G-749, a new-type FLT3 inhibitor, exhibits effective and sustained inhibition of the FLT3 wild type and mutants.

Mer:1 nM, FLT3 (D835Y):0.6 nM, RET:9 nM, FLT3:0.4 nM, Aurora B:6 nM

在Molm-14细胞骨髓移植模型中,口服 G-749（20 mg/kg）,能够抑制肿瘤生长.在MV4-11异种移植的小鼠模型中,口服 G-749（30 mg/kg）通过抑制FLT3通路,并显著抑制肿瘤细胞生长.

在AML患者的原始细胞中,G-749通过诱导细胞凋亡,能够抑制细胞增殖。在稳定表达FLT3-ITD/N676D,FLT3-ITD/F691L,FLT3-D835Y,或FLT3-D835Y/N676D的BaF3细胞系中（IC50 <10 nM）,G-749具有显著的抑制活性。

Kinase assay: Activity assays are conducted using Lance Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) technology from Perkin-Elmer. Briefly, 10 ng/mL FLT3 enzyme, a serial diluted G-749, 80 nM substrate of ULight-poly-GT peptide and variable amounts of ATP (8.5 μM to 1088 μM) are mixed in kinase assay buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT and 0.01% Tween-20) and are added to a 384-well OptiPlate-384 in a volume of 10 μL. Kinase reactions are incubated at room temperature for up to 1 h and then stopped by the addition of 5 μL of 10 mM EDTA. A volume of 5 μL of the specific Eu-labeled-anti-phosphopeptide antibody diluted in LANCE Detection Buffer is then added to a final concentration of 2 nM. After 30-minute incubation, assay plates are incubated at 23°C and the LANCE signal is measured on an EnVision Multilabel Reader. Excitation wavelength is set at 320 nm and emission monitored at 615 nm (donor) and 665 nm (acceptor). The IC50 is calculated using nonlinear regression analysis analysis by GradPad Prism 5.

Cells are seeded at a density of 2 ×104 cells per well and treated with the indicated concentrations of test inhibitor for 72 hours at 37℃. The conditioned medium (CM) is prepared from HS-5 cell culture for 5 days under routine culture conditions, clarified by centrifugation, and used immediately. The CM is added to complete medium at a final concentration of 35%. In coculture experiments, 5 ×104 AML blast cells are plated in 24-well plates containing 1 ×104 HS-5 monolayers and then cultured for at least 48 hours before the exposure of inhibitors. Cell viability is determined by an ATPLite assay. (Only for Reference)