Figure 4

Identification of inhibitors of Nef:Hck-YEEI signaling in yeast. A) Assay validation. Liquid cultures of yeast expressing wild-type Hck (WT), Hck-YEEI (YEEI), and Hck-YEEI plus wild-type Nef or the PA mutant were grown in 96-well plates for 22 h at 30°C. Cultures expressing Hck-YEEI and Nef were also grown in the presence of the broad-spectrum SFK inhibitor A-419259 at 1 and 5 μM under the same conditions. Growth was recorded as change in optical density at 600 nm, and data are normalized to the percentage of growth observed relative to cells transformed with the empty expression plasmids. Each condition was repeated in triplicate, and the bargraph shows the mean percentage of control growth ± S.D. The statistical significance of the values obtained with Hck-YEEI plus Nef alone was compared to the same cultures grown in the presence of 1 or 5 μM A-419259 (Student’s t-test; *p = .01). B) Yeast cultures expressing Hck-YEEI alone or Hck-YEEI plus Nef in the presence (+) or absence (−) of 5 μM A-419259 were grown in liquid medium in the presence of galactose at 30°C for 18 h. Protein extracts were separated via SDS-PAGE, and immunoblotted for tyrosine-phosphorylated proteins (pTyr), Hck and Nef. C) Fifteen initial hits from the chemical library screen were retested over a range of concentrations for rescue of growth arrest in comparison to A-419259 (5 μM). The plot shows a ranking of the results as a percentage of the growth reversion observed with A-419259. Optimal concentrations varied between compounds, which most likely reflects an effect on the Nef:Hck target vs. cytotoxicity at higher concentrations for some compounds. Data shown were obtained at 30 μM with the exception of compounds 3 and 10 (10 μM), 4 and 6 (3 μM), and 9 (1 μM).