In vitro activity: SMI-4a is a novel benzylidene-thiazolidine-2, 4-dione small molecule, potent and selective inhibitor of Pim1 with IC50 of 17 nM, it is modestly potent to Pim-2, and does not significantly inhibit any other serine/threonine- or tyrosine-kinases. SMI-4a blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma. SMI-4a was found to induce phosphorylation of extracellular signal-related kinase1/2 (ERK1/2). The serine/threonine Pim kinases are up-regulated in specific hematologic neoplasms, and play an important role in key signal transduction pathways, including those regulated by MYC, MYCN, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. SMI-4a kills a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre-T-LBL/T-ALL) being highly sensitive. Incubation of pre-T-LBL cells with SMI-4a induced G1 phase cell-cycle arrest secondary to a dose-dependent induction of p27(Kip1), apoptosis through the mitochondrial pathway, and inhibition of the mammalian target of rapamycin C1 (mTORC1) pathway based on decreases in phospho-p70 S6K and phospho-4E-BP1, 2 substrates of this enzyme. In addition, treatment of these cells with SMI-4a was found to induce phosphorylation of extracellular signal-related kinase1/2 (ERK1/2), and the combination of SMI-4a and a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor was highly synergistic in killing pre-T-LBL cells. In immunodeficient mice carrying subcutaneous pre-T-LBL tumors, treatment twice daily with SMI-4a caused a significant delay in the tumor growth without any change in the weight, blood counts, or chemistries. These data suggest that inhibition of the Pim protein kinases may be developed as a therapeutic strategy for the treatment of pre-T-LBL. SMI-4a is an ATP competitive inhibitor of Pim1 with IC50 of 17 nM. SMI-4a shows high selectivity for Pim1 against a panel of kinases. SMI-4a inhibits the in vitro phosphorylation by Pim-1 of the known substrate, the translational repressor 4E-BP1. SMI-4a (5μM) inhibits pancreatic and leukemic cells growth. SMI-4a reduces phosphorylation of the Pim target Bad in prostate and hematopoietic cells. SMI-4a causes cell cycle arrest and reverses the antiapoptotic activity of Pim-1. SMI-4a increases the amount of p27Kip1 in the nucleus. SMI-4a treatment of pre-T-LBL inhibits the mTOR pathway. SMI-4a reduces MYC protein expression in pre-T-LBL. SMI-4a treatment induces up-regulation of MAPK pathway

Kinase Assay: SMI-4a is a selective ATP-competitive Pim-1 kinase inhibitor with an IC50 of 21 nM for Pim-1 compared to an IC50 of 100 nM for Pim-2 and with little or no activity against a panel of 50 other kinases tested.

Cell Assay: 6812/2 cells were incubated for 24 hours and Jurkat cells, for 48 hours with SMI-4a (10μM) or dimethyl sulfoxide (DMSO) in serum-free medium. After incubation, cells were harvested, washed once in phosphate-buffered saline (PBS), fixed in cold 70% ethanol for 45 minutes, stained with propidium iodide solution that contains RNaseA for 30 minutes, and analyzed by flow cytometry.