Screens for off-patent drugs with unrecognized anticancer activity identify the antifungal CPX. (A) Compounds (n = 45) identified as hits in a primary screen for inhibitors of the survivin promoter were tested for their effects on survivin transactivation and cytotoxicity in HeLa cells stably overexpressing the survivin promoter driving luciferase. HeLa cells were treated with the hit compounds (5 μM) for 24 hours. After incubation, luciferase expression was measured as described in “Luciferase assay,” and cell viability was measured by the MTS assay. Data represent the percentage of viable cells (y-axis) and the percentage luciferase decrease (x-axis) compared with buffer control. (B) Chemical structure of CPX. (C) HeLa cells stably overexpressing the survivin promoter driving luciferase were treated with increasing concentrations of CPX. Twenty-four hours after incubation, luciferase activity was measured. Data represent the mean percentage of luciferase expression ± SD after CPX treatment compared with buffer control from one of 3 representative experiments. (D) HeLa cells were treated with CPX (5 μM). At increasing times after incubation, cells were harvested and total proteins were isolated. Expression of survivin and GAPDH was measured by immunoblotting. (E) TEX and M9-ENL1 cells were treated with increasing concentrations of CPX. Seventy-two hours after incubation, cell viability was measured by the Alamar Blue assay. Data represent the mean percentage of viable cells ± SD from 1 of 3 representative experiments.