Fig. 1.

Pfizer-identified small molecules target NS5A. (A) Replicon 1b cells were serially passaged in the presence of NS5Ai 1 at 20× the IC₅₀ (labeled “resistant”); control cells were passaged in 1% DMSO only. After 6 weeks, cells were expanded, and the IC₅₀ of NS5Ai 1 was determined for both cell populations. (B) Transient replication assays using chimeric replicons encoding genotype 1a NS5A sequences (dark shading) in a genotype 1b background were used to determine the IC₅₀s for BILN 2061 and NS5Ai 2. (C) Biotin-tagged S- and R-form NS5A-targeting molecules were exposed to Replicon 1b cells or Huh-7.5 cells (500 nM final assay concentration), and bound NS5A was detected following the capture of protein-compound complexes, within cell lysates, on streptavidin-coated ELISA plates (Abs, NS5A antibodies). (D) Huh-7.5 cells were infected with a recombinant lentivirus (LV) expressing a polyhistidine-tagged NS5A species and then treated with the biotin-tagged NS5A-targeting compounds. The NS5A bound to the compound was assessed as described above (C). Cont., control.