AZD1208 is a novel, orally bioavailable, highly selective PIM kinase inhibitor with single nanomolar potency against all three PIM kinases.

Pim1:0.4 nM, Pim3:1.9 nM, Pim2:5 nM

AZD1208剂量依赖性抑制MOLM-16和KG-1a异种移植肿瘤在体内的生长.

AZD1208在培养的MOLM-16细胞中引起细胞周期停滞和细胞凋亡。 这伴随着BAD,4EBP1和p70S6K磷酸化的剂量依赖性降低。 另外,AZD1208导致有效抑制来自骨髓抽吸物的原代AML细胞的集落生长并下调Pim靶标的磷酸化。

The activity of purified human PIM-1, PIM-2 and PIM-3 enzymes on substrate FL-Ahx-Bad (FITC-(AHX)RSRHSSYPAGT-COOH) is determined using a mobility shift assay on a Caliper LC3000 reader. The PIM-1 assay is performed in a 12 mL reaction containing 50 mM HEPES (pH 7.5), 1 mM DTT, 0.01% Tween 20, 50 mg/mL BSA, 10 mM MgCl2, 1.5 mM FL-Ahx-Bad peptide, 100 mM ATP, 2.5 nM PIM-1 and various amount of inhibitor. The reaction is quenched after 90 minute incubation at 25?C with?5 mL of stop mix consisting of 100 mM HEPES, 121 mM EDTA, 0.8% Coating Reagent 3 and 0.01% Tween 20. The ATP and enzyme concentrations for the PIM-2 assay are 5 mM and 2.5 nM, respectively, while 50 mM of ATP and 0.33 nM of enzyme is used for PIM-3 assays. For high [ATP] screenings, 5 mM ATP is used with 0.67 nM enzyme for both PIM-1 and PIM-2 or 0.11 nM PIM-3. Fluorescence of phosphorylated and unphosphorylated substrate is detected and a ratiometric value is calculated to determine percent turnover. IC50 values are determined from dose-response data using IDBS ActivityBase software.

AZD1208 is dissolved in DMSO. MOLM-16 cells, purchased from DSMZ and cultured in RPMI containing 10% fetal bovine serum (FBS) and 1% L-glutamine, are plated at 20,000 cells per well in 96 well plates overnight. Cells are treated for 72 hours with compound or control vehicle (dimethyl sulfoxide) and cell viability is measured after the addition of Cell Titer-Blue for 4 hours at 37?C and reading of fluorescence on a Tecan Infinite? 200. The GI50 is determined by calculating growth at each dose relative to vehicle treated cells and cell viability at the time of treatment.