In vitro activity: SMI-16a has excellent potency for inhibition of both Pim-1 and Pim-2. Treatment with Pim-2 short-interference RNA as well as the Pim inhibitor SMI-16a successfully restores osteoblastogenesis suppressed by all the above inhibitory factors and MM cells. The SMI-16a treatment potentiates BMP-2-mediated anabolic signaling while suppressing TGF-β signaling.

Kinase Assay: Recombinant human Pim-1 (Upstate) is incubated with S6 kinase/Rsk-2 peptide 2 (KKRNRTLTK) as the substrate in the presence 100 μM of compounds from the screening library, 1 μM ATP and 10 mM MgCl2 for 1 h. The Kinase-Glo luciferase kit is used to measure residual ATP levels after the kinase reaction.

Cell Assay: Human prostate cancer PC3 cells are seeded in 96-well tissue culture dishes at approximately 10% confluency and allowed to attach and recover for 24 h. Varying concentrations of the test compounds (SMI-16a) are then added to each well, and the plates are incubated for an additional 48 h. The number of surviving cells is determined by the MTS assay. The percentage of cells killed is calculated as the percentage decrease in MTS metabolism compared with control cultures.