Kinase experiment:

The FLT3 Kinase-Glo kinase assays are carried out in 96-well plates at 30℃ for 4 h in a final volume of 50 μL, including 25 mM Tris pH 7.4, 10 mM MgCl2, 4 mM MnCl2, 1 mM DTT, 0.02% Triton X-100, 0.01% BSA, 1 μM ATP, 20 μM peptide (GGMEDIYFEFMGGKKK), 75 ng recombinant FLT3 proteins, and test compound (BPR1J-097) at the indicated concentration. After incubation, 50 μL Kinase-Glo Plus Reagent is added and incubated at 25℃ for 20 min. A 70 μL aliquot of each reaction mixture is transferred to a black microtiter plate and the luminescence is measured on a multilabel counter. Each IC50 value is determined by three different experiments[1].

Cell experiment:

Proliferation assays are performed by seeding 10 000 cells per well in a 96-well culture plate. After 16 h, cells are then treated with vehicle or BPR1J-097 Hydrochloride at various concentrations in medium for 72 h. Cell viability is quantitated using the MTS method. The results are determined by measuring absorbance at 490 nm using a plate reader. The GC50 value is defined as the amount of compound that causes 50% reduction in cell viability in comparison with DMSO-treated (vehicle) control and is calculated using Prism version 4 software[1].

Animal experiment:

Male nude mice of 8 weeks of age are used. Nude mice (n=5 to 7 per group) are inoculated subcutaneously with MOLM-13 (1×106 per flank) or MV4-11 cells (5×106 per flank). When the tumour size reaches 100 to 200 mm3, animals are grouped and treated with BPR1J-097 Hydrochloride at various doses in a 2-week treatment period as indicated. Animals are treated with BPR1J-097 Hydrochloride (10 and 25 mg/kg, i.v.) or vehicle as control at once daily for 5 days per week for 2 weeks. Tumour volumes are measured and calculated with the formula length×width2/2 after initiation of treatments. Tumour size and animal body weight are measured twice a week after tumour cell inoculation. At the end of the study, animals are killed by carbon dioxide inhalation followed by cervical dislocation[1].

BPR1J-097 is a novel potent FLT3 inhibitor with an IC50 of 11 nM.

References:
[1]. Lin WH, et al. BPR1J-097, a novel FLT3 kinase inhibitor, exerts potent inhibitory activity against AML. Br J Cancer. 2012 Jan 31;106(3):475-81.