Identification of N10-substituted phenoxazines as potent and specific inhibitors of Akt signaling.

A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 mum, 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'-[(beta-hydroxyethyl)piperazino]butyl]-2-chlorophenoxazine (15B) (in vitro, IC50 approximately 1-2 microM) 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 Akt deltaPH, 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 microM), whereas "inactive" derivatives were > or = 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 1 mediates a variety of physiological responses, including the inhibition of apoptosis and promotion of cell survival (reviewed in Ref. 1). Three members, Akt1, Akt2, and Akt3, have been identified in this subfamily, and extensive evidence has demonstrated the crucial role of Akt1 as a central player in tumorigenesis (2). The Ser/Thr-protein kinase Akt is a downstream target of PI 3-kinase (2,3). PI 3-kinase phosphorylates the D-3-hydroxyl position of the myo-inositol ring of PtdIns(4) to generate the PtdIns 3-phosphates, PtdIns(3)P, PtdIns(3,4)P 2 , and PtdIns(3,4,5)P 3 (5). PI 3-kinase is activated by many growth factor receptors and oncogenic protein-tyrosine kinases (6 -8), as well as by Ras (9), leading to increased cell growth and inhibition of apoptosis (10,11). PI 3-kinase expression is increased in ovarian cancer (12), and it is constitutively activated in human small cell lung cancer cell lines, where it leads to anchorage-independent growth and has been suggested to be a cause of metastasis (13). However, the major role for PI 3-kinase in cancer cell growth is its role in survival signaling mediated by Akt to prevent apoptosis (14). Akt is overexpressed in gastric adenocarcinoma (15), breast cancer (16), ovarian cancer (16,17), pancreatic cancer (18), estrogen receptor-deficient breast cancer, and in androgen-independent prostate cell lines (19). Activation of Akt is negatively regulated by the tumor suppressor protein, PTEN, a tyrosine-threonine/lipid phosphatase that dephosphorylates the 3-position of PtdIns-3-phosphate (20,21), thus inhibiting the PI 3-kinase/Akt signaling pathway (20,22). Mutations in PTEN, which lead to activation of the Akt pathway, have been identified in various tumors (23,24). Furthermore, activation of Akt has been shown to associate with tumor invasiveness and chemoresistance (25).
The design and development of small molecules that specifically inhibit the kinase activity of Akt and its signal transduction pathway in cancer cells are thus attractive approaches for the development of new cancer therapeutic agents. Currently, considerable effort is being placed into the development of novel specific inhibitors of Akt. The compounds include a number of phosphatidylinositol (PI) analogs (26 -29), H-89 analogs (30), azapane derivatives (31), peptide inhibitors (32), Akt/ protein kinase B signaling inhibitor-2 (33), and compounds containing planar aromatic heterocyclics (34). This latter group includes phenothiazine derivatives. Isozyme-selective inhibitors have been described and tested for their ability to inhibit both PI 3-kinase and Akt activities (35).
Previously, we have reported (36 -40) the chemistry and biology of N 10 -substituted phenoxazines synthesized originally as modulators of P-glycoprotein-mediated multidrug resistance (MDR). Some of these N 10 -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.

MATERIALS AND METHODS
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 (37,39,40). Each inhibitor was dissolved in Me 2 SO before being added to culture medium (final concentration 0.1%).
Cellular Screening for Inhibitors-Rh1, Rh18, or Rh30 cells were seeded at a density of 4 ϫ 10 6 /10-cm plate in serum-free medium for overnight attachment. Cells were exposed to 0.1% Me 2 SO 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 TM mini protease inhibitor tablet (Roche Applied Science), 1 mM phenylmethylsulfonyl fluoride, 1 mM Na 3 VO 4 , and 1 mM NaF. Cellular debris was pelleted by centrifugation at 17,500 ϫ 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).
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 ␤-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 ␤-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 ϫ 10 6 per 10-cm plate. After 24 h, cells were exposed to Me 2 SO (0.1%) or phenoxazine at 5 M for 1 h. Cells were then stimulated with Ϯ IGF-I (10 ng/ml) for 10 min and washed once with ice-cold PBS. Cells were lysed in 200 l of ice-cold 1ϫ 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 ␤-glycerol phosphate; 1 mM Na 3 VO 4 , 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 ϫ g at 4°C. The volumes of the supernatants were adjusted so that each sample contained an equal amount of protein (150 g); 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 ␤-glycerol phosphate; 2 mM dithiothreitol; 0.1 mM Na 3 VO 4 ; and 10 mM MgCl 2 ). The pellets were suspended in 40 l of kinase buffer containing 200 M ATP and 1 g 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 3ϫ 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-3␣/␤ (Ser-21/9) antibody.
In Vitro Inhibition of Recombinant Akt, Akt⌬PH, and SGK1-In vitro kinase assays were performed using active recombinant full-length Akt1/PKB␣ (Upstate Biotechnology, Inc.), active recombinant Akt lacking the pleckstrin homology domain, Akt1⌬PH (Upstate Biotechnology, Inc.), or active recombinant SGK1 (Upstate Biotechnology, Inc.). Briefly, 10 ng of recombinant enzyme in 25 l of 1ϫ kinase buffer (25 mM Tris, pH 7.5; 5 mM ␤-glycerol phosphate; 2 mM dithiothreitol; 0.1 mM Na 3 VO 4 ; and 10 mM MgCl 2 ), was mixed with 2.5 l of Me 2 SO or phenoxazine derivative (5 M). Samples were incubated on ice for 1 h at which time 1 g of GSK-3 fusion protein, which served as the substrate, was added followed by ATP (200 M) to each reaction mixture. After the suspensions were incubated at 30°C for 30 min, the reaction was terminated by the addition of 3ϫ 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-3␣/␤ (Ser-21/9) antibody.
Competition Experiments with ATP and Phenoxazines-Concentrations of compound 15B were prepared as 10ϫ stocks in Me 2 SO ranging from 25 M to 50 mM to give a final reaction concentration range of 2.5 M to 5 mM. An ATP master mix was prepared containing 0.75 l of [␥-33 P]ATP (PerkinElmer Life Sciences NEG302H), 0.5 l of 10 mM ATP, and 1.25 l of 1ϫ kinase buffer for each sample. An enzyme/ substrate master mix was prepared containing 10 l of 1ϫ kinase buffer, 5 l of Akt peptide substrate (670 ng/l) (Upstate Biotechnology, Inc.), and 5 l of active Akt (10 ng/l) (Upstate Biotechnology, Inc.) diluted from stock using 1ϫ kinase buffer. The reactions were set up by adding 2.5 l of the phenoxazine to the bottom of the tube followed by the addition of 2.5 l of ATP mix near the bottom of the tube. The reaction was initiated by the addition of 20 l 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) (42) 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 ϩ 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 Յ1.5.
Photoaffinity ATP was added and allowed to bind for 1 min. In the sample containing ATP (200 M or 1 mM), the ATP was premixed with 8N 3 [␣-32 P]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 l of 3ϫ 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 H 2 O and overnight fixation (50% MeOH, 40% H 2 O, 10% acetic acid). After fixation the gel was washed two times for 30 min with H 2 O. 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 p110␥ enzyme (AG Scientific, San Diego, CA), Me 2 SO (5 l), phenoxazine derivative (5 M), or wortmannin (5 M) were placed on ice for 1 h in 100 l of 1ϫ kinase buffer (10 mM Tris, pH 7.4; 100 mM NaCl; and 5 mM MgCl 2 ). 10 g 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 M containing 30 Ci of [␥-32 P]ATP) was added to each sample, and the reaction mixtures were incubated at 37°C for 10 min. Reactions were terminated by adding 20 l of 6 N hydrochloric acid. The sample was vortexed, and the lipids were extracted into 300 l of MeOH/CHCl 3 (1:1) mixture. After mixing gently and spinning at 10,000 ϫ g for 5 min, 50 l 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 CHCl 3 /MeOH/H 2 O/NH 4 OH (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, Me 2 SO (5 l), or phenoxazines (5 M) were incubated in 80 l of 1ϫ 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 M) followed by ATP (40 M containing 10 Ci of [␥-32 P]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 l 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 ϫ 10 5 per chamber) were grown on 2-well glass chamber slides (Falcon, Franklin Lakes, NJ) in serum-free medium containing fibronectin (10 g/ml). After 20 h, cells were exposed to Me 2 SO (0.1%, vehicle control) or phenoxazine derivative (5 M) for 1 h and then stimulated with IGF-I (10 ng/ml) for 20 min. Cells were washed twice with PBS and fixed in 4% formaldehyde for 30 min at room temperature. The samples were then rinsed twice with PBS and permeabilized with 1% Triton X-100 for 5 min at room temperature. After rinsing twice with PBS, the cells were incubated with an anti-Akt antibody (Rockland, West Chester, PA; 1:50 dilution) for 45 min at 37°C. After rinsing three times with PBS, the slides were then incubated with an anti-IgG rabbit secondary antibody coupled to Alexa 488 (Molecular Probes, Eugene, OR) at a dilution of 1:50. The slides were washed and incubated with RNase. After rinsing twice with PBS, the slides were mounted in media containing TOPRO-3 (Molecular Probes, Eugene, OR) and then analyzed on a Leica confocal microscope.
Cell Growth Inhibition-Rh1, Rh18, and Rh30 cells at a density of 6000, 50,000 and 10,000 cells, respectively, were plated per well in 6-well flat-bottom tissue culture plates (Falcon) in complete medium. After 24 h at 37°C, medium was replaced with fresh medium containing Me 2 SO (0.1%) or phenoxazines at concentrations ranging from 100 nM to 25 M. The cells were further incubated for 6 days. Growth was assessed after lysing cells and counting nuclei. All measurements were made in triplicate.
Determination of Apoptosis-We used the ApoAlert TM annexin V-FITC apoptosis kit (Clontech) to evaluate the extent of apoptosis within cell populations. Cells (Rh1, 350,000 per 75-cm 2 flask; Rh18, 800,000 per 75-cm 2 flask; or Rh30, 500,000 per 75-cm 2 flask) were grown overnight in complete medium. On day 1, cells were treated with Me 2 SO (0.1%; vehicle control) or phenoxazine derivative. After 4 days, the cells were trypsinized, washed with PBS, and resuspended in 200 l of binding buffer. Cells were incubated with 10 l of annexin V-FITC (final concentration, 1 g/ml) and 500 ng of propidium iodide in a final volume of 410 l. Cells were incubated at room temperature in the dark for 10 min before flow cytometric analysis (FACSCalibur, BD Biosciences) was performed as described (41).

Phenoxazine Derivatives Inhibit Akt Phosphorylation in
Cells-In order to identify novel inhibitors of Akt, phenoxazines shown in Table I, were investigated to determine whether they would inhibit the phosphorylation of Akt (Ser-473 or Thr-308) in cancer cells. For these assays, serumstarved Rh1 cells were exposed to 1-5 M phenoxazine derivatives for 1 h before stimulating with IGF-I (10 ng/ml) for 10 min. Akt or Erk-1/2 phosphorylation was detected using the phospho-specific anti-Akt antibody or anti-Erk-1/2 antibody. As anticipated, IGF-I stimulated phosphorylation of Akt (Ser-473) and Erk-1/2 (Thr-202/Tyr-204) but had no effect on the overall protein levels of Akt or Erk-1/2. Initially, we screened compounds 4B, 5B, 10B, and 15B at 5 M (Fig. 1A); subsequently, an additional 18 2-chlorophenoxazines were screened (Fig. 1B). Ring unsubstituted phenoxazines and 2-triflourophenoxazine derivatives were examined at both 1 and 5 M (Fig. 1C). As shown in Fig. 1, A-C, to varying degrees, all of the compounds except 5C, 2B, 5B, 9B, 12B, and 16B-21B inhibited the phosphorylation of Akt at Ser-473 at a concentration of 5 M (summarized in Table I). In contrast, none of the phenoxazines inhibited IGF-I-stimulated phosphorylation of Erk-1/2. These data imply that the phenoxazines are not inhibiting the IGF-I receptor, insulin receptor substrate proteins, or PI 3-kinase, as these pathways are necessary for IGF-I-mediated activation of Erk-1/2. Further examination of the data reveals that the inhibition of Akt follows the order butyl Ͼ propyl series. Of note was that the morpholino and acetyl derivatives of phenoxazine exhibited little or no inhibition of cellular Akt activation at the concentrations examined. To determine the concentration of the various phenoxazines where Akt phosphorylation is inhibited, cells grown under serum-free conditions were exposed to phenoxazines at 1, 2.5, or 3.5 M, and phospho-Akt was detected after stimulating with IGF-I. The results revealed that 1 M caused about 60% and 3.5 M caused maximum inhibition for most of the compounds. However, compounds 10B and 15B showed complete inhibition at 2.5 M (data not shown). Thus, further studies concentrated upon the two most active compounds, 10B and 15B.
To determine whether these effects were general, the studies were extended to other cell lines. Rh18 or Rh30 rhabdomyosarcoma cells grown in serum-free medium were exposed to compounds 10B, 14B, 15B, or 20B at 5 M for 1 h and then stimulated with IGF-I for 10 min. Phospho-Akt (Ser-473), phospho-mTOR (Ser-2448 or Ser-2481), or phospho-S6 (Ser-235/236) was checked using the respective phospho-specific antibody as shown in Fig. 2C for Rh18 or Fig. 2D for Rh30 cells. In both cell lines, the IGF-I-induced phosphorylation of Akt, mTOR, and S6 was effectively blocked by all of the compounds except 20B. After the membrane was stripped of bound antibody, it was incubated with the anti-Akt antibody to determine the total amount of Akt. The data confirm that equal amounts of protein were loaded. Taken together, these results further confirm that Akt-mediated activation of mTOR/p70 S6 kinase/S6 pathway in three cancer cell lines is blocked by phenoxazine derivatives.
Phenoxazine Derivatives Inhibit Akt Kinase Activity in Cells-These studies strongly suggest that phenoxazines inhibit Akt activation. To determine this directly, we studied the effects of compounds 10B and 15B on the Akt kinase activity in cells. Activation of Akt by IGF-I was evaluated by assessing the phosphorylation of Akt (Ser-473) or the in vitro kinase activity of protein immunoprecipitated by the anti-Akt antibody. To determine whether changes in Akt phosphorylation were correlated with alterations in kinase activity, we examined the phosphorylation status of a target downstream from Akt, e.g. GSK-3␤. Rh1 cells grown in serum-free medium were exposed to 0.1% Me 2 SO or 5 M of 10B or 15B for 1 h and then stimulated with IGF-I for 10 min. Cell lysates were then immunoprecipitated with immobilized Akt antibody. Immunoprecipi-   (Fig. 3). These results indicate that phenoxazines effectively block the activity of endogenous Akt in Rh1 cells.
Phenoxazines Do Not Inhibit PI 3-Kinase Activity in Vitro-As discussed above, the finding that the phenoxazine derivatives do not inhibit IGF-I-induced phosphorylation of Erk1/2 implies that they do not inhibit PI 3-kinase. However, cells treated with phenoxazines 10B or 15B exhibit many of the effects observed in cells treated with PI 3-kinase inhibitors such as wortmannin (data not shown). This is not surprising because PI 3-kinase is required for both the association of Akt with the cell membrane via the PH domain of Akt and activation of the Akt kinase function through the phosphorylation of Ser-308 by the 3-phosphoinositide-dependent protein kinase PDK1. To verify that the phenoxazines were not targeting PI 3-kinase, we performed in vitro kinase assays using recombinant enzyme. By using phosphatidylinositol (PI) as the substrate and [␥-32 P]ATP as the phosphate donor, the kinase activity of samples treated with wortmannin, 10B, or 15B at 5 M was compared with an untreated sample. The lipids were resolved by thin layer chromatography and quantitated using a PhosphorImager. As expected, the untreated sample showed robust phosphorylation of PI as can be seen by the levels of phosphatidylinositol 3-phosphate detected (Fig. 4, 1st lane). The PI 3-kinase activity in samples incubated with 10B or 15B was comparable with the untreated sample (Fig. 4, 2nd and 3rd  lanes), whereas the wortmannin-treated sample had barely detectable levels of activity as shown by the lack of visible phosphatidylinositol phosphate (Fig. 4, 4th lane) and the low level of detectable counts. These results indicate that the phenoxazines do not inhibit the activity of PI 3-kinase.
Phenoxazines Do Not Inhibit the Kinase Activity of PDK-1-Although direct inhibition of Akt by the phenoxazines would be consistent with the observed effects on the Akt signaling pathway, inhibition of PDK1 and the consequent failure of PDK1 to phosphorylate and activate Akt would also be consistent with these effects. In addition, both Akt and PDK1 are members of the AGC family of serine-threonine kinases. To address the question of whether or not PDK1 was inhibited by 10B or 15B, an in vitro coupled-kinase assay was performed. Recombinant PDK1 was preincubated for 1 h with phenoxazines 10B, 15B, or 0.1% Me 2 SO (control). After this preincubation, the PDK1 substrate SGK1 was added along with ATP and incubated for 15 min followed by the addition of an SGK1 substrate peptide and [␥-32 P]ATP. PDK1 activity was measured by the extent to which SGK1 transferred phosphate from [␥-32 P]ATP to the substrate peptide. The amount of incorporation of phosphate into the substrate was determined by binding the substrate to phosphocellulose filters and quantitating with scintillation counting. The results indicate that PDK1 is not inhibited by the addition of either 10B or 15B (Fig. 5).
Phenoxazines Inhibit Recombinant Akt Kinase Activity in Vitro-To study the Akt inhibitory activity of phenoxazines, we have used the phosphorylation status of GSK-3 protein, a substrate of Akt, as a read out. Akt1/PKB␣ active enzyme (recombinant protein expressed in Sf21 cells, 10 ng/reaction) was preincubated with compound 10B or 15B at 5 M for 2 h on ice prior to initiating the kinase assay. The results show that compound 15B completely blocked the phosphorylation of GSK-3 (Fig. 6, 2nd and 3rd lanes), whereas inhibition by 10B was nearly complete. These results indicate that phenoxazine derivatives are targeting Akt directly and inhibit its kinase function.
Phenoxazines Do Not Block Activation of Akt via Its PH Domain-All three Akt isoforms consist of a conserved domain structure as follows: an amino-terminal pleckstrin homology (PH) domain, a central kinase domain, and a carboxyl-terminal regulatory domain that contains the hydrophobic motif, a characteristic of AGC kinases. The PH domain is a phosphoinositide-binding motif found in a number of signal-transducing proteins, including Akt that gives membrane-binding properties to the host proteins. The PH domain interacts with membrane lipid products such as PtdIns(3,4,5)P 3 produced by PI 3-kinase (45). Biochemical analysis revealed that the PH domain of Akt binds preferentially PtdIns(3,4,5)P 3 (46,47), resulting in the recruitment of Akt to the plasma membrane (22,47,48). Binding induces a conformational change in Akt, exposing the critical Thr-308 residue in the activation loop to phosphorylation by PDK-1. For full activation, Akt is subsequently phosphorylated at Ser-473 by an as yet unidentified kinase termed PDK-2 (3,22,47). The observation that the phenoxazines do not inhibit PDK1 activity suggests that interaction with the PH domain of Akt may not be a requirement for the inhibitory effect observed with these compounds. To determine whether the absence of the PH domain of Akt affects the ability of 10B or 15B to inhibit Akt kinase activity, in vitro kinase assays were performed using a recombinant Akt lacking the PH domain. Because GSK-3 is one of the major downstream phosphorylation targets of Akt, we used GSK-3 phosphorylation as an indication of Akt activity. In agreement with previous studies, the deletion of the PH domain results in a higher level of kinase activity than the full-length protein (Fig. 6,  compare 1st and 4th lanes). Despite the higher kinase activity in the control sample, the ability of 10B and 15B to inhibit Akt kinase activity was unaffected by the deletion of the PH domain (Fig. 6, 5th and 6th lanes), indicating that these compounds are unlikely to mediate their effects by blocking association of Akt with the membrane.
Phenoxazines Do Not Inhibit Recombinant SGK1 Kinase Activity in Vitro-Among the AGC family of kinases to which Akt belongs, SGK1 has the highest degree of homology to Akt. In addition to conservation of homologous sites at threonine 308 and serine 473, SGK1 also has a high degree of homology in the catalytic kinase domain and the carboxyl-terminal Ser/Thr kinase extension. To determine whether SGK1 was also a target of phenoxazines 10B or 15B, a kinase assay using 10 ng/reaction of recombinant active SGK1 was performed under the identical conditions described for the Akt kinase experiments. The results indicate that neither 10B nor 15B interfere with SGK1 kinase activity (Fig. 6B). This result is consistent with the results for the coupled enzyme assay measuring PDK1 activity where no difference was observed in the phosphorylation of the substrate peptide by SGK1 in the phenoxazinetreated versus -untreated samples.
ATP Competition with Phenoxazines-One possible mechanism of inhibition of Akt by phenoxazines is competition with ATP for binding to the kinase. To address this question, a number of activity assays were performed at various ATP and phenoxazine concentrations. These data were then modeled using equations for various types of inhibition and an experimentally determined ATP K m of 195 M. The modeling was unable to distinguish between competitive and noncompetitive inhibition with 50% probability for each type. However, other types of inhibition such as mixed inhibition and uncompetitive cells. Rh30 cells were treated and processed as in C. The results show that 10B or 15B effectively blocked the Akt pathway in Rh30 cells. In contrast, phenoxazine 20B was inactive. Similar results were obtained in two independent experiments.

FIG. 2.
A, phenoxazines inhibit IGF-I-mediated activation of Akt kinase pathway in Rh1 cells. Cells were plated in serum-free medium and allowed to attach overnight. Monolayers were treated with 0.1% Me 2 SO or 3B, 8B, 10B, 12B, or 15B at 5 M for 1 h. Cells were harvested after stimulating with IGF-I for 10 min, and phospho-Akt, phospho-mTOR, or phospho-S6 was detected using the respective phospho-specific antibody. The results show that all the compounds tested except 12B inhibit the phosphorylation of Akt and components of the downstream signaling pathway. The blots are representative of experiments that were replicated at least three times. B, phenoxazine derivatives inhibit the phosphorylation of p70 S6 kinase in Rh1 cells. Rh1 cells were seeded in serum-free medium for overnight attachment, exposed to 0.1% Me 2 SO,or phenoxazines at 5 M for 1 h, and stimulated with IGF-I for 10 min. Phosphorylation of p70 S6 kinase (Thr-389) was detected using phospho-specific antibody. The results show that Akt inhibitors 10B or 15B effectively blocked the phosphorylation of p70 S6 kinase, whereas compound 12B did not. The results are representative of two independent experiments. C, phenoxazines block the phosphorylation Akt, mTOR, and S6 in Rh18 cells. Rh18 cells seeded under serum-free conditions were exposed to 0.1% Me 2 SO, 10B, 14B, 15B, or 20B at 5 M for 1 h, stimulated with IGF-I for 10 min, and processed as in A. The data show that Akt kinase pathway is completely inhibited by 10B, 14B, or 15B in Rh18 cells. Results are representative of two independent experiments. D, phenoxazines inhibit phosphorylation of Akt in Rh30   FIG. 3. Phenoxazines inhibit Akt kinase activity in Rh1 cells. Rh1 cells grown in serum-free medium were exposed to 0.1% Me 2 SO, 5 M 10B or 15B for 1 h and then stimulated with IGF-I for 10 min. Cell lysates were then immunoprecipitated with Akt antibody. Immunoprecipitates were used in vitro to phosphorylate a GSK-3 fusion protein. inhibition were predicted to have a low probability as the mechanism by which phenoxazines inhibit kinase activity (data not shown).
Docking of Phenoxazines into the ATP Site of Akt-Based on the possibility that the phenoxazines might be inhibiting Akt by binding to the kinase domain, we carried out docking simulations to determine their ability to bind at the ATP site of the enzyme. The docking mode of the two most active compounds 10B and 15B is shown in Fig. 7, which was the binding mode preferred by the active compounds. In this mode, the phenoxazine ring occupies the adenine-binding region of ATP and the long hydrocarbon chain bridge, and its accompanying distal substituted amine (tertiary/cyclic) binds in the groove below the glycine-rich loop, i.e. the phosphate-binding area of ATP. The phenoxazine ring has favorable hydrophobic interactions between it and the following residues: Leu-158, Ala-179, Thr-213, Met-229, Tyr-231, Ala-232, Gly-235, and Met-282. The oxygen atom of the phenoxazine ring is placed close to the backbone amine of Ala-232, indicating a possible hydrogenbonding interaction with this hinge residue. In other serinethreonine and tyrosine kinases, inhibition is associated with a hydrogen bond interaction with an equivalent residue. In this case, the distance of 3.6 Å between the heavy atoms is close to a hydrogen bond but could very well be a true hydrogen bond (3.0 Å) if the ligand induces a fit to the receptor. Our docking did not simulate induced fit with changes in the protein conformation receptor. The hydrocarbon chain bridge interacts with residues of the glycine-rich loop as follows: Gly-159, Gly-161, and Val-166. The distal tertiary amine occupies vicinity with an electronegative electrostatic environment contributed to mainly by Asn-280 and Asp-293. The substituents on the amine interact with residues in this area through hydrogen bonds and/or hydrophobic interactions.
Generally, reducing the hydrocarbon chain bridge from four to three results in slight reduction in activity. A shorter hydrocarbon chain causes the substituted amino moiety to position the group into the narrow part of the groove resulting in less favorable binding, which might be due, at least in part, to less favorable steric and/or electrostatic interactions compared with the four carbon chain bridge counterparts.
The presence of electronegative groups in the distal amine group at the end of the hydrocarbon chain bridge as in compounds 2B, 5B, 5C, 9B, 12B, and 18B renders the molecules inactive due possibly to repulsive interactions. The loss of activity in analogs where the flexible hydrocarbon bridge is replaced by a shorter and rigid amidomethyl moiety as in compounds 16B, 17B, 18B, 19B, 20B and 21B may also stem from unfavorable steric and electrostatic interactions in the narrow groove below the glycine-rich loop.
Phenoxazines Enhance the Binding of 8-Azidoadenosine 5Ј-[␣-32 P]Triphosphate to Akt-To examine further the relationship between the inhibitory phenoxazines, and the ATP-binding site of Akt, photoaffinity labeling experiments were performed using 254 nm wavelength UV light to cross-link 8N 3 [␣-32 P]ATP to Akt. In this experiment, 200 ng of recombinant Akt was incubated on ice for 30 min either with or without phenoxazine 15B prior to the addition of 20 M 8N 3 [␣-32 P]ATP. Because this assay measures binding as opposed to activity, the samples were incubated only briefly (1 min) on ice after the addition of 8N 3 [␣-32 P]ATP and then UV cross-linked for 1 min. Photoactivation of 8N 3 [␣-32 P]ATP resulted in only very low levels of cross-linking with Akt (Fig. 8A, 1st lane). Changes in the duration of photoactivation, pH of the buffer, incubation time, and temperature or the inclusion of divalent cations did not increase the extent of cross-linking (data not shown). The low level of cross-linking observed in these reactions may be because of the 8N 3 [␣-32 P]ATP modification of ATP, which has been shown previously to adopt a conformation that fits poorly into the ATP binding domain of some proteins (50). The addition of a 10-fold excess of ATP (200 M) relative to 8N 3 [␣-32 P]ATP does not further reduce the extent of cross-linking (Fig. 8A, 2nd lane). Most surprisingly, the addition of a 10-fold excess of 15B (200 M) dramatically increases the extent of covalent 8N 3 [␣-32 P]ATP-Akt complexes formed by photoactivation (Fig. 8, A, 3rd lane, and B, 2nd lane). Furthermore, if a 5-fold excess of ATP (1 mM) relative to 15B is included in the reaction, the extent of cross-linking is reduced to almost the same level as is observed in the sample without 15B (Fig. 8B,  and 3rd lanes; 5th and 6th lanes). The results were similar in two independent experiments. B, phenoxazines do not inhibit SGK1 kinase activity in vitro. Reaction conditions were identical to those described for Akt (see A), but 10 ng of recombinant SGK1 was substituted for Akt. The results indicate the treatment with phenoxazines 10B or 15B (2nd and 3rd lanes) has no effect on SGK1 kinase activity when compared with the control (1st lane).
3rd and 1st lanes, respectively). These results clearly indicate that 15B facilitates the interaction of 8N 3 [␣-32 P]ATP with Akt and that ATP is able to compete with 8N 3 [␣-32 P]ATP or possibly 15B to reduce the interaction of 8N 3 [␣-32 P]ATP with Akt.
Phenoxazine Derivatives Block the Translocation of Akt from Cytoplasm to the Nucleus-Upon activation Akt translocates to the nucleus (51)(52)(53)(54). Therefore, one of the predicted effects of inhibiting Akt phosphorylation by phenoxazines would be a decrease in localization to the nucleus in response to growth factor stimulation. To address this question, confocal microscopy experiments were performed using an anti-Akt antibody to examine cellular localization in response to treatment with phenoxazines. Rh1 cells were placed in chamber well slides in MN2E medium for 20 h followed by the addition of 5 M phenoxazine (10B or 15B) or Me 2 SO (0.1%) vehicle control for 1 h, after which time 10 ng/ml of IGF-1 was added for 20 min. The cells were then fixed and incubated with anti-Akt antibody as well as anti-TOPRO-3 to identify the nucleus. As can be seen in Fig. 9, when compared with IGF-I-stimulated control (0.1% Me 2 SO) cells, exposure to either 10B or 15B led to a reduction in the level of Akt present in the nucleus of Rh1 cells. These results are consistent with a block in the nuclear localization when the activation of Akt is inhibited by phenoxazines.

Phenoxazine Derivatives Inhibit Cell Growth and Induce
Apoptosis-To assess the effect of phenoxazines on cell growth, Rh1, Rh18, and Rh30 cells grown in complete medium were exposed to graded concentrations (0.1-25 M) of 10B or 15B for 6 days. All three cell lines were sensitive to both the compounds with an IC 50 value of 2, 5, and 6 M for Rh1, Rh18, and Rh30 cells, respectively (Fig. 10A). Thus growth inhibition appears to correlate well with the concentration of phenoxazines that inhibit Akt in cells. In contrast, compounds 12B and 20B were at least 10-fold less inhibitory, consistent with their lack of Akt inhibition. Because Akt is an "anti-apoptotic kinase," we investigated the effects of several phenoxazine derivatives on apoptosis. We exposed Rh1, Rh18, or Rh30 cells grown in complete medium with 0.1% Me 2 SO, 10B, 11B, 13B, 14B, or 15B at 6.5 M (Rh1) or 7.5 M (Rh18 and Rh30) for 4 days. Cells were harvested, and the extent of apoptosis was evaluated by the ApoAlert flow cytometric assay. Within the apoptotic population, cells in the early stages of apoptosis were annexin Vpositive and propidium iodide-negative, whereas those in the late stages were annexin V-positive and propidium iodide-positive (Fig. 10B). These populations are combined and presented in Table II. Approximately 10 -19% of cells in the control population were undergoing spontaneous apoptosis (Table II). Treatment with 10B and 15B resulted, respectively, in ϳ52 and 75% apoptosis in Rh1, 34 and 33% apoptosis in Rh18, and 43 and 89% apoptosis in Rh30 cells. Cells exposed to phenoxazines appeared to lose membrane integrity late in apoptosis yet remained attached to the culture dish. Furthermore, a significant increase in the proportion of apoptotic cells was evident after treatment with 11B, 13B, and 14B (data not shown). To assess whether the apoptosis of these cells in response to 10B or 15B was because of a general toxic effect of the compound rather than because of inhibition of Akt, we compared 10B or 15B with other phenoxazines that poorly inhibit Akt in vitro (12B or 20B). Because of their overall chemical similarity with the Akt inhibitory phenoxazines, 12B and 20B might also be expected to exhibit cellular toxicity if the mechanism was general and independent of Akt. But in contrast to the effect observed with 10B and 15B, neither 12B nor 20B induced apoptosis (data not shown). Thus, there is a correlation between the ability of a compound to inhibit Akt in cells and its ability to induce apoptosis upon treatment of intact cells. DISCUSSION There is increasing evidence to support dysregulation of the PI 3-kinase-mTOR pathway in the pathogenesis and progression of human cancers (55)(56)(57). Thus, activation of Akt, downstream of PI 3-kinase, becomes a nodal point in the signaling pathway. Akt indirectly regulates mTOR signaling through suppression of the tuberous sclerosis complex, hence it regulates cap-dependent translation (56). Akt also regulates apoptosis in response to cellular stresses, including cytotoxic chemotherapy. Thus, Akt has become a focus for developing small molecule inhibitors. Here we report identification of N 10substituted phenoxazines, some of which appear to inhibit Akt at low concentrations.
These compounds were originally synthesized as modulators of MDR, hence the required characteristics were for nontoxic analogs that modulated P-glycoprotein-mediated drug resistance. Consequently, analogs that exerted intrinsic cytotoxicity were excluded from further study. The observation that certain phenothiazines (structurally related to phenoxazines), such as trifluoperazine, were identified as putative weak Akt inhibitors (34) prompted us to re-evaluate some of these phenoxazines as showed no inhibitory activity in this assay. Consistent with inhibition of Akt, the most potent inhibitors blocked IGF-I-stimulated phosphorylation of mTOR, p70 S6 kinase, and S6, downstream of Akt in the signaling cascade. More importantly, neither 12B nor 20B exerted any detectable effect on phosphorylation of Akt or downstream effectors. In serum-starved cells, IGF-I rapidly activated Akt, as demonstrated by in vitro kinase assay of immunoprecipitated enzyme using a GSK-3 peptide as substrate. In contrast, Akt immunoprecipitated from cells exposed to 10B or 15B the enzyme was inactive. Although we have not undertaken an exhaustive study of other kinases, neither 10B nor 15B was found to inhibit recombinant PI 3-kinase, PDK1, or the closest member of the AGC kinase family SGK1. However, these phenoxazines potently inhibited the activity of recombinant Akt and the mutant enzyme lacking the PH domain (Akt⌬PH). Thus, we conclude that the phenoxazines are not like the PH domain-dependent isozyme-specific Akt inhibitors reported recently (35).
The precise mechanism by which phenoxazines inhibit Akt activity is unclear. Initial studies do not rule out the possibility that phenoxazines may be competing with ATP for binding to the enzyme. However, phenoxazines may also be locking the enzyme in an inactive conformation or acting in an allosteric manner. Modeling studies suggest that the inhibitory phenoxazines may bind in the ATP site, whereas those with shorter, more rigid amidomethyl side chains (i.e. 16B-20B) may be sterically hindered from binding. For compounds with electronegative moieties (i.e. 2B, 5B, etc.), there may be repulsion from the site.
The results from the photoaffinity cross-linking experiments, however, complicate the interpretation of the modeling data. The binding of 15B greatly facilitates the interaction of 8N 3 [␣-32 P]ATP with Akt. The simplest interpretation of the data is that 15B binding to Akt results in the enzyme assuming a conformation that is more favorable for the binding of 8N 3 [␣-32 P]ATP. The observation that ATP competes effectively with the 8N 3 [␣-32 P]ATP would argue against the phenoxazines acting as competitive inhibitors for ATP as the modeling data suggest but rather as noncompetitive inhibitors acting allosterically.
Clearly, further studies will be required to determine the specific interaction of the phenoxazines with Akt. Additional studies involving co-crystallization of Akt along with 10B or 15B are planned to resolve the mode of phenoxazine binding to Akt.
Consistent with inhibition of Akt activation, 10B and 15B suppressed IGF-I-stimulated nuclear translocation of Akt. At low micromolar concentrations, these compounds were effective inhibitors of cell growth in three lines examined (Rh1, Rh18, and Rh30). In contrast, compounds 12B and 20B were at least 10-fold less potent. Of interest is that both Akt inhibitors induced very significant levels of apoptosis, even under serumcontaining conditions of cell growth. This is important, as inhibition of mTOR signaling by rapamycin induces significant apoptosis only under serum-free conditions in Rh1 and Rh30 cells. Thus, these results demonstrate qualitative differences in cellular responses when different kinases are targeted within the same pathway. mTOR in the rictor-Lst8 (rapamycin-insensitive) complex has been proposed as the putative PDK2 (49). Our data do not formally address whether Akt-inhibitory phenoxazines inhibit this complex. However, preliminary studies show that in vitro neither 10B nor 15B inhibits mTOR kinase activity. Also of interest is that although treatment of cells with rapamycin increases the proportion of cells in the G 1 phase of the cell cycle, no such accumulation was observed in cells exposed to either 10B or 15B (data not shown), further demonstrating the differences between Akt and mTOR inhibitors. Very preliminary in vivo toxicity testing with one compound (11B) suggests that it is well tolerated, causing sedation similar to that induced by phenoxazine, the parent compound (data not shown). Studies to determine the in vivo pharmacokinetics as well as initial studies to determine whether these compounds have anti-tumor properties are currently being undertaken.
In summary, we have identified a series of N 10 -substituted phenoxazines that are potent inhibitors of Akt activity in vitro and inhibit Akt signaling in cells. These compounds are growth inhibitory and induce significant apoptosis in cell lines under serum-containing conditions of culture and hence have differential cellular activities from mTOR inhibitors that induce only low level apoptosis under similar culture conditions.