In vitro activity: AZD1208 is an orally available, potent and highly selective Pim inhibitor that effectively inhibits all three isoforms. AZD1208 inhibits the growth of several AML cell lines and sensitivity correlates with the level of Pim-1 expression, STAT5 activation and presence of protein tyrosine kinase mutation. AZD1208 causes cell cycle arrest and apoptosis in MOLM-16 cells in culture. This is accompanied by a dose-dependent reduction in phosphorylation of BAD, 4EBP1 and p70S6K. In addition, AZD1208 leads to potent inhibition of colony growth of primary AML cells from bone marrow aspirates and downregulates phosphorylation of Pim targets.

Kinase Assay: The activity of purified human Pim-1, -2, and -3 enzymes on a BAD peptide substrate was determined as previously described. To determine inhibition constants (Ki), 50% inhibition concentration (IC50) values were acquired at a series of adenosine triphosphate (ATP) concentrations and compound doses with 1 nM enzyme and 1.5 μM full-length BAD substrate in 50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 1 mM dithiothreitol, 0.01% Tween 20, 50 μg/mL bovine serum albumin, and 10 mM MgCl2. The Ki values were calculated by global data fitting using the Cheng-Prusoff equation or the Morrison equation for tight-binding inhibitors. To assess selectivity, 442 kinases were screened by using DiscoveRx KINOMEscan technology at a single concentration of 1 μM. Kinases inhibited by more than 50% were retested at DiscoveRx with a full dose-response to determine binding affinity.

Cell Assay: 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(R) 200. The GI50 is determined by calculating growth at each dose relative to vehicle treated cells and cell viability at the time of treatment.