Schema of the molecular screening approach. ATP-driven luminescence values are shown for the compounds in the Prestwick Chemical Library. Values are normalized for maximum luminescence observed on positive-control (guanidine HCl) wells in each 384-well plate. Negative-control wells on each plate included DMSO; note the low levels of luminescence. Primary hit compounds are indicated, which produced normalized luminescence levels above a threshold of 50% of the luminescence levels of the positive controls. All compounds were tested at a final concentration of 12.5 μM.

Fluoxetine and norfluoxetine antiviral activity. (A) The antiviral EC₅₀s for fluoxetine (Fluox) and norfluoxetine (Norf) were determined by comparison of viabilities 48 h after infection with CVB3 (normalized by comparison to cells infected with CVB3 in the presence of guanidine). A similar assay to the primary HTS assay based on ATPLite 1-Step was applied to determine EC₅₀ and CC₅₀ of the compounds. The final concentration range was 0.38 nM to ∼200 μM (numbers next to symbols represent concentrations in μM), and each concentration was tested in triplicate. Averages and standard deviations of triplicates are shown as symbols with error bars. (B) Similar to the EC₅₀ determination, CC₅₀ determinations for fluoxetine and norfluoxetine were determined by determination of relative luciferase luminescence of uninfected HeLa cells compared to that of untreated control wells (DMSO only). (C) Estimated EC₅₀ for fluoxetine as determined by plaque reduction assay. Cells in replicate wells were infected with CVB3 30 min after addition of fluoxetine at the concentrations indicated. After 6 h, the cells were harvested and the titer of intracellular virus was determined by limiting dilution plaque assay. Symbols represent single determinations, and a representative of two separate experiments is shown. (D) Reduction of viral titer by treatment of infected cells with fluoxetine and norfluoxetine. Cells in replicate wells were infected with CVB3 30 min after addition of the drugs at an effective concentration of 6.25 μM. After 6 h, the cells were harvested and the titer of intracellular virus was determined by limiting dilution plaque assay. Averages and standard deviations of triplicates are shown as shaded bars and error bars, respectively.

Assays of antiviral activity. (A) Immunoblot detection of viral capsid protein 6 h after infection with CVB3-H3; no viral protein is detected in cells treated with fluoxetine (Fluox) and norfluoxetine (Norf). Immunoblot detection of glycyl-tRNA synthease protein (GlyRS) (a housekeeping protein) is shown as a loading control. (B) RT-PCR detection of CVB3 RNA in cells treated with guanidine (Guan) or fluoxetine beginning 30 min before inoculation of cell cultures. Symbols represent single determinations, and a representative of two separate experiments is shown. (C) Dual-luciferase assay of Renilla and firefly luciferase in cells transfected with plasmid encoding a bicistronic RNA with firefly luciferase expressed under the control of the CVB3 IRES. The values next to the bars are average luminescence readings of duplicate samples for Renilla luciferase and firefly luciferase, respectively. The values of mock-transfected HeLa cells are 0.3 and 0.1 for Renilla luciferase and firefly luciferase, respectively. A representative of two separate experiments with similar results is shown.

Fluoxetine inhibits replication of clinical isolates of coxsackieviruses B1 to B3. HeLa-RW cells were infected with clinical isolates of three serotypes of coxsackieviruses at a multiplicity of infection of approximately 1 with or without pretreatment 30 min prior with 6 μM fluoxetine (Fluox). After 6 h, cells were harvested and the titer of intracellular virus was determined by limiting dilution plaque assay. Averages of triplicate determinations are shown in the bar graph, with standard deviations shown by error bars.