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Characterization of Resistance Mutations Selected In Vitro by Non-Nucleoside HCV Polymerase Inhibitors ABT-333 and ABT-072
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Reported by Jules Levin
44th Annual Meeting of the European Association for the Study of the Liver, Copenhagen, Denmark, April 22-26, 2009
Gennadiy Koev*, R. Mondal, J. Beyer, T. Reisch, S. Masse, W. Kati, D. Hutchinson, C. Flentge, J. Randolph, P. Donner, A.
Krueger, P. Yan, C.T. Lin, R. Wagner, C. Maring, A. Molla
Abbott Laboratories, Abbott Park, IL, USA
AUTHOR CONCLUSIONS
Resistance selection studies with ABT-333 and ABT-072 in subgenomic replicons revealed the following common resistance mutations: C316Y, S368T, M414T/I/V, Y448H/C, or S556G.
Levels of potency losses by the compounds against the mutants ranged from less than 10-fold to more than 1000-fold.
Most mutants exhibited reduced replication capacity compared to the wild type replicon.
In short-term replicon combination assays, both ABT-333 and ABT-072 exhibited additive to synergistic interactions with IFN.
In long-term replicon assays, combinations of either ABT-333 or ABT-072 with IFN exhibited greater RNA inhibitory effect than the inhibitor or IFN alone.
See graphs below showing in vitro HCV RNA declines of mono and combination with IFN.
INTRODUCTION
HCV infection is one of the major healthcare problems in the world, with co-administration of pegylated interferon and ribavirin being the current standard of care for treating the disease. We have discovered two potent HCV NS5B RNA polymerase inhibitors, ABT-333 and ABT-072. In this study, we have selected and characterized replicon mutants resistant to these inhibitors using HCV subgenomic replicons. We have also examined the antiviral activity of each of the inhibitors alone and in combination with interferon in both short- and long-term HCV replicon assays.
METHODS
Huh7 cells with the 1a and 1b subgenomic replicons containing a firefly luciferase reporter were maintained in DMEM supplemented with 10% FBS and Pen/Strep solution. Antiviral activity was determined by monitoring the levels of HCV RNA and/or luciferase activity.
To select drug-resistant replicon variants, genotype 1a or 1b replicon cells were plated in tissue culture plates and grown in supplemented DMEM medium in the presence of the inhibitors (10-fold or 100-fold above their EC50s) and G418 (400 µg/ml). Resistant replicon colonies were picked after 3 weeks of selection and characterized for genotype and phenotype.
Single-mutant transient replicon constructs were made by site-directed mutagenesis. RNA was transcribed using a TranscriptAid T7 transcription kit (Fermentas) or a MegaScript kit (Ambion) and electroporated into Huh7 cells.
In short-term replicon combination assays, an inhibitor was serially diluted two-fold, and six dilutions of the inhibitor were combined with six dilutions of human leukocyte interferon (IFN) (PBL Biomedical Laboratories) in a checkerboard manner. Drug interactions were analyzed using Loewe additivity and Bliss independence models (Greco et al., 1995, The search for synergy: a critical review from a response surface perspective. Pharmacol Rev 47(2): 331-85; Thisted, 1998, Elements of Statistical Computing: Numerical Computation. London, Chapman & Hall).
In long-term replicon combination assays, 106 replicon cells were plated in a T75 flask in 15 µl of supplemented DMEM without G418. INF and/or inhibitor were added, and the cells were grown to ~95% confluency (4 days). At each passage, 106 cells were frozen in 350 µl of RLT RNA lysis buffer (QIAGEN), and an additional 106 cells were passed into another T75 flask with fresh media and inhibitors.
RNA was extracted from 106 cells using QIAGEN RNeasy spin kit. Total RNA was eluted in 50 µl of nuclease-free water. RNA representing 105 cells (5 µl) was analyzed in a Real-Time RT-PCR assay (Applied Biosystems).
RESULTS
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