In vitro selection and characterization of HCV replicons resistant to multiple non-nucleoside polymerase inhibitors Leen Delang, Inge Vliegen, Pieter Leyssen, Johan Neyts ⇑ Rega Institute for Medical Research, KU Leuven, Belgium See Editorial, pages 11–13 Background & Aims: To delay or prevent the selection of HCV drug-resistant variants, combination therapy will be needed. Our aim was to determine the antiviral efficacy of various combi- nations of non-nucleoside polymerase inhibitors (NNI) (that have a different allosteric binding site) and the barrier towards resis- tance development of such combinations. Methods: Short-term antiviral combination assays were per- formed in a checkerboard format. Resistance selection experi- ments employing HCV replicons were performed using two different protocols: (i) a short-term treatment with fixed concen- trations and (ii) a long-term treatment with increasing concentrations. Results: All pair-wise combinations of NNI resulted in an addi- tive antiviral effect in short-term antiviral assays. Combination treatment of two NNIs markedly reduced or even prevented the emergence of double resistant colonies. However, double and even triple NNI-resistant variants emerged readily when relatively low starting concentrations were used in a long-term selection protocol. Genotyping confirmed the presence of the previously published resistance mutations. For some NNI, differ- ent signature mutations appeared depending on the other NNI in the particular combination. Remarkably, variants that were selected to be resistant to three different classes of NNIs [a thiophene carboxylic acid (TCA), a benzimidazole (JT-16), and a benzofuran (HCV-796)] proved resistant to yet a fourth class of NNIs (benzothiadiazines). Conclusions: Double and even triple NNI-resistant HCV replicons can be readily selected with a stepwise resistance selection protocol. Depending on the particular combination, different signature mutations may arise for some NNI. Resistance to three classes of NNI resulted in resistance to yet a fourth class. Ó 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Introduction Hepatitis C virus is a single stranded (+) RNA virus that belongs to the family of the Flaviviridae. Worldwide an estimated 170 million people are chronically infected with the hepatitis C virus (HCV) and thus at high risk of developing life-threatening liver disease [1]. In developed countries, HCV infection is the major cause of liver transplantation. Standard therapy consists of the combination of pegylated interferon-a (pegIFN) and ribavirin. However, the effi- cacy of standard therapy is limited, particularly in genotype 1 infected patients, and is associated with serious adverse effects. Hence, potent and well tolerated HCV inhibitors are urgently needed; the first drugs, i.e. the protease inhibitors Telaprevir and Boceprevir, are expected to receive market authorization in 2011/2012. The RNA-dependent RNA polymerase (RdRp) of HCV, the NS5B protein, is an essential enzyme for viral RNA replication and is therefore an attractive antiviral target. The structure of NS5B is characteristic for RNA polymerases and can be compared with a right hand [2]. The palm domain contains the catalytic site and the fingers and the thumb domain are responsible for the interac- tion with the RNA. Both (i) nucleoside polymerase inhibitors and (ii) non-nucleoside polymerase inhibitors have been identified. Nucleoside polymerase inhibitors (NI) act as virtual chain terminators. In this class of inhibitors, R7128, a prodrug of b-D-2 0 -deoxy-2 0 -fluoro-2 0 -C-methylcytidine, is the most advanced. Non-nucleoside polymerase inhibitors (NNI) act as allosteric inhibitors and are non-competitive with NTPs. Their binding sites are located outside the catalytic site of the polymer- ase. At least four different allosteric binding sites have been reported: (i) thumb domain 1, (ii) thumb domain 2, (iii) palm domain 1, and (iv) palm domain 2. Benzimidazole and indole derived structures can inhibit HCV replication by interacting with the thumb domain 1. Thiophene carboxylic acid, dihydropyra- nones, and pyranoindoles were identified to target thumb domain 2, a hydrophobic cavity located at the base of the thumb domain. Palm domain 1 and 2 are overlapping regions located at the junction of the palm and thumb domain in close proximity of the catalytic site. NNIs that bind to palm domain 1 are benzothi- Journal of Hepatology 2012 vol. 56 j 41–48 Keywords: Hepatitis C virus; Resistance; Non-nucleoside polymerase inhibitors; Combination. Received 12 January 2011; received in revised form 7 March 2011; accepted 2 April 2011; available online 20 May 2011 q DOI of original article: 10.1016/j.jhep.2011.09.001. ⇑ Corresponding author. Address: Minderbroedersstraat 10, 3000 Leuven, Belgium. Tel.: +32 16337353; fax: +32 16337340. E-mail address: Johan.Neyts@rega.kuleuven.be (J. Neyts). Abbreviations: HCV, hepatitis C virus; pegIFN, pegylated interferon; RdRp, RNA- dependent RNA polymerase; NI, nucleoside inhibitor; NNI, non-nucleoside inhi- bitor; NTP, nucleoside triphosphate; DAA, direct-acting antivirals; PI, protease inhibitor; TCA, thiophene carboxylic acid; EC 50 , 50% effective concentration; 2 0 -C- MC, 2 0 -C-methylcytidine. Research Article