A novel small-molecule FLT3 inhibitor. (A) Chemical structure of G-749. (B) Five human leukemia cell lines were incubated with increasing concentrations of G-749 for 72 hours. Cell viability was determined using ATPLite assay. G-749 inhibited the proliferation of MV4-11 and Molm-14 with 50% inhibition concentration (IC₅₀) values of 3.5 and 7.5 nM, respectively. The IC₅₀ values were calculated using nonlinear regression. (C) Molm-14 cells were incubated with the indicated concentrations of G-749 for 2 hours. The phosphorylation levels of FLT3 (Tyr 591), STAT5 (Tyr 694), ERK1/2 (Tyr 204), AKT (Ser 473), and FoxO3a (Thr 32) were detected by western blot. Each total protein was used as a loading control. (D) MV4-11 cells were incubated with 100 nM inhibitors for 2 hours in 10% serum and then washed with fresh medium. The autophosphorylation levels of FLT3 were determined for 24 hours by p-FLT3 ELISA. (E) After washout as in panel D, the phosphorylation levels of STAT5 and ERK1/2 were monitored for 8 hours with treatment of G-749 or AC220. (F) After 18 hours, caspase-3/7 activities were measured in MV4-11 and Molm-14 cells treated with the indicated concentration of G-749. (G) MV4-11 cells were treated with the indicated concentrations of G-749 for 36 hours. Cells were stained with propidium iodide/annexin V and then analyzed by flow cytometry. The percentages of early and late apoptotic cells were indicated in the right lower and right upper quadrants, respectively. (H) MV4-11 cells were treated with cytarabine, G-749, or a combination of cytarabine and G-749 (ratio 100:1), and cell viability was measured. Error bars show standard deviation (SD).