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Blood, Vol. 106, Issue 4, 1432-1440, August 15, 2005
Sequential activation of class IB and class IA PI3K is important for the primed respiratory burst of human but not murine neutrophils
Blood Condliffe et al.
106: 1432
Supplemental materials for: Condliffe et al, Vol 106, Issue 4, 1432-1441
Files in this Data Supplement:
- Table S1. Comparison of PI3K inhibitors in identical in vitro assays (PDF, 56KB) -
Measurement of compound IC50s against the 4 class I PI3K isoforms was performed using a commercially available homogeneous time-resolved fluorescence assay (Upstate), as described previously.1 All assays were performed at 2 µM ATP and a concentration of purified class I PI3K known to generate product within the linear range of the assay. PI3Kα (accession number [acc no.] U79143)2 and PI3Kβ possessing a C-terminal His-tag were produced as heterodimers with p85α in baculovirus-infected High 5 cells and purified by nickel-affinity chromatography followed by anion-exchange chromatography. The PI3-kinase δ/p85 heterodimer was purchased from Upstate, and PI3Kγ was produced according to previously published methods.3
- Figure S1. Structures of PI3K inhibitors (PDF, 39.9 KB) -
Synthesis of AS-252424 (5-[5-(4-Fluoro-2-hydroxy-phenyl)-furan-2-ylmethylene]-thiazolidine-2,4-dione). Intermediate 1: 2-benzyloxy-1-bromo-4-fluoro-benzene (1) 2-Bromo-5-fluorophenol (1.16 mL; 10 mM) was dissolved in THF (25 mL) and potassium carbonate (2.89 g; 21 mM) was added at room temperature and stirred for 10 minutes. Benzyl bromide (1.5 mL; 12.6 mM) was then added slowly, and the reaction was left overnight at 25°C. The reaction mixture was hydrolyzed and extracted with ethylacetate and washed several times with Na2CO3 aqueous (aq). The crude was purified by flash chromatography using cyclohexane as eluent, affording 2.0 g (68%) title compound (1) in 98% purity. Intermediate 2: 5-(2-Benzyloxy-4-fluoro-phenyl)-furan-2-carbaldehyde (2). 2-(Benzyloxy)-1-bromo-4-fluorobenzene (1) (1.6 g; 5.7 mM) was dissolved in toluene (100 mL) and ethanol (25 mL) at 80°C. Reaction was degassed with nitrogen for 10 minutes prior to addition of triphenylphosphine-tetrakis-palladium (0.6 g). Potassium carbonate (5.5 mL; 2.00 m; 11 mM) was then added, and the reaction was left for 5 minutes. A solution of 5-formyl-2-furylboronic acid (880 mg; 6.2 mM) dissolved in 30 mL toluene/ethanol (4:1) was then added at 80°C for 3.5 hours, and the reaction was left at 80°C for one night. The crude reaction mixture was purified by flash chromatography using cyclohexane/ethyl acetate (9:1) to give 0.6 g (36%) of a yellow solid in 91% purity. Intermediate 3: 5-(4-fluoro-2-hydroxy-phenyl)-furan-2-carbaldehyde (3). 600 mg of 5-[2-(benzyloxy)-4-fluorophenyl]-2-furaldehyde; 2 mM) (2) was dissolved in DCM (250 mL) and cooled to –70°C under nitrogen. Boron trichloride (3.65 mL; 1.00 M; 3.6 mM) was then added slowly at –70°C, and the reaction was stirred at –70°C for 30 minutes. Reaction was finished after 40 minutes. The crude was hydrolised and extracted with dichloromethane. The organic layer was extracted with K2CO3 aq (30 mL) several times. The water layer was neutralized slowly with citric acid (25%) to pH 7. Ethylacetate was added, and the product was extracted with ethylacetate several times to afford a colorless solid (200 mg), which was used directly for the next step without any further purification. (5-[5-(4-fluoro-2-hydroxy-phenyl)-furan-2-ylmethylene]-thiazolidine-2,4-dione), (AS-252424): 5-(4-fluoro-2-hydroxyphenyl)-2-furaldehyde (200 mg; 0.97 mM) (3), 2,4-thiazolidinedione (227 mg; 1.9 mM) and β-alanine (172 mg; 1.9 mM) were heated at 90°C in AcOH (5 mL) for 2 hours. Water was added, and the corresponding brown precipitate was left in the refrigerator at 4°C and then filtered, washed with water, and dried with ether, affording AS-252424 of 96% purity. The parent compound was suspended in THF/water, and 1.05 mL KOH (1 M) was added while the solution cleared. After lyophilisation, 203 mg as a red powder of bis-potassium salt of AS-252424 was obtained (82%). LC/MS: [M-1]-: 304; [M+1]+:306. 1H NMR (DMSO-d6) d9.82 (s, 1H), 7.60 (s, 1H), 7.55 (d, J = 8.7 Hz, 1H), 7.18 (d, J = 6 Hz, 1H), 6.99 (d, J = 6 Hz, 1H), 6.46 (d, J = 8.7 Hz, 1H).
- Figure S2. Effects of PI3K inhibitors on the levels of [32P] phosphoinositides in fMLP-stimulated, TNF-α–primed human neutrophils (PDF, 127 KB) -
See Figure 2 legend in the main article. The data are expressed as a percentage of the value with no inhibitor, with no unstimulated values subtracted; inspection of the data in Figure 1 will allow a comparison between unstimulated and fMLP-stimulated samples. For example, PtdIns(3,4)P2 values at 6seconds are only approximately double the unstimulated values, so substantial reduction in the levels of this lipid in the presence of the relevant compounds at this time must reflect inhibition of both basal and fMLP-stimulated generation of this lipid.
- Figure S3. Time course of changes in the levels of [32P] phosphoinositides in fMLP-stimulated, TNF-α–primed mouse neutrophils (PDF, 96 KB) -
See Figure 5 legend in the main article. The human neutrophil experiments gave excellent separations of the relevant GroPInsPs, but the GroPIns(3,4)P2 data collected from mouse samples were compromised by the presence of an unusually high amount of material in the control samples, possibly reflecting other compounds running in the same region of the gradient.
- Figure S4. Expression of PI3Kδ in various tissues (PDF, 83 KB) -
Twenty micrograms of protein from lysates derived from human peripheral blood T cells (lane 1), wild-type mouse thymocytes (lane 2), wild-type mouse lymph nodes (lane 3), human peripheral blood neutrophils from 2 separate donors (lanes 4 and 5), wild-type mouse bone marrow neutrophils (lane 6), wild-type mouse peritoneal neutrophils (lane 7), and PI3Kδγ–/– bone marrow neutrophils (lane 8) were analyzed by Western blotting using an antibody to PI3Kδ. The membrane was stripped and reprobed with antibody to GAPDH to demonstrate equal protein loading. Murine neutrophils were harvested from the peritoneal cavity by lavage with PBS 6 hours after the intraperitoneal injection of 0.25 mL 4% thioglycollate; purity was enhanced to 90% by centrifugation over Percoll gradients, as described in “Materials and methods.” Murine thymocytes and lymphocytes were obtained by disruption of freshly isolated thymus and lymph node tissue.
- Figure S5. Effect of PI3K inhibitors on oxidase activity in mouse neutrophils (PDF, 25 KB) -
See the legend to Figure 7 in the main article. Bone marrow neutrophils obtained from wild-type (closed triangle) and PI3KδD910A/D910A (open triangle) mice were prepared in parallel, primed with 500 U/mL TNF-α and pre-incubated with luminol and HRP in the presence of 0 to 10 µM of the indicated inhibitors, as described in “Materials and methods.” We stimulated 3.75 × 105 cells per well with 10 µM fMLP and recorded chemiluminescence using a Berthold MicroLumat Plus luminometer. Data represent the accumulated light emission over 3 minutes and are expressed as a percentage of the value with no inhibitor (mean ± SEM from 3 separate experiments).
1Gray A, Olsson H, Batty I, Priganica L, Downes CP. Nonradioactive methods for the assay of phosphoinositide 3-kinases and phosphoinositide phosphatases and selective detection of signalling lipids in cell and tissue extracts. Anal Biochem. 2003;313:234-245.
2Stirdivant SM, Ahern J, Conroy RR, et al. Cloning and mutagenesis of the p110 alpha subunit of human phosphoinositide 3'-hydroxykinase. Bioorg Med Chem. 1997;5:65-74.
3Walker EH, Pacold ME, Perisic O, et al. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercitin, myricetin and staurosporine. Mol Cell. 2000;6:909-919.
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