We have identified a new chemotype of Pim protein kinases inhibitors, the benzylidene-thiazolidine-2,4-diones. In vitro
, these small molecules are capable of blocking the ability of Pim kinase to phosphorylate peptides with IC50
values in the nanomolar range and inhibit the Pim protein kinase directed phosphorylation of two known substrates, 4E-BP1 and p27Kip1
. Members of this chemotype can be Pim-1, or Pim-2, specific or dual inhibitors blocking the activity of both of these enzymes (20
). As suggested by a screen of 60 additional serine, threonine, and tyrosine protein kinases (20
), benzylidene-thiazolidine-2,4-diones are highly specific inhibitors of the Pim protein kinase. Our compounds differ in structure from previously described small-molecule inhibitors of this enzyme. These inhibitors include ruthenium half-sandwich complexes, which mimic staurosporine (30
), substituted pyridones (33
), and specific flavinoids (34
). It is well known that staurosporine analogues and flavinoids inhibit several other kinases, leaving the usefulness of such compounds as Pim-1 inhibitors unclear.
The addition of 4a or 16a to two different prostate cancer cell lines inhibited the ability of Pim kinase to phosphorylate the proapoptotic Bad protein on Ser112
. This is consistent with the previous observation that either Pim-1 or Pim-2 is capable of phosphorylating this residue and thus inhibiting apoptotic cell death of growth factor-starved FDCP1 cells (12
). Phospho-Bad protein is sequestered by 14-3-3 proteins, which blocks its ability to cause apoptotic cell death. Possibly through this mechanism, Pim promotes survival of chemotherapy-treated prostate cancer (21
) and regulates cardiomyocyte survival and T-cell survival (36
). We find that this inhibitor chemotype is capable of reversing the prosurvival phenotype induced by Pim overexpression, thus suggesting that these compounds could be useful as chemotherapeutic agent in tumors with enhanced survival secondary to overexpression of this enzyme.
Our data show that addition of Pim inhibitors to several prostate and leukemic cell lines induces a G1
cell cycle block, although, in other cell lines, especially in low-serum conditions, these inhibitors are capable of inducing apoptosis (data not shown). The cell cycle block induced by these agents is consistent with our previous observation that PC3 cells overexpressing Pim express fewer cells in the G1
phase of the cell cycle (9
). Recently, Morishita et al. also showed that overexpression of Pim in K562 cells decreased the number of cells in G1
and increased the cells found in S phase. Further, they showed that Pim is capable of phosphorylating p27Kip1
, decreasing the cellular levels of this protein and translocating this protein to the cytosol, thus stimulating cell growth (19
). In leukemic cells, the benzylidene-thiazoli-dine-2,4-diones inhibit the activity of Cdk2, and in both leukemic and prostate cancer cells, the Pim inhibitors reverse the effects of Pim by transferring at least a portion of the cytoplasmic enzyme to the nucleus.
Both 4a and 16a were shown to be highly selective for Pim-1 based on profiling a total of 60 kinases; however, in this in vitro
assay, 16a, but not 4a, showed significant inhibition of the DYRK1a (20
). Other kinase inhibitors have shown potent inhibition of all members of the Pim kinase family (Pim-1, Pim-2, and Pim-3) and members of the DYRK family (DYRK1a, DYRK2, and DYRK3; ref. 38
). Based on these results, it is expected that the Pim and DYRK kinases would share some structural similarity within their ATP-binding domains because inhibitors common to both Pim and DYRK kinases were shown to be ATP competitive (38
). An amino acid alignment of Pim-1, Pim-2, and DYRK1a shows a considerable number of conserved residues, including residues known to facilitate inhibitor interactions in the ATP-binding domain of Pim-1 (Supplementary Fig. S2
; ref. 39
Interestingly, both 4a and the recently identified Pim-1 inhibitor K00135 share a trifluoromethyl substituent, which was suggested to interact with Arg122 (23
). This Arg corresponds to Met or Leu in the DYRKs and may contribute to the observed lack of inhibitory activity of 4a toward DYRK1a.
The phosphatidylinositol/Akt/mTOR pathway is often activated in leukemia and lymphoma downstream of a variety of oncogenes including receptor tyrosine kinases (40
). Therefore, several clinical trials have investigated using the mTOR inhibitor rapamycin and second-generation rapamycin analogues for the treatment of hematologic malignancies including acute myelogenous leukemia, chronic myelogenous leukemia, B-cell lymphoma, mantle cell lym-phoma, and multiple myeloma (41
). The results of one clinical trial showed that (a
) mTOR is activated in 23 acute myelogenous leukemia patient samples and the downstream mTOR target 4E-BP1 is phosphorylated, (b
) rapamycin treatment of these samples reduced 4E-BP1 phosphorylation, and (c
) rapamycin showed significant clinical response in 4 of 9 patients with acute myelogenous leukemia (42
). We have shown that the Pim inhibitors 4a and 16a act synergistically with the mTOR inhibitor rapamycin to reduce the level of phospho-4E-BP1 and inhibit leukemic cell growth in FDCP1 cells and the MV4;11 cell line containing the activating internal tandem duplication mutation of the tyrosine kinase FLT3. Additionally, we have shown that 4a and 16a decrease the level of c-Myc, the transcription and translation of which are known to be rapamycin sensitive. Based on the observations that the Pim kinases have been shown to promote rapa-mycin resistance in hematopoietic cells (26
) and are frequently up-regulated along with c-Myc in leukemia and lymphoma (1
), the combination of Pim kinase and mTOR inhibitors represents a potential novel treatment option for hematologic malignancies and other tumor types that show reduced sensitivity to rapamycin.