Characterization of Aurintricarboxylic Acid as a Potent Hepatitis C Virus Replicase Inhibitor

• Published in: Antiviral chemistry & chemotherapy Vol. 20; no. 1; pp. 19 - 36
• Main Authors: Chen, Ye, Bopda-Waffo, Alain, Basu, Amartya, Krishnan, Ramalingam, Silberstein, Erica, Taylor, Deborah R, Talele, Tanaji T, Arora, Payal, Kaushik-Basu, Neerja
• Format: Journal Article
• Language: English
• Published: London, England SAGE Publications 01.08.2009; Sage Publications Ltd
• Subjects: Allosteric properties; Antiviral Agents; Aurintricarboxylic acid; Aurintricarboxylic Acid - pharmacology; Aurintricarboxylic Acid - therapeutic use; Cell Line; Cytotoxicity; Genotypes; Hepacivirus - drug effects; Hepacivirus - enzymology; Hepatitis; Hepatitis C; Hepatitis C - drug therapy; Humans; Inhibitory Concentration 50; Kinetics; Molecular modelling; Polymerization; Replicase; Replication; Ribonucleic acid; RNA; RNA-Dependent RNA Polymerase - antagonists & inhibitors; RNA-directed RNA polymerase; Structure-Activity Relationship; Viral Nonstructural Proteins - antagonists & inhibitors
• ISSN: 2040-2066; 0956-3202; 2040-2066
• DOI: 10.3851/IMP1286

Background: Hepatitis C virus (HCV) NS5B is an essential component of the viral replication machinery and an important target for antiviral intervention. Aurintricarboxylic acid (ATA), a broad-spectrum antiviral agent, was evaluated and characterized for its anti-NS5B activity in vitro and in HCV replicon cells. Methods: Recombinant NS5B, HCV replicase and Huh-7 cells harbouring the subgenomic HCV replicon of genotype 1b were employed for biochemical and mechanistic investigations. Results: Analysis of ATA activity in vitro yielded equipotent inhibition of recombinant NS5B and HCV replicase in the submicromolar range (50% inhibition concentration [IC50] approximately 150 nM). Biochemical and mechanistic studies revealed a bimodal mechanism of ATA inhibition with characteristics of pyrophosphate mimics and non-nucleoside inhibitors. Molecular modelling and competition displacement studies were consistent with these parameters, suggesting that ATA might bind to the benzothiadiazine allosteric pocket 3 of NS5B or at its catalytic centre. Kinetic studies revealed a mixed mode of ATA inhibition with respect to both RNA and UTP substrates. Under single-cycle assay conditions, ATA inhibited HCV NS5B initiation and elongation from pre-bound RNA, but with ≥fivefold decreased potency compared with continuous polymerization conditions. The IC50 value of ATA for the native replicase complex was 145 nM. In HCV replicon cells, ATA treatment ablated HCV RNA replication (50% effective concentration =75 nM) with concomitant decrease in NS5B expression and no apparent cytotoxic effects. Conclusions: This study identified ATA as a potent anti-NS5B inhibitor and suggests that its unique mode of action might be exploited for structural refinement and development of novel anti-NS5B agents.