A novel small molecule antagonist of choline kinase-α that simultaneously suppresses MAPK and PI3K/AKT signaling

Abstract

Choline kinase-α expression and activity are increased in multiple human neoplasms as a result of growth factor stimulation and activation of cancer-related signaling pathways. The product of choline kinase-α, phosphocholine, serves as an essential metabolic reservoir for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the production of lipid second messengers. Using in silico screening for small molecules that may interact with the choline kinase-α substrate binding domain, we identified a novel competitive inhibitor, N-(3,5-dimethylphenyl)-2-[[5-(4-ethylphenyl)-1H-1,2,4-triazol-3-yl]sulfanyl] acetamide (termed CK37) that inhibited purified recombinant human choline kinase-α activity, reduced the steady-state concentration of phosphocholine in transformed cells, and selectively suppressed the growth of neoplastic cells relative to normal epithelial cells. Choline kinase-α activity is required for the downstream production of phosphatidic acid, a promoter of several Ras signaling pathways. CK37 suppressed mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling, disrupted actin cytoskeletal organization, and reduced plasma membrane ruffling. Finally, administration of CK37 significantly decreased tumor growth in a lung tumor xenograft mouse model, suppressed tumor phosphocholine, and diminished activating phosphorylations of extracellular signal-regulated kinase and AKT in vivo. Together, these results further validate choline kinase-α as a molecular target for the development of agents that interrupt Ras signaling pathways, and indicate that receptor-based computational screening should facilitate the identification of new classes of choline kinase-α inhibitors.

Figure 1. Computational identification of a novel small molecule inhibitor of choline kinase-α, CK37

a. Molecular structure of CK37 and the secondary structure of choline kinase-α with CK37 (rod) depicted within the active site of the protein. b. Recombinant choline kinase activity assays were performed with 2μM ¹⁴C-choline chloride in the presence of 10, 25, 50, and 100μM CK37. Representative thin layer chromatography (t.l.c.) plate examining choline and phosphocholine levels with several concentrations of CK37. Data are represented as % of control activity for each CK37 concentration. Mean ± STD of three independent experiments. p < 0.05. c. Recombinant choline kinase activity assays were performed with different total choline concentrations (2, 10, 25, 50, 100, 150, and 200μM) in the presence or absence of 25μM CK37. Data are represented as % of control activity for each concentration of choline, and shown are mean ± STD from two separate experiments. p < 0.05.

Figure 2. CK37 exposure inhibits endogenous choline kinase-α activity and decreases levels of downstream choline metabolites

Hela cells were labeled with ¹⁴C-choline chloride treated with or without (a) several concentrations of CK37 or (b) siRNA silencing of choline kinase-α, and metabolites were analyzed by thin layer chromatography (t.l.c.). Shown is a representative t.l.c. plate examining choline and P-choline levels. Data are represented as % of control activity and mean ± STD of three independent experiments. p < 0.05. c. Intracellular phosphocholine levels from HeLa cells treated with vehicle or several concentrations of CK37 for 1, 6, or 12 hours were analyzed by 1-D NMR spectrometry. d. Phosphatidylcholine and phosphatidic acid levels were determined by lipidomic analysis from methanol extracted lipids from HeLa cells treated with different concentrations of CK37 for 12 hours.

Figure 3. CK37 decreases activating phosphorylations of ERK and AKT

a. Analysis of ERK1/2 and AKT activation in the presence of CK37 was performed by Western blot analysis. Representative immunoblot depicting p-ERK1/2 (T202/Y204), ERK1/2, p-AKT (S473), and AKT levels from two independent experiments. b. Densitometry analysis was performed to determine the phospho / total ratio for AKT, ERK1, and ERK2.

Figure 4. Inhibition of choline kinase by CK37 or specific siRNA silencing disrupts the actin cytoskeleton and causes ultrastructural changes in the plasma membrane

a. Immunofluorescence confocal microscopy was performed to analyze the effect of CK37 or siRNA silencing on the actin cytoskeleton arrangement in HeLa cells. Representative images for vinculin, phalloidin and merged staining from vehicle or 10μM CK37 treated cells or cells that were either transfected with scrambled control (Ctrl siRNA) or choline kinase-α specific siRNA (CK siRNA) for 48 hrs. b. Electron microscopy was performed to evaluate the membrane structure of HeLa cells in the presence of CK37 or choline kinase-specific siRNA. Represented are HeLa cell electron micrographs at 15000× and 44000× magnification from vehicle or 10μM CK37 treated samples, or cells that were either transfected with scrambled control (Ctrl siRNA) or choline kinase-α specific siRNA (CK siRNA) for 48 hrs.

Figure 5. CK37 selectively suppresses tumor cell proliferation and anchorage-independent growth

a. Cell proliferation assays were performed after 48 hours of treatment with or without CK37. Data are represented as % of cell growth of vehicle control as log₁₀ of CK37 from duplicate values in three independent experiments. b. Target specificity of CK37 is demonstrated by the loss of CK37 suppression of HeLa cell proliferation by over-expression of choline kinase-α. Data is represented as % of cell growth of vehicle control of each cell line for each concentration of CK37 from duplicate values from three independent experiments. p < 0.05. c. Anti-proliferation assays were performed on MDA-MB-231 breast tumor cells compared to primary human mammary epithelial cells (HMEC) to analyze the selective inhibition of CK37 for transformed versus normal cells. d. Densitometry analysis of soft-agar colony formation between different CK37 treated samples. Colonies from three separate one cm² boxes per plate were enumerated, and shown are mean ± STD colonies from two different experiments. * p < 0.5. e. Anchorage-independent growth of HeLa cells treated with several concentrations of CK37 was assessed by colony formation in soft agar. Shown are representative images from control, vehicle, 1μM CK37, or 5μM CK37 treated soft-agar plates from two separate experiments.

Figure 6. Intraperitoneal administration of CK37 suppresses tumor growth, phosphocholine levels, and activating phosphorylations of ERK and AKT in vivo

a. Tumors were measured daily using blunt-end Vernier calipers, and mice with established tumors were blindly randomized into either Vehicle (filled circles) or CK37 treatment (open circles) groups. Mice were administered daily intraperitoneal doses of either 50 μL DMSO (n=10) or 0.08 mg / g of CK37 (n=10) in 50 μL DMSO at the indicated time points. Data are presented as mean ± SEM. A significant difference (p < 0.01) was observed at day 2 of administration. b. Phosphocholine was measured in resected tumors by 1D-NMR analysis. Phosphocholine levels were normalized to the stable metabolite valine and to dry weight of the extracted tumor section. * p < 0.5 c. Tumors from vehicle or CK37 treated groups were analyzed by immunohistochemistry for p-ERK (T202/Y204) and p-AKT (S473). Shown are representative 400× field images of tumor sections from vehicle and CK37 treated groups as well as the corresponding isotype controls. Positive controls: phospho-ERK – testes, phospho-AKT – rat tail skin. d&e. Positive cells from three 400× image fields were quantified from three different tumors for both DMSO and CK37 treated groups. Shown is the mean expression score of p-ERK and p-AKT protein. The intensity of the immunoreactivity was graded as weakly positive (1), moderately positive (2), or strongly positive (3). * p < 0.5.