Volume 132, Issue 1, Pages 311-320 (January 2007) A Cell-Based, High-Throughput Screen for Small Molecule Regulators of Hepatitis C Virus Replication  Sun Suk Kim, Lee F. Peng, Wenyu Lin, Won-Hyeok Choe, Naoya Sakamoto, Stuart L. Schreiber, Raymond T. Chung  Gastroenterology  Volume 132, Issue 1, Pages 311-320 (January 2007) DOI: 10.1053/j.gastro.2006.10.032 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Luciferase activity of different cell inoculation concentrations in the 384-well plate format. Different concentrations of PEG-IFN were used as a positive control and to determine signal-to-background (S/B) ratio. HCV RNA replication levels were determined by luciferase activity, as described in Material and Methods. Each point represents the average of 3 data points, with the standard deviation represented as data bars. Gastroenterology 2007 132, 311-320DOI: (10.1053/j.gastro.2006.10.032) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 A graphical summary of the primary screening for the known bioactives library using Huh7/Rep-Feo cell. Each point represents 1 compound. The X-axis shows HCV replication as measured by normalized luciferase signal, expressed as the composite Z-score. The Y-axis shows cell viability as measured by normalized CellTiterGlo (Promega) signal, expressed as the composite Z-score. Gastroenterology 2007 132, 311-320DOI: (10.1053/j.gastro.2006.10.032) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 (A) Anti-HCV activity of PEG-IFN and ribavirin on HCV RNA replication in OR6 cell system. OR6 cells were cotreated with PEG-IFN (0, 0.001, 0.002, 0.007, 0.03, and 0.07 ng/mL) and ribavirin (0, 25, 50, 100, 200, and 400 μM) for 48 hours. Luciferase activity for HCV RNA replication levels is shown as a percentage of control. Each bar represents the average of triplicate data points with standard deviation represented as the error bar. (B) A normalized isobologram generated by CalcuSyn using the data from Figure 3A. Points below and to the left of the line represent synergy. Thirteen of the 14 concentration ratios examined demonstrate the synergistic effect of the combination of PEG-IFN and ribavirin. Gastroenterology 2007 132, 311-320DOI: (10.1053/j.gastro.2006.10.032) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Results of secondary screening with corticosteroids. (A) Luciferase activity for HCV RNA replication levels is shown as a percentage of control. (B) Cell viability is also shown as a percentage of control. Each bar represents the average of triplicate data points with standard deviation represented as the error bar. *Denotes a significant difference from control of at least P < .05. Gastroenterology 2007 132, 311-320DOI: (10.1053/j.gastro.2006.10.032) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Results of secondary screening with several hit compounds. Luciferase activity for HCV RNA replication levels is shown as a percentage of control (A, C, E). Cell viability is also shown as a percentage of control (B, D, F). Each bar represents the average of triplicate data points with standard deviation represented as the error bar. (A, B) MY-5445 and trequinsin (C, D) SB 203580 (E, F) tetrandrine. *Denotes a significant difference from control of at least P < .05. Gastroenterology 2007 132, 311-320DOI: (10.1053/j.gastro.2006.10.032) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Results of secondary screening with the statins. (A) Luciferase activity for HCV RNA replication levels is shown as a percentage of control. (B) Cell viability is shown as a percentage of control. Each bar represents the average of triplicate data points with standard deviation represented as the error bar. *Denotes significant difference from control of at least P < .05. Gastroenterology 2007 132, 311-320DOI: (10.1053/j.gastro.2006.10.032) Copyright © 2007 AGA Institute Terms and Conditions