PSI-352938

GT 1a, 1b, and 2a replicon cells are cultured in the presence of G418 (0.75 mg/mL for GT 1a, 0.25 mg/mL for GT 1b and 2a) and increasing concentrations of PSI-352938 (PSI-938) or PSI-353661 starting at their respective EC₅₀ or EC₉₀. As a no-compound control, replicon cells are maintained in parallel in the equivalent percent volume (0.2%) of DMSO. Cells are passaged whenever they reach ∼80% confluence and replenished with G418 medium containing fresh compound. At various passages, cells are tested for sensitivity to PSI-352938 (PSI-938) and PSI-353661. For each assay, 3-fold dilutions of test compound are added to cells in duplicate and incubated at 37°C in a humidified 5% CO₂ atmosphere for 4 days. Inhibition of HCV replicon RNA replication is determined by real-time PCR (RT-PCR) using primers that anneal to the 5′ untranslated region or by measuring the levels of luminescence expressed via the firefly or Renilla luciferase reporter gene using the Bright-Glo or Renilla-Glo reagent, respectively. EC₅₀ and EC₉₀, the concentrations at which 50% and 90% inhibition are achieved, are determined using GraphPad Prism software. Aliquots of cells are also saved for RNA isolation, cDNA synthesis, and PCR amplification for sequencing analysis^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.