SMI-16a

Kinase experiment:

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

Cell experiment:

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[1].

Animal experiment:

Mice: Female Balb/C mice are injected subcutaneously with JC cells suspended in PBS. After palpable tumor growth, animals are treated five days per week by intraperitoneal injection of vehicle alone or 50 mg/kg of SMI-16a.Whole body weights and tumor volume measurements are performed three times per week[1].

References:

[1]. Xia Z, et al. Synthesis and evaluation of novel inhibitors of Pim-1 and Pim-2 protein kinases. J Med Chem. 2009 Jan 8;52(1):74-86.
[2]. Hiasa M, et al. Pim-2 kinase is an important target of treatment for tumor progression and bone loss in myeloma. Leukemia. 2015 Jan;29(1):207-17.