Downregulation of SK-1 induces endoreduplication.

<p>(A) MDA-MB-231 wt cells and SK-1 kd cells were stained with propidium iodide and DNA content was measured by flow cytometry using a FACSCalibur flow cytometer and the Cell Quest software for data processing. (B) confocal microscopy of MDA-MB-231 wt and SK-1 kd cells upon actin (red) and nuclear staining (blue) using TRITC-labeled phalloidin (dilution 1∶1000) and DAPI, respectively. (C) calculation of the nuclear size of SK-1 kd cells relative to wt cells. (D) quantification of cells with nuclei in different size ranges (<10 µM, >10 µM, >15 µM). Data are means ± SD of three independent experiments; **p<0.01, ***p<0.001 compared to MDA-MB-231 wt cells values.</p

Downregulation of SK-1 results in sphingosine accumulation.

<p>(A) MDA-MB-231, NCI-H358, and HCT 116 cells were transduced with lentiviral SK-1 shRNA to downregulate SK-1 expression (SK-1 kd) or left untreated (wild-type, wt). MDA-MB-231 cells transduced with empty virus or transfected with the SK-1 cDNA for overexpression (SK-1 ov) are shown for comparison. Cell lysates were prepared and SK-1 protein expression was detected by Western blotting using antibodies against SK-1 (dilution 1∶1000) or GAPDH (dilution 1∶2000) as loading control. The two SK-1 splice variants SK-1a and SK-1b run at 43 and 51 kDa, respectively. The films were digitized and for each protein lane a density blot was measured. Each value in the graph represents the mean ± SD band density for each group (<i>n = </i>3); ***p<0.001 compared to wt cells. (B) cellular sphingosine was determined in the genetically modified SK-1 kd carcinoma cell lines, in the respective wt cells, and additional MDA-MB-231 control cells as described above (empty virus transfected, SK-1 ov). Lipid extractions were performed and sphingosine was quantified by mass spectrometry. Data are means ± SD (<i>n = </i>3); *p<0.05, **p<0.01, ***p<0.001 compared to wt cells. (C) endogenous sphingosine in the wt carcinoma cell lines upon treatment with the SK-1 inhibitor SK I II (10 µM) or DMSO as vehicle control for 24 h. Quantification was done as above. Data are means ± SD (n = 3); *p<0.05, **p<0.01 compared to DMSO treated cells.</p

Downregulation of SK-1 increases sphingosine which inhibits PKC.

<p>(A) lysates were prepared from MDA-MB-231, NCI-H358, and HCT 116 wt and SK-1 kd cells, and analyzed for PKC activity by Western blotting using antibodies against various p(Ser) PKC substrates (dilution 1∶1000) or GAPDH (dilution 1∶2000) as loading control. MDA-MB-231 cells treated with sphingosine or DMSO vehicle control were analyzed for comparison. (B) cells were treated with the PKC inhibitors CGP 41-251 (300 nM) or Ro 31-8220 (1 µM) or with DMSO as control for 24 h before collection of lysates or quiescent MDA-MB-231 wt cells were pretreated for 4 h with the PKC inhibitors Ro 31-8220 (1 µM), CGP 41-251 (300 nM) or DMSO as control. Thereafter, cells were stimulated with the PKC activator TPA (200 nM) for 15 min. and lysates were analyzed for p(Ser) PKC substrates as described above. (C) quiescent MDA-MB-231 wt and SK-1 kd cells were stimulated with the PKC activator TPA (200 nM) for 15 min, treatment of SK-1 kd cells with lactacystin (20 µM for 24 h) was used to control for protein degradation. Lysates were analyzed for p(Ser) PKC substrates as described above. (D) MDA-MB-231 wt and SK-1 overexpressing (SK-1 ov) cells were analyzed for p(Ser) PKC substrates as described above.</p

Inhibition of PKC decreases colony formation of carcinoma cells.

<p>MDA-MB-231 wt cells were seeded in 60 mm diameter dishes at a density of 700 cells per dish in cell culture medium. After 24 h cells were treated with various concentrations of the PKC inhibitors CGP 41-251, Ro 31-8220 or with sphingosine, DMSO was used as vehicle control. Colony formation of SK-1 kd cells (untreated) incubated under identical conditions is shown for comparison. Cells were incubated for another 14 d before colonies were stained with 2% crystal violet and counted using a ColCountTM (Mammalian Cell Colony Counter, Oxford Optronix). Only colonies containing more than 50 cells were evaluated. Data are means ± S.D. (n = 3); *p<0.05, **p<0.01, ***p<0.001 compared to the DMSO control values.</p

Downregulation of SK-1 compromises M phase arrest and sensitizes cells to taxol.

<p>(A) MDA-MB-231 wt and SK-1 kd cells were treated with various concentrations of taxol or DMSO as vehicle control for 24 h before apoptosis was measured by annexin V staining using flow cytometry. Data are means ± SD (n = 3); ***p<0.001 compared to DMSO treated cells. (B)<b>,</b> detection of phospho-histone H3(Ser10) as a marker of the mitotic index in MDA-MB-231 wt and SK-1 kd cells at different time points upon treatment with taxol (100 nM). Lysates were subjected to Western blotting using antibodies against total histone H3 (dilution 1∶2000) and phospho-histone H3(Ser10) (dilution 1∶1000). The Density of phospho-histone H3(Ser10) (pH3)/H3 ratio in taxol-treated MDA-MB-231 cells is presented as % of total H3. Each value in the graph represents the mean ± SD band density for each group (<i>n = </i>3); ***p<0.001 compared to wt cells. (C) MDA-MB-231 wt cells were treated with taxol (100 nM), the SK-1 inhibitor SK I II (10 µM), sphingosine (20 µM) or the various combinations for 48 h. DMSO was used as vehicle control. Cell viability was determined in colorimetric MTT assays. Data are means ± S.D. (n = 3); *p<0.05, ***p<0.001 compared to DMSO treated cells; ^#p<0.05, ^###p<0.001 compared to the respective taxol treated cells; ^¥¥¥p<0.001 compared to the respective SK I II treated cells.</p

Downregulation of SK-1 inhibits cdc2 activity and decreases Chk1 expression.

<p>(A) lysates from MDA-MB-231 wt, SK-1 kd, and SK-1 overexpressing cells (SK-1 ov) were subjected to Western blotting using antibodies against total cdc2 (dilution 1∶2000), phospho-cdc2(Tyr15) (dilution 1∶1000), total cyclin B1, and phospho-cyclin B1(Ser133) (dilution 1∶1000). The films were digitized and for each protein lane a density blot was measured. Each value in the graph represents the mean ± SD band density for each group (<i>n = </i>3); ***p<0.001 compared to wt cells (B) MDA-MB-231 wt cells were treated with the cdc2 inhibitor RO 3306 (9 µM) or DMSO as vehicle control for 24 h, stained with propidium iodide and DNA content was measured as described in Fig. 4. (C) Chk1 mRNA expression in MDA-MB-231 wt and SK-1 kd cells was measured by quantitative real-time PCR and data were normalized to 18S rRNA. ***p<0.001 compared to wt values. (D) Chk1 protein expression in MDA-MB-231 wt and SK-1 kd cells detected by Western blotting using antibodies against Chk1 (dilution 1∶500) or GAPDH (dilution 1∶2000) as loading control. The films were digitized and for each protein lane a density plot was measured. Each value in the graph represents the mean ± SD band density for each group (<i>n = </i>3); **p<0.01 compared to wt cells.</p

7,8-Dichloro-1-oxo-β-carbolines as a Versatile Scaffold for the Development of Potent and Selective Kinase Inhibitors with Unusual Binding Modes

Development of both potent and selective kinase inhibitors is a challenging task in modern drug discovery. The innate promiscuity of kinase inhibitors largely results from ATP-mimetic binding to the kinase hinge region. We present a novel class of substituted 7,8-dichloro-1-oxo-β-carbolines based on the distinct structural features of the alkaloid bauerine C whose kinase inhibitory activity does not rely on canonical ATP-mimetic hinge interactions. Intriguingly, cocrystal structures revealed an unexpected inverted binding mode and the presence of halogen bonds with kinase backbone residues. The compounds exhibit excellent selectivity over a comprehensive panel of human protein kinases while inhibiting selected kinases such as the oncogenic PIM1 at low nanomolar concentrations. Together, our biochemical and structural data suggest that this scaffold may serve as a valuable template for the design and development of specific inhibitors of various kinases including the PIM family of kinases, CLKs, DAPK3 (ZIPK), BMP2K (BIKE), and others

5‑(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)­pyridin-2-amine (PQR309), a Potent, Brain-Penetrant, Orally Bioavailable, Pan-Class I PI3K/mTOR Inhibitor as Clinical Candidate in Oncology

Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound <b>1</b> (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for <b>1</b> in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, <b>1</b> did not bind tubulin, which was observed for the structurally related <b>4</b> (BKM120, buparlisib). Compound <b>1</b> is orally available, crosses the blood–brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound <b>1</b> demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compound’s safety profile, identifies <b>1</b> as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound <b>1</b> is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma

Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor

The discovery of inhibitors targeting novel allosteric kinase sites is very challenging. Such compounds, however, once identified could offer exquisite levels of selectivity across the kinome. Herein we report our structure-based optimization strategy of a dibenzodiazepine hit <b>1</b>, discovered in a fragment-based screen, yielding highly potent and selective inhibitors of PAK1 such as <b>2</b> and <b>3</b>. Compound <b>2</b> was cocrystallized with PAK1 to confirm binding to an allosteric site and to reveal novel key interactions. Compound <b>3</b> modulated PAK1 at the cellular level and due to its selectivity enabled valuable research to interrogate biological functions of the PAK1 kinase