Zuping Xia

Synthesis and Evaluation of Novel Inhibitors of Pim-1 and Pim-2 Protein Kinases

The Pim protein kinases are frequently overexpressed in prostate cancer and certain forms of leukemia and lymphoma. 5-(3-Trifluoromethylbenzylidene)thiazolidine-2,4-dione (4a) was identified by screening to be a Pim-1 inhibitor and was found to attenuate the autophosphorylation of tagged Pim-1 in intact cells. Although 4a is a competitive inhibitor with respect to ATP, a screen of approximately 50 diverse protein kinases demonstrated that it has high selectivity for Pim kinases. Computational docking of 4a to Pim-1 provided a model for lead optimization, and a series of substituted thiazolidine-2,4-dione congeners was synthesized. The most potent new compounds exhibited IC(50)s of 13 nM for Pim-1 and 2.3 microM for Pim-2. Additional compounds in the series demonstrated selectivities of more than 2500-fold and 400-fold for Pim-1 or Pim-2, respectively, while other congeners were essentially equally potent toward the two isozymes. Overall, these compounds are new Pim kinase inhibitors that may provide leads to novel anticancer agents.

A small molecule inhibitor of Pim protein kinases blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma

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. We demonstrate that SMI-4a, a novel benzylidene-thiazolidine-2, 4-dione small molecule inhibitor of the Pim kinases, 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. Our data suggest that inhibition of the Pim protein kinases may be developed as a therapeutic strategy for the treatment of pre-T-LBL.

The Pim protein kinases regulate energy metabolism and cell growth

The serine/threonine Pim kinases are overexpressed in solid cancers and hematologic malignancies and promote cell growth and survival. Here, we find that a novel Pim kinase inhibitor, SMI-4a, or Pim-1 siRNA blocked the rapamycin-sensitive mammalian target of rapamycin (mTORC1) activity by stimulating the phosphorylation and thus activating the mTORC1 negative regulator AMP-dependent protein kinase (AMPK). Mouse embryonic fibroblasts (MEFs) deficient for all three Pim kinases [triple knockout (TKO) MEFs] demonstrated activated AMPK driven by elevated ratios of AMP∶ATP relative to wild-type MEFs. Consistent with these findings, TKO MEFs were found to grow slowly in culture and have decreased rates of protein synthesis secondary to a diminished amount of 5′-cap–dependent translation. Pim-3 expression alone in TKO MEFs was sufficient to reverse AMPK activation, increase protein synthesis, and drive MEF growth similar to wild type. Pim-3 expression was found to markedly increase the protein levels of both c-Myc and the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), enzymes capable of regulating glycolysis and mitochondrial biogenesis, which were diminished in TKO MEFs. Overexpression of PGC-1α in TKO MEFs elevated ATP levels and inhibited the activation of AMPK. These results demonstrate the Pim kinase-mediated control of energy metabolism and thus regulation of AMPK activity. We identify an important role for Pim-3 in modulating c-Myc and PGC-1α protein levels and cell growth.

Discovery and characterization of inhibitors of human palmitoyl acyltransferases

The covalent attachment of palmitate to specific proteins by the action of palmitoyl acyltransferases (PAT) plays critical roles in the biological activities of several oncoproteins. Two PAT activities are expressed by human cells: type 1 PATs that modify the farnesyl-dependent palmitoylation motif found in H- and N-Ras, and type 2 PATs that modify the myristoyl-dependent palmitoylation motif found in the Src family of tyrosine kinases. We have previously shown that the type 1 PAT HIP14 causes cellular transformation. In the current study, we show that mRNA encoding HIP14 is up-regulated in a number of types of human tumors. To assess the potential of HIP14 and other PATs as targets for new anticancer drugs, we developed three cell-based assays suitable for high-throughput screening to identify inhibitors of these enzymes. Using these screens, five chemotypes, with activity toward either type 1 or type 2 PAT activity, were identified. The activity of the hits were confirmed using assays that quantify the in vitro inhibition of PAT activity, as well as a cell-based assay that determines the abilities of the compounds to prevent the localization of palmitoylated green fluorescent proteins to the plasma membrane. Representative compounds from each chemotype showed broad antiproliferative activity toward a panel of human tumor cell lines and inhibited the growth of tumors in vivo. Together, these data show that PATs, and HIP14 in particular, are interesting new targets for anticancer compounds, and that small molecules with such activity can be identified by high-throughput screening. [Mol Cancer Ther 2006;5(7):1647–59]

Novel benzylidene-thiazolidine-2,4-diones inhibit Pim protein kinase activity and induce cell cycle arrest in leukemia and prostate cancer cells

The Pim protein kinases play important roles in cancer development and progression, including prostate tumors and hematologic malignancies. To investigate the potential role of these enzymes as anticancer drug targets, we have synthesized novel benzylidene-thiazolidine-2,4-diones that function as potent Pim protein kinase inhibitors. With IC50 values in the nanomolar range, these compounds block the ability of Pim to phosphorylate peptides and proteins in vitro and, when added to DU145 prostate cancer cells overexpressing Pim, inhibit the ability of this enzyme to phosphorylate a known substrate, the BH3 protein BAD. When added to prostate cancer cell lines, including PC3, DU145, and CWR22Rv1, and human leukemic cells, MV4;11, K562, and U937 cells, these compounds induce G1-S cell cycle arrest and block the antiapoptotic effect of the Pim protein kinase. The cell cycle arrest induced by these compounds is associated with an inhibition of cyclin-dependent kinase 2 and activity and translocation of the Pim-1 substrate p27Kip1, a cyclin-dependent kinase 2 inhibitory protein, to the nucleus. Furthermore, when added to leukemic cells, these compounds synergize with the mammalian target of rapamycin inhibitor rapamycin to decrease the phosphorylation level of the translational repressor 4E-BP1 at sites phosphorylated by mammalian target of rapamycin. Combinations of rapamycin and the benzylidene-thiazolidine-2,4-diones synergistically block the growth of leukemic cells. Thus, these agents represent novel Pim inhibitors and point to an important role for the Pim protein kinases in cell cycle control in multiple types of cancer cells. [Mol Cancer Ther 2009;8(6):1473–83]

Characterization of human palmitoyl-acyl transferase activity using peptides that mimic distinct palmitoylation motifs.

The covalent attachment of palmitate to proteins commonly occurs on cysteine residues near either N-myristoylated glycine residues or C-terminal farnesylated cysteine residues. It therefore seems likely that multiple palmitoyl-acyl transferase (PAT) activities exist to recognize and modify these distinct palmitoylation motifs. To evaluate this possibility, two synthetic peptides representing these palmitoylation motifs, termed MyrGCK(NBD) and FarnCNRas(NBD), were used to characterize PAT activity under a variety of conditions. The human tumour cell lines MCF-7 and Hep-G2 each demonstrated high levels of PAT activity towards both peptides. In contrast, normal mouse fibroblasts (NIH/3T3 cells) demonstrated PAT activity towards the myristoylated substrate peptide but not the farnesylated peptide, while ras -transformed NIH/3T3 cells were able to palmitoylate the FarnCNRas(NBD) peptide. The kinetic parameters for PAT activity were determined using membranes from MCF-7 cells, and indicated that the K (m) values for palmitoyl-CoA were identical for PAT activity towards the two substrate peptides; however, the K (m) for MyrGCK(NBD) was 5-fold lower than the K (m) for FarnCNRas(NBD). PAT activity towards the two substrate peptides was dose-dependently inhibited by 2-bromopalmitate and 3-(1-oxo-hexadecyl)oxiranecarboxamide (16C; IC(50) values of approx. 4 and 1.3 microM, respectively); however, 2-bromopalmitate was found to be uncompetitive with respect to palmitoyl-CoA, whereas 16C was competitive. To seek additional evidence for multiple PATs, the effects of altering the assay conditions on the palmitoylation of MyrGCK(NBD) and FarnCNRas(NBD) were compared. PAT activity towards the two peptide substrates was modulated similarly by changing the ionic strength or incubation temperature, or by the addition of dithiothreitol. In contrast, the enzymic palmitoylation of the two peptides was differentially affected by N -ethylmaleimide and thermal denaturation. Overall, these data demonstrate that the enzymic palmitoylation of farnesyl- and myristoyl-containing peptide substrates can be differentiated, suggesting that multiple motif-specific PATs exist.

Discovery and Evaluation of Inhibitors of Human Ceramidase

The ceramide/sphingosine-1-phosphate (S1P) rheostat has been hypothesized to play a critical role in regulating tumor cell fate, with elevated levels of ceramide inducing death and elevated levels of S1P leading to survival and proliferation. Ceramidases are key enzymes that control this rheostat by hydrolyzing ceramide to produce sphingosine and may also confer resistance to drugs and radiation. Therefore, ceramidase inhibitors have excellent potential for development as new anticancer drugs. In this study, we identify a novel ceramidase inhibitor (Ceranib-1) by screening a small molecule library and describe the synthesis of a more potent analogue (Ceranib-2). In a cell-based assay, both compounds were found to inhibit cellular ceramidase activity toward an exogenous ceramide analogue, induce the accumulation of multiple ceramide species, decrease levels of sphingosine and S1P, inhibit the proliferation of cells alone and in combination with paclitaxel, and induce cell-cycle arrest and cell death. In vivo, Ceranib-2 was found to delay tumor growth in a syngeneic tumor model without hematologic suppression or overt signs of toxicity. These data support the selection of ceramidases as suitable targets for anticancer drug development and provide the first nonlipid inhibitors of human ceramidase activity. Mol Cancer Ther; 10(11); 2052–61. ©2011 AACR.

Cellular palmitoylation and trafficking of lipidated peptides.

Many important signaling proteins require the posttranslational addition of fatty acid chains for their proper subcellular localization and function. One such modification is the addition of palmitoyl moieties by enzymes known as palmitoyl acyltransferases (PATs). Substrates for PATs include C-terminally farnesylated proteins, such as H- and N-Ras, as well as N-terminally myristoylated proteins, such as many Src-related tyrosine kinases. The molecular and biochemical characterization of PATs has been hindered by difficulties in developing effective methods for the analysis of PAT activity. In this study, we describe the use of cell-permeable, fluorescently labeled lipidated peptides that mimic the PAT recognition domains of farnesylated and myristoylated proteins. These PAT substrate mimetics are accumulated by SKOV3 cells in a saturable and time-dependent manner. Although both peptides are rapidly palmitoylated, the SKOV3 cells have a greater capacity to palmitoylate the myristoylated peptide than the farnesylated peptide. Confocal microscopy indicated that the palmitoylated peptides colocalized with Golgi and plasma membrane markers, whereas the corresponding nonpalmitoylatable peptides accumulated in the Golgi but did not traffic to the plasma membrane. Overall, these studies indicate that the lipidated peptides provide useful cellular probes for quantitative and compartmentalization studies of protein palmitoylation in intact cells.

Improved synthesis of a fluorogenic ceramidase substrate

Substantial interest has focused on the roles of sphingolipid metabolizing enzymes in a variety of hyperproliferative and inflammatory diseases. A key family of enzymes involved in these pathologies is the ceramidases. Ceramidases cleave the pro-apoptotic lipid ceramide into a long-chain fatty acid and sphingosine, which can then be further metabolized to the mitogenic and inflammatory lipid sphingosine 1-phosphate. Consequently, development of ceramidase inhibitors would provide useful pharmacologic probes for further studies of sphingolipid metabolism, as well as lead compounds for drug development. This effort has been hampered by the lack of in vitro and cellular ceramidase assays that are amenable to high-throughput screening. Recently, a fluorogenic ceramide analog has been described as a substrate for use in ceramidase assays. The synthesis of this compound has now been substantially improved in terms of both the required effort and the overall yield of the process. Key improvements include: reduction in number of required steps, use of a hydroboration reaction; incorporation of a Mitsunobu reaction; improved acylation by the addition of triethylamine; together providing a fourfold increase in the overall yield. In addition, it has been demonstrated that the ceramide analog can be used in high-throughput assays to identify ceramidase inhibitors. Overall, the improved efficiency in the preparation of this ceramidase substrate should accelerate discovery efforts relating to sphingolipid metabolism.

Abstract 2533: Synergistic leukemic killing when combining a small molecule Pim protein kinase inhibitor and standard chemotherapeutic agents

The serine/threonine Pim protein kinases, originally cloned from a M-MuLV integration site, are up-regulated and associated with poor outcomes in multiple human hematologic malignancies including chronic lymphocytic leukemia, myeloid leukemia, diffuse large B-cell lymphoma, and T-cell lymphoma. We have developed novel benzylidene-thiazolidine-2, 4-diones inhibitors of Pim kinases that kill a wide range of both myeloid and lymphoid cell lines and shown that one of those inhibitors, SMI-4a inhibits subcutaneous tumor growth of T-cell acute lymphoblastic leukemia (T-ALL) in nude mice by an average of 47.9% (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2533.