icon-folder.gif   Conference Reports for NATAP  
  EASL 45th Annual Meeting
April 14-18, 2010
Vienna, Austria
Back grey_arrow_rt.gif
Characterization of resistant mutants selected in vitro by the HCV NS3/4A protease inhibitor BI 201335
  Reported by Jules Levin
45th EASL, Apr 14-18 2010 Vienna Austria
George Kukolj, Christiane Bousquet, Nathalie Dansereau, Lisette Lagace, Montse Llinas-Brunet, Marie-Josee Massariol, Martin Marquis, Roger Maurice, Florence Do, Catherine Spickler, Diane Thibeault, Songping Zhao, Peter W. White Boehringer Ingelheim (Canada) Ltd, Laval, Quebec, Canada
· In vitro BI 201335 resistance studies identified amino acid substitutions in the NS3 protease domain that affect BI 201335 binding yet remain susceptible to inhibition by other drug classes
· The major genotype 1a and genotype 1b variants encode for substitutions at R155 and D168, respectively, and correspond to the prevalent variants observed in early clinical trials
· BI 201335 maintains activity against NS3/4A variants such as V36M or T54A that confer low-level resistance to other HCV protease inhibitors
· A combination of BI 201335 and IFN-α effectively suppresses the emergence of resistant variants
Background and Aims:
BI 201335 is a potent HCV NS3/4A protease inhibitor currently in large phase IIb clinical trials in combination with pegylated interferon and ribavirin. In this study, resistance to BI 201335 was examined in vitro through selection and characterization of variants in both genotype 1a and genotype 1b subgenomic replicons.
Methods: Drug selection studies were performed in stable cell lines that maintain derivatives of subgenomic replicons from HCV genotypes 1a-H77c and 1b-Con1. The two replicon cell lines were treated for 3 weeks with BI 201335 concentrations that corresponded to 100-fold and 1,000-fold their respective EC50 values in order to select for resistant colonies. The NS3/4A coding regions were sequenced to identify substitutions by population sequencing. Both single amino acid substituted replicons and purified NS3/4A proteases were constructed to characterize the susceptibility of the emergent mutants to BI 201335 and other HCV replication inhibitors.
Results: The most frequently observed genotype 1b mutations encoded amino acid changes at D168 with the G variant more prevalent at the lower concentration and the V or A variant at higher drug concentration. R155K was selected most frequently at 100X EC50 with genotype 1a replicon, whereas D168V was the predominant variant observed in genotype 1a with higher drug levels. Phenotypic characterization of the mutants revealed shifts in sensitivity to inhibition by BI 201335: the genotype 1a R155K and genotype 1b D168V variants reduced sensitivity to BI 201335 by 400-fold and 1,000-fold, respectively. BI 201335 maintained activity against NS3/4A variants such as V36M that confers resistance to other HCV protease inhibitors.
Conclusions: HCV NS3 genotype 1a and 1b variants that confer resistance to BI 201335 were selected in vitro and correspond to the predominant subtype specific variants that were identified in early clinical trials with BI 201335 as well as other HCV protease inhibitors. These resistant variants do not alter the sensitivity to IFN-α.
· The treatment of chronic hepatitis C virus (HCV) infection may be improved with the emergence of NS3/4A protease inhibitors that have progressed in clinical development in combination with pegylated interferon and ribavirin
· The first assessment of NS3/4A protease inhibitors in monotherapy revealed the potential for rapid selection of drug resistant variants that encode for amino acid substitutions in the NS3 protease domain - Many of these variants were first selected in vitro with subgenomic replicon studies
· BI 201335 is a potent NS3/4A protease inhibitor currently in large phase IIb clinical studies
· Here we describe the selection and characterization of NS3 protease amino acid substitutions that alter sensitivity to BI 201335

· Two BI 201335 concentrations were used for selection: ∼100X and 1,000X the EC50 of genotype 1a and genotype 1b replicons
· The predominant genotype 1b variant encoded a substitution at D168 - D168G was most prevalent at the lower concentration and D168A or D168V were most prevalent at the higher concentration (Table 1)

· The genotype 1b mutant replicons encoded for phenotypic changes that resulted in fold-changes which ranged from 50-fold for D168G variants, up to 720-fold for the D168V variant (Figure 1)

The EC50 fold-change values are the mean ± SE of n>/=3 determinations. The mean EC50 for each variant is listed on top of the error bar

· Select mutants reconstructed as clones in the isogenic genotype 1b replicon provided phenotypic changes in sensitivity that were specific to BI 201335 and did not change sensitivity to interferon-alfa (IFN-α) (Table 3)

· Mutant genotype 1b and 1a NS3/4A proteins displayed Km values that differ by less than 3-fold relative to the WT enzymes (Tables 5 and 6)
· The affinity of the mutant enzymes for BI 201335 was affected by individual substitutions
- The R155K mutant had a similar effect on BI 201335 binding for both the genotype 1a and 1b enzymes with 420- and 210-fold reductions in Ki, respectively
- The A156T and A156V genotype 1b mutants shifted Ki values by 710- and 1,100-fold - D168V also had a major impact on inhibitor binding for both genotype 1a and 1b enzymes with >690-fold shift in the Ki value relative to WT enzyme
· NS3 protease mutants, such as V36M or T54A3,4 that have been selected with other HCV protease inhibitors remain susceptible to inhibition by BI 201335

· Individually, BI 201335 and IFN-α weakly suppressed the emergence of resistant HCV replicons, whereas the combination of these inhibitors was extremely effective at reducing the emergence of resistant replicons (Figure 2)
· The structure of the NS3 protease domain with the BI 201335 binding site is shown in Figure 3 - Substitutions at residues that confer resistance to BI 201335 are identified: Arg155 (blue), Ala156 (yellow) and Asp168 (red)
FIGURE 2. Selection of HCV resistant replicons is reduced with a combination of BI 201335 and IFN-α

FIGURE 3. The location of major amino acids in the NS3 protease where substitutions confer resistance to BI 201335


1. Lagace L, et al. BILN 2061 and beyond: pre-clinical evaluation of HCV subgenomic replicon resistance to a NS3 protease inhibitor. In: ER Schiff, RF Schinazi, editors. Framing the Knowledge of Viral Hepatitis. IHL Press, College Park, Georgia, 2006.
2. Thibeault D, et al. Sensitivity of NS3 serine proteases from hepatitis C virus genotypes 2 and 3 to the inhibitor BILN 2061. J Virol. 2004;78:7352-7359.
3. Tong X, et al. Identification and analysis of fitness of resistance mutations against the HCV protease inhibitor SCH 503034. Antivir Res. 2006;70:28-38.
4. Sarrazin C, et al. Dynamic hepatitis C virus genotypic and phenotypic changes in patients treated with the protease inhibitor telaprevir. Gastroenterology. 2007;132:1767-1777.