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Genotype 1a (H77, Licensed from Apath LLC) HCV Replicon Cells Were Resuspended to a Concentration of 1.4 × 105 Cells/ml by Addition of Pre-Warmed Assay Medium (DMEM + 10% FCS). 45 μl of This Suspension Was Added to Each Well of a 384-Well Assay Plate (Lumitrac, Greiner), Already Containing 0.5 l of Test Compound. All Plates Were Covered with Gas Permeable Seals and Incubated at 37oC, 5% CO2 for 48 Hours. After 48 Hours, the Assay Plate Was Removed from the Incubator and Left to Cool to Room Temperature for 15-30 mins. Medium Was Removed from the Wells and 5 μl Lysis Buffer (Renilla Luciferase Assay Kit, Promega) Was Added to Each Well. The Plate Was Incubated at Room Temperature on a Rocker for 15 mins then 15 μl Assay Substrate was Added to Each Well. Luminescence Was Read Immediately Using an EnVision Plate Reader.

has been cited by the following article:

• TITLE: Identification of HCV Inhibitors from a Cell-Based Sub-Genomic Replicon Screen

  AUTHORS: David C. Pryde, Thien-Duc Tran, Mark Gardner, Chris Pickford, Stephen M. Shaw, Mike Westby, Tanya Parkinson, Caroline Smith-Burchnell, Rob Webster, Satish Dayal

  KEYWORDS: HCV; Replicon; Antiviral; Cell-Based Screen

  JOURNAL NAME: Open Journal of Medicinal Chemistry, Vol.3 No.1, March 29, 2013

  ABSTRACT: A high throughput screen of the Pfizer compound collection was carried out using a hepatitis C virus (HCV) genotype 1b subgenomic replicon cell line. Those confirmed hits that demonstrated broad spectrum activity without overt cytotoxicity were further evaluated, leading to the identification of a series of pyrrolopyridines with excellent antiviral activity in a fully infectious HCV cell-based assay and pharmacokinetic properties.