Figure 5.

Specificity of novel p7 inhibitors. Screening lead hits LDS19 and 21 were examined for specificity against wild-type and rimantadine resistant HCV. (A) Titration of LDS 19 and 21 against Huh7 cells transfected with both wild-type (diamonds) and Leu20Phe (squares) J4/JFH-1 RNA, showing effects on released infectivity. Graph plots the average released titers from duplicate wells from at least two separate experiments, normalized to the 100% untreated value. Error bars represent normalized percent error of the mean between experiments (*P < 0.05). (B) Effects of p7 inhibitors on HCV spread following infection at low M.O.I (0.0001 FFU/cell). Huh7 cells were infected in triplicate overnight with wild-type (black) and Leu20Phe (white) J4/JFH-1 and then media replaced after extensive washing with that containing inhibitors. Fluorescent NS5A foci were counted at 72 hours postinfection. Histogram shows the average counts for two experiments with triplicate wells normalized to 100% untreated value to allow comparison between viruses. Error bars represent normalized percent error of the mean (*P < 0.05, **P < 0.01). (C) The northern and southern R-groups of LDS21 were rationally altered to determine tolerances for molecular structures and fit within the structure-guided channel model peripheral binding site. Activities of a limited series of LDS21 structural analogs against both wild-type (black) and Leu20Phe (white) J4/JFH-1, normalized to untreated values. Results are the average of two separate experiments with duplicate wells and error bars are normalized percent error of the mean between experiments. (D) Immunofluorescence staining for core (green Alexa-fluor 488 nm) and NS5A (red, Alexa-fluor 565 nm) in huh7 cells 72 hours posttransfection of J4/JFH-1 RNA following treatment with 4 µM LDS21, 21.8, and 21.9.