuvir (Najera. Curr Opin Virol 2013; 3: 508-513). C316N In vivo Appears together with L159F in some patients failing treatment with sofosbuvir (Donaldson et al. Hepatology 2015; 61: 56-65). C316Y In vivo Selected in patients treated with an interferon-free drug combination including desabuvir (ABT-333) (Poordad et al. N Engl J Med 2014; 370: 1973-1982). In vitro It confers high-level resistance to nesbuvir (HCV-796) and A-782759 and moderate resistance to tegobuvir (Delang et al. Antimicrob Agents Chemother 2011; 55: 4103-4113; Shi et al. Antimicrob Agents Chemother 2011; 55: 4196- 4203). Selected in vitro with JTK-853, it confers significant resistance to the drug (Ando et al. Antimicrob Agents Chemother 2012; 56: 4250-4256). L320I In vivo Occassionally selected in clinical trials, in patients treated with sofosbuvir (Lee et al. Hepatol Int 2017; 11: 161-170). In vitro See C223H and C223Y. L320F In vivo See L159F. V321A In vitro Found in patients failing sofosbuvir treatment (Zeuzem et al. N Engl J Med 2014; 370: 1993-2001; Svarovskaia et al. Clin Infect Dis 2014; 59: 1666-1674; Donaldson et al. Hepatology 2015; 61: 56-65). The mutation has no effect on drug susceptibility in in vitro assays. V321I In vitro See C223H and C223Y.

716 Amino acid In vivo / Comments and references substitution In vitro A338V In vitro See M423V. S368T In vitro Selected in HCV genotype 1a and 1b replicons passaged in the presence of the inhibitor A-848837 (Molla et al. Antivir Ther 2006; 11: S6). T389A In vitro Selected in vitro with the benzimidazole nonnucleoside T389S polymerase inhibitor JT-16 using HCV genotype 1b replicons. Confer moderate levels of resistance to the drug

(7- to 13-fold increase of the EC50) (Delang et al. Antiviral Res 2012; 93: 30-38). L392I In vitro Confers 5- to 8-fold increased resistance to TMC647055 and BMS-791325 in assays carried out with HCV genotype 1 replicons (Devogelaere et al. Antimicrob Agents Chemother 2012; 56: 4676-4684; Lemm et al. Antimicrob Agents Chemother 2014; 58: 3485-3495). Y392F In vitro Selected in HCV genotype 1b replicons passaged in the presence of the inhibitor A-848837. This mutation does not impair viral replication capacity (Molla et al. Antivir Ther 2006; 11: S6). N411S In vitro Associated with resistance to benzothiadiazines (A-782759) (Mo et al. Antimicrob Agents Chemother 2005; 49: 4305-4314). M414I In vivo Selected in patients treated with an interferon-free drug combination including desabuvir (ABT-333) (Poordad et al. N Engl J Med 2014; 370: 1973-1982). In vitro Confers resistance to the benzothiadiazine NNI-2 (Le Pougam et al. J Antimicrob Chemother 2008; 61: 1205-1216). M414L In vitro In combination with V499A, this amino acid substitution was found to confer resistance to the benzothiadiazine NNI-2 in phenotypic assays (Le Pougam et al. J Antimicrob Chemother 2008; 61: 1205-1216). See also M423T. Also identified in HCV genotype 1b replicons grown in the presence of JTK-853 (Ando et al. Intervirology 2013; 56: 302-309). M414T In vivo Identified in virus from patients receiving JTK-853 (Ogura et al. Antimicrob Agents Chemother 2013; 57: 436-444) and interferon-free therapies containing dasabuvir and other drugs (Poordad et al. N Engl J Med 2014; 370: 1973-1982).

717 Amino acid In vivo / Comments and references substitution In vitro In vitro Selected in HCV genotype 1a and 1b replicons passaged in the presence of the inhibitor A-848837 (Molla et al. Antivir Ther 2006; 11: S6). It confers significant resistance to nonnucleoside inhibitors such as benzothiadiazines (Nguyen et al. Antimicrob Agents Chemother 2003; 47: 3525-3530; Tomei et al. J Virol 2004; 78: 938-946; Mo et al. Antimicrob Agents Chemother 2005; 49: 4305-4314; Le Pougam et al. J Antimicrob Chemother 2008; 61: 1205-1216; Delang et al. Antimicrob Agents Chemother 2011; 55: 4103-4113) and JTK-853 (Ando et al. Antimicrob Agents Chemother 2012; 56: 4250-4256; Ando et al. Intervirology 2013; 56: 302-309; Ogura et al. Antimicrob Agents Chemother 2013; 57: 436-444). F415Y In vivo Identified in ribavirin-treated patients infected with HCV genotype 1a (Young et al. Hepatology 2003; 38: 869-878). Its relevance in resistance has been confirmed using the HCV replicon. L419C In vitro These amino acid substitutions decrease lomibuvir L419S susceptibility by >200-fold (Jiang et al. Antimicrob Agents Chemother 2014; 58: 5456-5465). L419M In vitro This amino acid substitution confers 10-fold increased resistance to the pyranoindole HCV-371 (Howe et al. Antimicrob Agents Chemother 2006; 50: 4103-4113). As shown for other mutations at this position (e.g. L419I and L419S), L419M has a minor impact on filibuvir susceptibility (<2.3-fold increase in the EC50) (Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341), but confers moderate to high-level resistance to lomibuvir and GS-9669 (Fenaux et al. Antimicrob Agents Chemother 2013; 57: 804-810; Dvory-Sobol et al. Antimicrob Agents Chemother 2014; 58: 6599-6606). R422K In vitro It confers high-level resistance to filibuvir, lomibuvir and GS-9669 (Fenaux et al. Antimicrob Agents Chemother 2013; 57: 804-810; Jiang et al. Antimicrob Agents Chemother 2014; 58: 5456-5465; Dvory-Sobol et al. Antimicrob Agents Chemother 2014; 58: 6599-6606). However, it has a deleterious effect on viral fitness (Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341; Fenaux et al. Antimicrob Agents Chemother 2013; 57: 804-810). M423A In vitro Confers low-level resistance to filibuvir in replicon-based assays (Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341).

718 Amino acid In vivo / Comments and references substitution In vitro M423I In vivo Selected under treatment with filibuvir in patients infected with HCV genotype 1a strains (Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341). In vitro It confers high-level resistance to lomibuvir, filibuvir and GS-9669, but diminishes viral replication capacity (Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341; Fenaux et al. Antimicrob Agents Chemother 2013; 57: 804-810; Dvory-Sobol et al. Antimicrob Agents Chemother 2014; 58: 6599-6606). M423T In vivo Found in patients treated with filibuvir (Wagner et al. Hepatology 2011; 54: 50-59; Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341). In vitro Primary mutation associated with filibuvir resistance in HCV genotypes 1a and 1b, it confers >500-fold increased resistance to the inhibitor (Shi et al. Antimicrob Agents Chemother 2009; 53: 2544-2552; Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341). M423T produces >37-fold increase

in the EC50 obtained for thiophene carboxylic acid derivative (TCA) (Delang et al. Antimicrob Agents Chemother 2011; 55: 4103-4113), and for lomibuvir and filibuvir (Fenaux et al. Antimicrob Agents Chemother 2013; 57: 804-810). This mutation was also selected in the presence of GS-9669 (Dvory-Sobol et al. Antimicrob Agents Chemother 2014; 58: 6599-6606), and in the presence of thiophene-2-carboxylic acid, together with M414L. The double mutant showed reduced replication capacity (Le Pogam et al. Antivir Ther 2006; 11: S5). M423V In vivo Found in patients treated with filibuvir (Wagner et al. Hepatology 2011; 54: 50-59; Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341). In vitro This amino acid substitution confers 8-fold increased resistance to the pyranoindole HCV-371, and in combination with T19P, M71V, A338V and A442T, this value can go up to 17-fold (Howe et al. Antimicrob Agents Chemother 2006; 50: 4103-4113). It confers high-level resistance to filibuvir and lomibuvir (Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341; Fenaux et al. Antimicrob Agents Chemother 2013; 57: 804-810), and moderate resistance to GS-9669 (Fenaux et al. Antimicrob Agents Chemother 2013; 57: 804-810; Dvory-Sobol et al. Antimicrob Agents Chemother 2014; 58: 6599-6606).

719 Amino acid In vivo / Comments and references substitution In vitro M426A In vitro This amino acid change confers 9.6-fold increased resistance to filibuvir (Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341). M426T In vitro This amino acid substitution confers about 4-fold increased re- sistance to filibuvir and the pyranoindole HCV-371 (Howe et al. Antimicrob Agents Chemother 2006; 50: 4103-4113; Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341). M426V In vitro This amino acid substitution confers 3-fold increased resistance to the pyranoindole HCV-371 (Howe et al. Antimicrob Agents Chemother 2006; 50: 4103-4113). It has a minor effect on filibuvir resistance (Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341). A442T In vitro See M423V. C445R In vivo Identified in virus from patients receiving JTK-853. Confers 5-fold increased resistance to the inhibitor when introduced in HCV replicons (Ogura et al. Antimicrob Agents Chemother 2013; 57: 436-444). Y448H In vivo Tegobuvir resistance mutation found in less than 3% of treatment-naïve patients (Bae et al. J Clin Microbiol 2011; 49: 3168-3174). Identified in patients treated with tegobuvir, sometimes in combination with Y452H (Mo et al. J Viral Hepat 2016; 23: 644-651). Also found together with Y448C in virus from patients treated with JTK-853 (Ogura et al. Antimicrob Agents Chemother 2013; 57: 436-444). In vitro Selected in HCV genotype 1a and 1b replicons passaged in the presence of the inhibitor A-848837 (Molla et al. Antivir Ther 2006; 11: S6). It confers high-level resistance to tegobuvir and the inhibitor A-782759 (Shih et al. Antimicrob Agents Chemother 2011; 55: 4196-4203). Confers 6-fold increased resistance to JTK-853 when introduced in HCV replicons (Ogura et al. Antimicrob Agents Chemother 2013; 57: 436-444). C451R In vitro This amino acid substitution confers high-level resistance to benzothiadiazines designated as compounds 1 and 2 (Tomei et al. J Virol 2004; 78: 938-946).

720 Amino acid In vivo / Comments and references substitution In vitro Y452H In vivo Found in virus obtained from patients treated with tegobuvir (Mo et al. J Viral Hepat 2016; 23: 644-651). In vitro Selected in vitro with JTK-853, it confers high-level resistance to the drug (Ando et al. Antimicrob Agents Chemother 2012; 56: 4250-4256). L466V In vivo Identified in virus from patients receiving JTK-853 (Ogura et al. Antimicrob Agents Chemother 2013; 57: 436-444). In vitro Selected in HCV genotype 1b replicons grown in the presence of JTK-853 (Ando et al. Intervirology 2013; 56: 302-309). L466V confers high-level resistance in the replicon assay (Ogura et al. Antimicrob Agents Chemother 2013; 57: 436-444). L466I In vitro Identified in HCV genotype 1a replicons selected in the presence of JTK-853 (Ando et al. Intervirology 2013; 56: 302-309). I482L In vitro Located in the base of the thumb of the polymerase, it has I482S been selected with filibuvir and with the inhibitor VCH-759 I482T (Le Pogam et al. J Virol 2006; 80: 6146-6154; Cooper et al. I482V J Hepatol 2009; 51: 39-46). I482S and I482T produce an

82- and a 12-fold increase of the filibuvir EC50, respectively, when assayed using the HCV genotype 1b replicon (Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341). I482L confers high-level resistance to lomibuvir, filibuvir and GS-9669 (Fenaux et al. Antimicrob Agents Chemother 2013; 57: 804-810; Dvory-Sobol et al. Antimicrob Agents Chemother 2014; 58: 6599-6606). V494A In vitro This mutation produces a 6.9-fold increase in the filibuvir

EC50. Other mutations at this position (i.e. V494I and V494T) had no effect on resistance (Troke et al. Antimicrob Agents Chemother 2011; 56: 1331-1341). Responsible in part for the reduced susceptibility to BMS-791325 of clinical isolates of HCV genotype 6a (Liu et al. Antimicrob Agents Chemother 2014; 58: 7416-7423).

721 Amino acid In vivo / Comments and references substitution In vitro P495A In vivo P495L and P495S have been observed in patients treated P495L with beclabuvir (BMS-791325) as part of combination P495S therapies (Sims et al. Antimicrob Agents Chemother 2014; P495T 58: 3496-3503; Muir et al. JAMA 2015; 313: 1736-1744). In vitro Selected in cell culture with the HCV genotype 1b replicon system, in the presence of benzimidazole 5-carboxamide compounds. P495A, P495S and P495L confer high-level

resistance (>25-fold increase of the IC50) to those non- nucleoside inhibitors (Kukolj et al. J Biol Chem 2005; 280: 39260-39267; Delang et al. Antimicrob Agents Chemother 2011; 55: 4103-4113). P495L confers moderate resistance to A-782759 (Delang et al. Antimicrob Agents Chemother 2011; 55: 4103-4113) and high-level resistance to deleobuvir (BI 207127) (Larrey et al. Antimicrob Agents Chemother 2013; 57: 4727-4735). P495S, P495L and P495T also confer high-level resistance to TMC647055 and BMS-791325 (Devogelaere et al. Antimicrob Agents Chemother 2012; 56: 4676-4684; Lemm et al. Antimicrob Agents Chemother 2014; 58: 3485-3495). P495L confers resistance to BMS-791325 (Sims et al. Antimicrob Agents Chemother 2014; 58: 3496- 3503; Rigat et al. J Biol Chem 2014; 289: 33456-33468). P496A In vitro Selected in cell culture in the presence of nonnucleoside P496S inhibitors. These mutations confer 3- to 10-fold increased resistance to benzimidazole 5-carboxamide derivatives (Kukolj et al. J Biol Chem 2005; 280: 39260-39267). V499A In vitro Found in wild-type HCV genotype 1a, and common polymorphism in genotype 1b. It confers low-level resistance to benzimidazole 5-carboxamide derivatives (Kukolj et al. J Biol Chem 2005; 280: 39260-39267), and is associated with decreased susceptibility to deleobuvir (BI 207127) (Larrey et al. Antimicrob Agents Chemother 2013; 57: 4727-4735; Berger et al. PLoS One 2016; 11: e0160668). See also M414L. Q514R In vitro Selected in HCV genotype 1b replicons passaged in the presence of the inhibitor A-848837 (Molla et al. Antivir Ther 2006; 11: S6). G554D In vitro Selected in HCV genotype 1a and 1b replicons passaged in the presence of the inhibitor A-848837 (Molla et al. Antivir Ther 2006; 11: S6).

722 Amino acid In vivo / Comments and references substitution In vitro S556G In vivo Frequent in genotype 1b isolates (Chen et al. Sci Rep 2016; 6: 20310). In vitro Selected with dasabuvir in replicon systems (Koev et al. J Hepatol 2009; 50: S346-S347). S556R In vivo Frequently found in genotype 6 isolates (Chen et al. Sci Rep 2016; 6: 20310). G558R In vitro This amino acid substitution confers high-level resistance to benzothiadiazines designated as compounds 1 and 2 (Tomei et al. J Virol 2004; 78: 938-946). D559A In vitro In enzymatic assays, these amino acid substitutions confer D559G decreased susceptibility to silibinin-hemisuccinate, a flavo- D559N noid inhibitor of the polymerase [Ahmed-Belkacem et al. Antivir Ther 2012; 17 (suppl. 1): A25]. D559G is a secondary mutation selected in cell culture in the presence of A-848837 (Molla et al. Antivir Ther 2006; 11: S6), and in the presence of dasabuvir (Koev et al. J Hepatol 2009; 50: S346-S347). I585V In vivo Associated with resistance to dasabuvir, this substitution is more prevalent in HCV genotype 1a isolates (Chen et al. Sci Rep 2016; 6: 20310). Y586C In vitro Selected in HCV genotype 1a and 1b replicons passaged in the presence of the inhibitor A-848837 (Molla et al. Antivir Ther 2006; 11: S6). a Underlined substitutions appear in the RNA polymerase of other genotypes different from 1a.

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