ABSTRACT
VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.
Subject(s)
Carbamates/pharmacology , Drug Resistance, Viral , Hepacivirus/enzymology , Macrocyclic Compounds/pharmacology , Mutation , Oligopeptides/pharmacology , Quinolines/pharmacology , Serine Proteinase Inhibitors/pharmacology , Thiazoles/pharmacology , Viral Nonstructural Proteins/pharmacology , Amino Acid Substitution , Amino Acids/chemistry , Aspartic Acid/chemistry , Binding Sites , Genes, Dominant , Hepacivirus/drug effects , Hepacivirus/genetics , Humans , In Vitro Techniques , Inhibitory Concentration 50 , Kinetics , Models, Chemical , Models, Molecular , RNA, Viral/physiology , Replicon/physiology , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/geneticsABSTRACT
We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3.4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3.4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3.4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp(168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061.
Subject(s)
Carbamates/pharmacology , Enzyme Inhibitors/pharmacology , Hepacivirus/enzymology , Macrocyclic Compounds , Oligopeptides/pharmacology , Quinolines , Serine Proteinase Inhibitors/pharmacology , Thiazoles/pharmacology , Viral Nonstructural Proteins/chemistry , Amino Acids/chemistry , Aspartic Acid/chemistry , Binding Sites , Dose-Response Relationship, Drug , Drug Resistance , Genes, Dominant , Inhibitory Concentration 50 , Kinetics , Models, Chemical , Models, Molecular , Mutation , Plasmids/metabolism , Protein Structure, Tertiary , Time Factors , Viral Nonstructural Proteins/antagonists & inhibitorsABSTRACT
Transfection of cDNA in 3'untranslated region of human nuclear factor for interleukin-6 (NF-IL6 3'UTR) induced tumor suppression in a human hepatoma cell line. cDNA array analysis was used to reveal changes in gene expression profile leading to tumor suppression The results indicate that this suppression was not due to activation of dsRNA-dependent protein kinase, nor to inactivation of oncogenes; rather, all the changes in expression of known genes, induced by NF-IL6 3'UTR cDNA may be ascribed to the suppression of cellular malignancy. Therefore, our results imply that this 3'untranslated region may have played role of a regulator of gene expression profile.
