Figure 1.

Identification of cell cycle phase specific inhibitors through a novel cell-based high-throughput small molecule screening approach. (a) Targeting the cell cycle in the treatment of cancer. (b) Cell-based high-throughput screening (HTS) of 79 827 drug-like molecules for cell cycle modulators. Twenty hours post compound treatment, HeLa cancer cells were fixed and stained with Vybrant DyeCycle green, and a high-content cytometer was used to generate a cell cycle profile for each compound. (c–e) Scatter plots of percent G1, S, and G2/M arrest for all compounds. The cutoffs for G1-phase and S-phase inhibitors were set at >4 and >5 S.D. from the mean, respectively. The cutoff for G2/M inhibitors was set at ≥ 67% G2/M arrest. (f–h) Examples of G1, S, and G2/M-phase arresting compounds and their cell cycle profiles. (i) Immunofluorescence HTS assay for distinguishing G2-phase inhibitors from M-phase inhibitors. Cells were co-stained with 1 μg/ml Hoechst 33342 (blue) and Alexa-488-phospho-histone-H3 (p-H3, green). Mitotic cells are positive for p-H3. Bar indicates 5 μm. (j) Summary of screen results indicating that 266 compounds arrest cells in mitosis and 7 compounds arrest cells in G2-phase. (k) Screen summary plot of percent hit rate indicates that M-phase inhibitors were the most abundant. (b–k) See also Supplementary Table 2