SGI-1776 (Pim-Kinase Inhibitor IX) has been used in trials studying the treatment of Prostate Cancer, Non-Hodgkins Lymphoma, and Relapsed/Refractory Leukemias.

Pim1:7 nM, FLT3:44 nM, Pim3:69 nM, Pim2:363 nM

Kinase Assays: Kinase inhibition is measured by the use of radiometric assays performed by KinaseProfiler service. Assays contain a peptide substrate, known purified recombinant human kinases, gamma-labeled ATP, magnesium ion, and a fixed concentration (1 μM) of SGI-1776. In a final reaction volume of 25 μL, 5 to 10 mU of Pim1/2/3 is incubated with 8 mM of MOPS, pH 7.0; 0.2 mM ethylene diamine tetraacetic acid; 100 μM KKRNRTLTV;10 mM MgAcetate; and [γ-32P-ATP] . The reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 μL of a 3% phosphoric acid solution. Then, 10 μL of the reaction is spotted onto a P30 filtermat and washed 3 times for 5 minutes in 75 mM phosphoric acid and once in methanol before it is dried and measured via a scintillation counter.

Cells are cultured in IMDM (ATCC) supplemented with 10% FBS and grown in a 37oC incubator with 5% CO2. Cells are routinely tested for Mycoplasma infection using a commercially available kit. Cells are treated with DMSO or various concentrations of SGI-1776 for 24 hours. Cells (1×106) are washed, then resuspended in 100 μL of annexin binding buffer, mixed with 5 μL of FITC solution and 5 μL of propidium iodide (PI; 50 μg/mL) solution. For each sample, 1×104 cells are measured using a Becton Dickinson FACSCalibur flow cytometer. (Only for Reference)