Identification of N10-Substituted Phenoxazines as Potent and Specific Inhibitors of Akt Signaling
2005; Elsevier BV; Volume: 280; Issue: 36 Linguagem: Inglês
10.1074/jbc.m507057200
ISSN1083-351X
AutoresKuntebommanahalli N. Thimmaiah, John Easton, Glen S. Germain, Christopher L. Morton, Shantaram Kamath, John K. Buolamwini, Peter J. Houghton,
Tópico(s)Quinazolinone synthesis and applications
ResumoA series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 μm, 17 compounds inhibited insulin-like growth factor 1 (IGF-I)-stimulated phosphorylation of Akt (Ser-473) by at least 50% but did not inhibit IGF-I-stimulated phosphorylation of Erk-1/2 (Thr-202/Tyr-204). Substitutions at the 2-position (Cl or CF3) did not alter inhibitory activity, whereas N10-substitutions with derivatives having acetyl (20B) or morpholino (12B) side chain lost activity compared with propyl or butyl substituents (7B and 14B). Inhibition of Akt phosphorylation was associated with the inhibition of IGF-I stimulation of the mammalian target of rapamycin phosphorylation (Ser-2448 and Ser-2481), phosphorylation of p70 S6 kinase (Thr-389), and ribosomal protein S6 (Ser-235/236) in Rh1, Rh18, and Rh30 cell lines. The two most potent compounds 10-[4′-(N-diethylamino)butyl]-2-chlorophenoxazine (10B) and 10-[4′-[(β-hydroxyethyl)piperazino]butyl]-2-chlorophenoxazine (15B) (in vitro, IC50 ∼1-2 μm) were studied further. Inhibition of Akt phosphorylation correlated with inhibition of its kinase activity as determined in vitro after immunoprecipitation. Akt inhibitory phenoxazines did not inhibit the activity of recombinant phosphatidylinositol 3′-kinase, PDK1, or SGK1 but potently inhibited the kinase activity of recombinant Akt and AktΔPH, a mutant lacking the pleckstrin homology domain. Akt inhibitory phenoxazines blocked IGF-I-stimulated nuclear translocation of Akt in Rh1 cells and suppressed growth of Rh1, Rh18, and Rh30 cells (IC50 2-5 μm), whereas "inactive" derivatives were ≥10-fold less potent inhibitors of cell growth. In contrast to rapamycin analogs, Akt inhibitory phenoxazines induced significant levels of apoptosis under serum-containing culture conditions at concentrations of agent consistent with Akt inhibition. Thus, the cellular responses to phenoxazine inhibitors of Akt appear qualitatively different from the rapamycin analogs. Modeling studies suggest inhibitory phenoxazines may bind in the ATP-binding site, although ATP competition studies were unable to distinguish between competitive and noncompetitive inhibition. A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 μm, 17 compounds inhibited insulin-like growth factor 1 (IGF-I)-stimulated phosphorylation of Akt (Ser-473) by at least 50% but did not inhibit IGF-I-stimulated phosphorylation of Erk-1/2 (Thr-202/Tyr-204). Substitutions at the 2-position (Cl or CF3) did not alter inhibitory activity, whereas N10-substitutions with derivatives having acetyl (20B) or morpholino (12B) side chain lost activity compared with propyl or butyl substituents (7B and 14B). Inhibition of Akt phosphorylation was associated with the inhibition of IGF-I stimulation of the mammalian target of rapamycin phosphorylation (Ser-2448 and Ser-2481), phosphorylation of p70 S6 kinase (Thr-389), and ribosomal protein S6 (Ser-235/236) in Rh1, Rh18, and Rh30 cell lines. The two most potent compounds 10-[4′-(N-diethylamino)butyl]-2-chlorophenoxazine (10B) and 10-[4′-[(β-hydroxyethyl)piperazino]butyl]-2-chlorophenoxazine (15B) (in vitro, IC50 ∼1-2 μm) were studied further. Inhibition of Akt phosphorylation correlated with inhibition of its kinase activity as determined in vitro after immunoprecipitation. Akt inhibitory phenoxazines did not inhibit the activity of recombinant phosphatidylinositol 3′-kinase, PDK1, or SGK1 but potently inhibited the kinase activity of recombinant Akt and AktΔPH, a mutant lacking the pleckstrin homology domain. Akt inhibitory phenoxazines blocked IGF-I-stimulated nuclear translocation of Akt in Rh1 cells and suppressed growth of Rh1, Rh18, and Rh30 cells (IC50 2-5 μm), whereas "inactive" derivatives were ≥10-fold less potent inhibitors of cell growth. In contrast to rapamycin analogs, Akt inhibitory phenoxazines induced significant levels of apoptosis under serum-containing culture conditions at concentrations of agent consistent with Akt inhibition. Thus, the cellular responses to phenoxazine inhibitors of Akt appear qualitatively different from the rapamycin analogs. Modeling studies suggest inhibitory phenoxazines may bind in the ATP-binding site, although ATP competition studies were unable to distinguish between competitive and noncompetitive inhibition. Akt 1The abbreviations used are: Akt, oncogene from AKR mouse thymoma; IGF-1, insulin-like growth factor 1; Erk-1/2, extracellular signal-regulated kinase 1/2; mTOR, mammalian target of rapamycin; Rh, rhabdomyosarcoma; PI, phosphoinositide; PDK1/2, phosphoinositide 3-phosphate-dependent kinase 1/2; SGK1, serum glucocorticoid-regulated kinase 1; PH, pleckstrin homology domain; PTEN, phosphatase and tensin homolog; MDR, multidrug resistance; GSK-3, glycogen synthase kinase 3; Me2SO, dimethyl sulfoxide; p70 S6K, p70 ribosomal protein S6 kinase; AGC, protein kinase A, protein kinase G, protein kinase C; S6, ribosomal protein S6; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; MES, 4-morpholineethanesulfonic acid; Ptd, phosphatidylinositol.1The abbreviations used are: Akt, oncogene from AKR mouse thymoma; IGF-1, insulin-like growth factor 1; Erk-1/2, extracellular signal-regulated kinase 1/2; mTOR, mammalian target of rapamycin; Rh, rhabdomyosarcoma; PI, phosphoinositide; PDK1/2, phosphoinositide 3-phosphate-dependent kinase 1/2; SGK1, serum glucocorticoid-regulated kinase 1; PH, pleckstrin homology domain; PTEN, phosphatase and tensin homolog; MDR, multidrug resistance; GSK-3, glycogen synthase kinase 3; Me2SO, dimethyl sulfoxide; p70 S6K, p70 ribosomal protein S6 kinase; AGC, protein kinase A, protein kinase G, protein kinase C; S6, ribosomal protein S6; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; MES, 4-morpholineethanesulfonic acid; Ptd, phosphatidylinositol. mediates a variety of physiological responses, including the inhibition of apoptosis and promotion of cell survival (reviewed in Ref. 1Kandel E.S. 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Channu B.C. Dass C. Horton J.K. Houghton P.J. Thimmaiah K.N. Asian J. Chem. 1999; 11: 878-905Google Scholar) the chemistry and biology of N10-substituted phenoxazines synthesized originally as modulators of P-glycoprotein-mediated multidrug resistance (MDR). Some of these N10-substituted phenoxazines demonstrated significant cytotoxicity per se (hence were not studied further), whereas several derivatives enhanced vincristine toxicity in cells with undetectable levels of P-glycoprotein. From these results, we concluded that at least part of the activity of some of these phenoxazine MDR modulators is mediated through an unknown, but P-glycoprotein-independent, mechanism. As it is now established that Akt signaling protects against cellular stress, including cytotoxic agents, we have investigated whether phenoxazine derivatives inhibit Akt and induce apoptosis. We screened a number of phenoxazine derivatives for their effects on Akt activation in cells derived from pediatric cancers. From among these compounds, we report the identification of a small group of novel lead compounds, which at low micromolar concentrations specifically block Akt activation and downstream signaling to substrates such as mTOR, p70 S6 kinase, and ribosomal protein S6 (S6). These agents do not affect activation of the upstream kinases, PDK-1, PI 3-kinase, or other kinases downstream of ras such as Erk-1/2. Furthermore, at low micromolar concentrations, under normal growth conditions, these small molecule inhibitors induce apoptosis in rhabdomyosarcoma cells. All the chemicals and supplies were obtained from standard commercial sources unless otherwise indicated. Wortmannin was purchased from Calbiochem. Phenoxazine derivatives were prepared in pure form according to our procedures published previously (37Thimmaiah K.N. Horton J.K. Seshadri R. Israel M. Houghton J.A. Houghton P.J. J. Med. Chem. 1992; 35: 3358-3364Crossref PubMed Scopus (42) Google Scholar, 39Eregowda G.B. Kalpan H.N. Hegde R. Thimmaiah K.N. Indian J. Chem. 2000; 39: 243-259Google Scholar, 40Eregowda G.B. Krishnegowda G. Kalpana H.N. Channu B.C. Dass C. Horton J.K. Houghton P.J. Thimmaiah K.N. Asian J. Chem. 1999; 11: 878-905Google Scholar). Each inhibitor was dissolved in Me2SO before being added to culture medium (final concentration 0.1%). Cell Lines and Growth Conditions—The human cell lines Rh1, Rh18, and Rh30 have been described (41Hosoi H. Dilling M.B. Shikata T. Liu L.N. Shu L. Ashmun R.A. Germain G.S. Abraham R.T. Houghton P.J. Cancer Res. 1999; 59: 886-894PubMed Google Scholar). Briefly, Rh1, Rh18, or Rh30 cells were grown in antibiotic-free RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT) and 2 mm l-glutamine (BioWhittaker) at 37 °C in an atmosphere of 5% CO2. For serum-free experiments, cells were cultured in modified N2E (MN2E) medium (DMEM/F-12; 1:1 mixture) supplemented with 1 μg/ml human holotransferrin, 30 nm sodium selenite, 20 nm progesterone, 100 μm putrescine, 30 nm vitamin E phosphate, and 50 μm ethanolamine. Cells in MN2E medium containing 5 μg/ml bovine fibronectin (Sigma) were plated and allowed to attach overnight at 37 °C in a humidified 5% CO2 atmosphere. Cellular Screening for Inhibitors—Rh1, Rh18, or Rh30 cells were seeded at a density of 4 × 106/10-cm plate in serum-free medium for overnight attachment. Cells were exposed to 0.1% Me2SO or each of the phenoxazine derivatives for 1 h and then stimulated with IGF-I (10 ng/ml) for 10 min. Western Blot Analysis—Cells were rapidly washed with ice-cold phosphate-buffered saline (PBS), placed on ice, and lysed in mammalian protein extraction reagent (Pierce) containing one Complete™ mini protease inhibitor tablet (Roche Applied Science), 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, and 1 mm NaF. Cellular debris was pelleted by centrifugation at 17,500 × g for 10 min at 4 °C. Protein concentration of the supernatants was measured by the bicinchoninic acid assay using bovine serum albumin as the standard (Pierce). For the analysis of Akt, Erk-1/2, mTOR, p70 S6 kinase, ribosomal protein S6, and GSK-3, equivalent amounts of protein were separated on a 12% SDS-polyacrylamide gel (Bio-Rad) by electrophoresis and subsequently transferred to a nitrocellulose membrane (Bio-Rad). After a 1-h incubation in 1× TBS containing 0.05% Tween 20 and 5% blocking reagent (skim milk) (Upstate Biotechnology Inc., Lake Placid, NY) at room temperature, the wet nitrocellulose membranes were then incubated with the appropriate antibodies from Cell Signaling Technologies (Waltham, MA) at the dilutions indicated: rabbit polyclonal antiserum specific for the phosphorylated Ser-473 or Thr-308 of Akt (dilution 1:1000); rabbit polyclonal antiserum specific for the phosphorylated Thr-202/Tyr-204 of Erk-1/2 (dilution 1:1000); rabbit polyclonal anti-serum specific for the phosphorylated Ser-2448 or Ser-2481 of mTOR (dilution 1:1000); rabbit polyclonal antiserum specific for the phosphorylated Thr-389 of p70 S6 kinase (dilution 1:4000); rabbit polyclonal antiserum specific for the phosphorylated Ser-235/236 of S6 (dilution 1:1000); or rabbit polyclonal antiserum specific for the phosphorylated Ser-21/9 of GSK-3α/β (dilution 1:1000). The secondary antibody was the horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (dilution, 1:10,000). Immunoreactive protein was visualized by using Western Lightning chemiluminescence reagent (PerkinElmer Life Sciences). To ensure that equivalent amounts of protein were loaded on each gel, all immunoblots were treated with stripping buffer (62.5 mm Tris-HCl, pH 6.7; 2% SDS; and 100 mm β-mercaptoethanol) for 30 min at 50 °C and then incubated with one of the appropriate antibodies as follows: rabbit polyclonal antibody to Akt (dilution 1:1000; Cell Signaling Technology) or mouse monoclonal antibody to β-tubulin (dilution 1:2000; Sigma). The secondary antibodies and detection of bound antibody were as described above. Determination of Cellular Akt Kinase Activity—Rh1 cells were seeded in serum-free medium at a density of 4 × 106 per 10-cm plate. After 24 h, cells were exposed to Me2SO (0.1%) or phenoxazine at 5 μm for 1 h. Cells were then stimulated with ± IGF-I (10 ng/ml) for 10 min and washed once with ice-cold PBS. Cells were lysed in 200 μl of ice-cold 1× lysis buffer (20 mm Tris, pH 7.5; 150 mm NaCl; 1 mm EDTA; 1 mm EGTA; 1% Triton X-100; 2.5 mm sodium pyrophosphate; 1 mm β-glycerol phosphate; 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride; and 1 mm leupeptin) and incubated for 10 min on ice. The cell lysates were centrifuged for 10 min at 17,500 × g at 4 °C. The volumes of the supernatants were adjusted so that each sample contained an equal amount of protein (150 μg); the supernatants were then incubated with immobilized (cross-linked) anti-Akt antibody for 3 h at 4 °C. The immunoprecipitates were pelleted and washed twice in ice-cold cell lysis buffer and twice in kinase buffer (25 mm Tris, pH 7.5; 5 mm β-glycerol phosphate; 2 mm dithiothreitol; 0.1 mm Na3VO4; and 10 mm MgCl2). The pellets were suspended in 40 μl of kinase buffer containing 200 μm ATP and 1 μg of a GSK-3 fusion protein, which served as the substrate. After the suspensions were incubated at 30 °C for 30 min, the reaction was terminated by the addition of 3× SDS sample buffer (187.5 mm Tris-HCl, pH 6.8; 6% SDS; 30% glycerol; 150 mm dithiothreitol; and 0.03% bromphenol blue). The samples were boiled for 5 min, and the proteins were separated on a 12% SDS-polyacrylamide gel and subsequently transferred to a nitrocellulose membrane. Membranes were incubated with rabbit polyclonal anti-phospho-GSK-3α/β (Ser-21/9) antibody. In Vitro Inhibition of Recombinant Akt, AktΔPH, and SGK1—In vitro kinase assays were performed using active recombinant full-length Akt1/PKBα (Upstate Biotechnology, Inc.), active recombinant Akt lacking the pleckstrin homology domain, Akt1ΔPH (Upstate Biotechnology, Inc.), or active recombinant SGK1 (Upstate Biotechnology, Inc.). Briefly, 10 ng of recombinant enzyme in 25 μl of 1× kinase buffer (25 mm Tris, pH 7.5; 5 mm β-glycerol phosphate; 2 mm dithiothreitol; 0.1 mm Na3VO4; and 10 mm MgCl2), was mixed with 2.5 μl of Me2SO or phenoxazine derivative (5 μm). Samples were incubated on ice for 1 h at which time 1 μg of GSK-3 fusion protein, which served as the substrate, was added followed by ATP (200 μm) to each reaction mixture. After the suspensions were incubated at 30 °C for 30 min, the reaction was terminated by the addition of 3× SDS sample buffer (187.5 mm Tris-HCl, pH 6.8; 6% SDS; 30% glycerol; 150 mm dithiothreitol; and 0.03% bromphenol blue). The samples were boiled for 5 min, and the proteins were separated on a 12% SDS-polyacrylamide gel and subsequently transferred to a nitrocellulose membrane. Membranes were incubated with rabbit polyclonal anti-phospho-GSK-3α/β (Ser-21/9) antibody. Competition Experiments with ATP and Phenoxazines—Concentrations of compound 15B were prepared as 10× stocks in Me2SO ranging from 25 μm to 50 mm to give a final reaction concentration range of 2.5 μm to 5 mm. An ATP master mix was prepared containing 0.75 μl of [γ-33P]ATP (PerkinElmer Life Sciences NEG302H), 0.5 μl of 10 mm ATP, and 1.25 μl of 1× kinase buffer for each sample. An enzyme/substrate master mix was prepared containing 10 μl of 1× kinase buffer, 5 μl of Akt peptide substrate (670 ng/μl) (Upstate Biotechnology, Inc.), and 5 μl of active Akt (10 ng/μl) (Upstate Biotechnology, Inc.) diluted from stock using 1× kinase buffer. The reactions were set up by adding 2.5 μl of the phenoxazine to the bottom of the tube followed by the addition of 2.5 μl of ATP mix near the bottom of the tube. The reaction was initiated by the addition of 20 μl of the enzyme/substrate master mix. After the master mix was added to all of the tubes, the samples were placed at 30 °C for 30 min. After incubation the samples were centrifuged briefly and spotted onto phosphocellulose in the same order as the addition of the master mix. After 2 min these samples were added to a beaker with 0.75% phosphoric acid in the same order as above. The samples were washed for 5 min three times in 0.75% phosphoric acid followed by 5 min in acetone. The squares were then placed on Whatman paper and allowed to dry. The radioactivity was quantitated by scintillation counting. Molecular Modeling—Structures of inhibitors were built, and energy was minimized by using SYBYL 6.9.1 two-dimensional modeling software (version 6.9.1, Tripos Associates, St. Louis, MO). The GOLD program (version 2.1.2) (42Jones G. Willet P. Glen R.C. Leach A.R. Taylor R. J. Mol. Biol. 1997; 267: 727-748Crossref PubMed Scopus (5160) Google Scholar) from Cambridge Crystallographic Data Center, UK, was used to dock the inhibitors into the ATP site of Akt, the three-dimensional coordinates of which were imported from the Protein Data Bank (code 1O6K). GOLD uses a genetic algorithm to explore ligand conformational flexibility. The program also optimizes the torsion angles of serine and threonine hydroxyl groups as well as lysine NH+3 groups to achieve limited receptor flexibility. The ATP site was defined as residues lying within 15 Å of Thr-292. Up to 10 different docking solutions were obtained for each molecule, the docking being terminated when the top three solutions were within a root mean square deviation of ≤1.5. Photoaffinity Labeling of Recombinant Akt—The photoaffinity labeling experiments were performed on ice in MES buffer (20 mm MES, pH 6.1) using 200 ng of recombinant active Akt (Upstate Biotechnology, Inc.) and 20 μm 8-azidoadenosine 5′-[α-32P] triphosphate (8N3[α-32P]ATP) (Altcorp) in a total reaction volume of 25 μl. All of the samples, including samples containing phenoxazine 15B (200 μm), were incubated on ice for 30 min. After 30 min of incubation, 8N3[α-32P]ATP was added and allowed to bind for 1 min. In the sample containing ATP (200 μm or 1 mm), the ATP was premixed with 8N3[α-32P]ATP prior to its addition to the reaction. Immediately after binding, the samples were photocross-linked using a handheld model UVLS-28 (UVP) UV lamp set at 254 nm at a distance of 4 cm from the sample surface for 1 min. After cross-linking, 15 μl of 3× SDS sample loading buffer was added to each sample. The entire sample was electrophoresed on a 4-20% Tris glycine gel followed by one rinse with H2O and overnight fixation (50% MeOH, 40% H2O, 10% acetic acid). After fixation the gel was washed two times for 30 min with H2O. The bands were detected using a STORM 860 PhosphorImager (Amersham Biosciences). To verify even loading of Akt, the gels were stained with Coomassie Brilliant Blue (Sigma). PI 3-Kinase Assay—20 ng of recombinant p110γ enzyme (AG Scientific, San Diego, CA), Me2SO (5 μl), phenoxazine derivative (5 μm), or wortmannin (5 μm) were placed on ice for 1 h in 100 μl of 1× kinase buffer (10 mm Tris, pH 7.4; 100 mm NaCl; and 5 mm MgCl2). 10 μg of phosphatidylinositol (Sigma) was then added to each sample, and the incubation was continued on ice for an additional 15 min. ATP (final concentration of 25 μm containing 30 μCi of [γ-32P]ATP) was added to each sample, and the reaction mixtures were incubated at 37 °C for 10 min. Reactions were terminated by adding 20 μl of 6 n hydrochloric acid. The sample was vortexed, and the lipids were extracted into 300 μl of MeOH/CHCl3 (1:1) mixture. After mixing gently and spinning at 10,000 × g for 5 min, 50 μl of the organic phase was spotted onto a silica-coated TLC plate (EMD Biosciences Inc., La Jolla, CA) and developed using a solvent system containing CHCl3/MeOH/H2O/NH4OH (60:47:11.3:2). The TLC plate was allowed to dry, and the bands were analyzed using a Storm 860 PhosphorImager (Amersham Biosciences). PDK1 Kinase Assays—In vitro PDK1 activity assays were performed using a PDK1 assay kit (Upstate Biotechnology, Inc.) with a slight modification of the manufacturer's instructions. Briefly, 10 ng of recombinant PDK1 enzyme, Me2SO (5 μl), or phenoxazines (5 μm) were incubated in 80 μl of 1× PDK assay dilution buffer on ice. After 1 h, 100 ng of SGK1 was added to each sample and incubated on ice for an additional 10 min. The samples were transferred to a 30 °C water bath and incubated for an additional 15 min. Then SGK1 substrate peptide (245 μm) followed by ATP (40 μm containing 10 μCi of [γ-32P]ATP) were added, and the reaction mixture was gently vortexed. Samples were incubated at 30 °C for 15 min with a gentle vortexing every 2 min. Samples were centrifuged, and 40 μl of the reaction mixture was spotted onto the center of a PE 81 phosphocellulose paper square. After 30 s, the filter was washed four times with 0.75% phosphoric acid and twice with acetone. The filter was drained and transferred into a scintillation vial to which 5 ml of scintillation mixture was added. The amount of incorporated radioactivity into the substrate was determined by scintillation counting. The assay for SGK1 kinase activity was performed as described above for the PDK1 assay. Translocation of Akt in Rh1 Cells—Rh1 cells (2 × 105 per chamber) were grown on 2-well glass chamber slides (Falcon, Franklin Lakes, NJ) in serum-free medium containing fibronectin
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