Specific Inhibition of the Akt1 Pleckstrin Homology Domain bymct.aacrjournals.org/content/molcanther/2/4/389.full.pdf · Specific Inhibition of the Akt1 Pleckstrin Homology Domain

Specific Inhibition of the Akt1 Pleckstrin Homology Domain byD-3-Deoxy-Phosphatidyl-myo-Inositol Analogues1

Emmanuelle J. Meuillet,2 Daruka Mahadevan,Hariprasad Vankayalapati, Margareta Berggren,Ryan Williams, Amy Coon, Alan P. Kozikowski, andGarth PowisArizona Cancer Center, University of Arizona, Tucson, Arizona 85724[E. J. M., D. M., H. V., M. B., R. W., A. C., G. P.], and Drug DiscoveryProgram, Department of Neurology, Washington, DC 20007-2197[A. P. K.]

AbstractActivation of Akt (protein kinase B), a Ser/Thr proteinkinase that promotes cell survival, has been linked totumorigenesis. Akt is activated by phosphorylationafter binding of its pleckstrin homology (PH) domainto plasma membrane phosphatidyl-myo-inositol-3-phosphates, formed by phosphoinositide-3-kinase. Wereport a novel strategy to inhibit Akt activation basedon the use of D-3-deoxy-phosphatidyl-myo-inositols(DPIs) that cannot be phosphorylated on the 3-positionof the myo-inositol ring. We have studied the DPIs, DPI1-[(R)-2,3-bis(hexadecanoyloxy)propyl hydrogenphosphate], its ether lipid derivative DPI 1-[(R)-2-methoxy-3-octadecyloxypropyl hydrogen phosphate](DPIEL), and its carbonate derivative DPI 1-[(R)-2-methoxy-3-octadecyloxypropyl carbonate]. Wedemonstrate in platelet-derived growth factor-stimulated mouse NIH3T3 cells that the DPIs bind tothe PH domain of Akt, trapping it in the cytoplasm andthus preventing Akt activation. DPIEL did not inhibitmyristylated-Akt, a constitutively active membrane-bound Akt expressed in NIH3T3 cells, and cell growthwas not inhibited, unlike in wild-type NIH3T3 cells.Molecular modeling and docking studies show thatDPIEL binds with much higher affinity to Akt’s PHdomain as compared with DPI and DPI 1-[(R)-2-methoxy-3-octadecyloxypropyl carbonate]. This studyshows that the DPIs are a novel class of growthinhibitory agents with a novel mechanism of actionthrough binding to the PH domain of Akt and inhibitionof Akt activation.

IntroductionThe Ser/Thr protein kinase Akt, also called protein kinase Bor related to A- and C-kinase, is a downstream target of PI-3kinase3 (1, 2). PI-3 kinase phosphorylates the D-3-hydroxylposition of the myo-inositol ring of PtdIns (3) to generatethe PtdIns-3-phosphates, PtdIns(3)P, PtdIns(3,4)P2, andPtdIns(3,4,5)P3 (4). PI-3 kinase is activated by many growthfactor receptors and oncogenic protein tyrosine kinases(5–7), as well as by p21Ras (8), leading to increased cellgrowth and inhibition of apoptosis (9, 10). PI-3 kinase ex-pression is increased in ovarian cancer (11), and it is consti-tutively activated in human small cell lung cancer cell lines,where it leads to anchorage-independent growth and hasbeen suggested to be a cause of metastasis (12). However,the major role for PI-3 kinase in cancer cell growth is its role insurvival signaling mediated by Akt to prevent apoptosis (13).

Akt mediates a variety of biological responses, includingthe inhibition of apoptosis and promotion of cell survival(reviewed in Ref. 2). There are three mammalian isoforms: (a)Akt1/�; (b) Akt2/�; and (c) Akt3/� (2). PtdIns-3-phosphatesformed by PI-3 kinase (14) present in the inner leaflet of theplasma membrane bind to the PH domain of Akt (15, 16),which causes the translocation of Akt from the cytoplasm tothe plasma membrane (17). Akt is then activated by phos-phorylation on Ser473 and Thr308 by PDK-1 and integrin-linked kinase, respectively (18, 19). Phosphorylated Akt canthen detach from the plasma membrane and move to thenucleus (17, 20). Activated Akt phosphorylates proteins,such as Bad, an inhibitor of apoptosis, FRAP, an activator ofp70S6k, which is required for cell cycle progression,caspase-9, forkhead transcription factors, and nuclear factor��, thereby regulating cell proliferation and promoting cellsurvival (reviewed in Ref. 1). Akt1 is overexpressed in gastricadenocarcinoma (21), and Akt2 is overexpressed in breastcancer (22), ovarian cancer (22, 23), and pancreatic cancer(24).

Activation of Akt is negatively regulated by the tumor sup-pressor protein PTEN/MMAC, a tensin homologue detectedin chromosome 10 and mutated in multiple advancedcancer/phosphatase (25). PTEN is a dual specificity tyrosine-threonine/lipid phosphatase that dephosphorylates the3-position of PtdIns-3-phosphate (26, 27), thus inhibiting thePI-3 kinase/Akt signaling pathway (26, 28). In PTEN-deficientmouse embryo fibroblasts, constitutively elevated Akt activ-

Received 4/30/02; revised 10/24/02; accepted 2/5/03.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 Supported by NIH Grants CA78277, CA48725, CA77204, and CA17094(G. P.) and a Research Fellowship CA90053-01A1 (E. J. M.).2 To whom requests for reprints should be addressed, at Arizona CancerCenter, University of Arizona, 1515 North Campbell Boulevard, Tucson,AZ 85704. Phone: (520) 626-6408; Fax: (520) 626-4848; E-mail:[email protected].

3 The abbreviations used are: PI, phosphoinositide; DAG, Diacylglycerol;DCIEL, D-3-deoxy-phosphatidyl-myo-inositol 1-[(R)-2-methoxy-3-octa-decyloxypropyl carbonate]; DPI, D-3-deoxy-phosphatidyl-myo-inositol;GSK, glycogen synthase kinase; DPIEL, D-3-deoxy-phosphatidyl-myo-inositol 1-[(R)-2-methoxy-3-octadecyloxypropyl hydrogen phosphate];FBS, fetal bovine serum; PDGF, platelet-derived growth factor; PDK,phosphatidylinositol-dependent kinase; PtdIns, phosphatidylinositol;CMV, cytomegalovirus; PH, pleckstrin homology; PKC, protein kinase C.

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ity together with decreased sensitivity to cell death in re-sponse to apoptotic stimuli demonstrate a role for PTEN asa negative regulator of cell survival (25). Somatic mutationsof PTEN have been reported in several types of humantumors, including those from brain, breast, endometrium,kidney, and prostate (29, 30). In addition, germ-line muta-tions of the PTEN gene have been shown to be associatedwith Cowden’s disease, an autosomal dominant cancer pre-disposition syndrome associated with an elevated risk fortumors of the breast, thyroid, and skin (31).

Inhibition of Akt signaling thus offers the opportunity toinhibit a major survival signaling pathway found in manytypes of human cancers, and it further provides a uniqueopportunity to replace the activity loss of the tumor suppres-sor protein PTEN. We have used a novel strategy to inhibitAkt based on the use of DPIs that cannot be phosphorylatedby PI-3 kinase. We demonstrate that the DPIs bind specifi-cally to the PH domain of Akt, probably preventing Akt’stranslocation to the plasma membrane and its activation byPDKs, thus inhibiting cell growth and inducing apoptosis.

Materials and MethodsReagents. DPI 1-[(R)-2,3-bis(hexadecanoyloxy)propyl hy-drogen phosphate], its ether lipid analogue DPIEL, and itscarbonate analogue DCIEL (Fig. 1) were synthesized as de-scribed previously (32, 33). L-�-PtdIns was obtained fromSigma Chemical Co. (St. Louis, MO). The cDNA encoding formyristylated HA-tagged human Akt1/� in the pCMV6 plas-

mid was a generous gift from Dr. L. Karnitz (Mayo Clinic, Roch-ester, MN). Human PDGF-BB homodimer was purchased fromGenzyme (Cambridge, MA). Polyclonal antibodiesto phospho-Ser473-Akt, phospho-Thr308-Akt, phospho-Ser136-BAD, pan-phospho-PKCs, and phosho-Ser241-PDK-1 were all purchasedfrom New England Biolabs-Cell Signaling (Beverly, MA); a goatpolyclonal antibody to Akt1 and mouse monoclonal p-Erk(E-4) antibody were from Santa Cruz Biotechnology (SantaCruz, CA). Recombinant Akt PH domain, polyclonal antibod-ies to Akt recognizing Akt1, 2 and 3 isoforms, a sheep poly-clonal antibody against the PH domain (amino acids 1–149)of human Akt1, and antiphosphotyrosine antibodies (clone4G10) coupled to agarose were purchased from UpstateBiotechnologies, Inc. (Lake Placid, NY). Anti-HA antibody(clone 12CA5) was purchased from Exalpha Biological, Inc.(Boston, MA). Donkey antirabbit IgG conjugated to peroxi-dase was purchased from Amersham Pharmacia Biotech(Piscataway, NJ), and rabbit antisheep IgG conjugated toperoxidase was obtained from Chemicon (Temecula, CA).The Renaissance chemiluminescence kit used for WesternBlot detection was obtained from NEN Life Science Prod-ucts, Inc. (Boston, MA). Akt/GSK, a peptide substrate relatedto the phosphorylation site of GSK-3, was obtained fromUpstate Biotechnolgies. The LipoTAXI system used for tran-sient transfection was purchased from Stratagene CloningSystems (La Jolla, CA).

Cells. Mouse NIH3T3 embryo-derived fibroblastic cells,human MCF-7 breast cancer cells, human HT-29 colorectaladenocarcinoma cancer cells, human DU-145, and LNCaPprostate cancer cells were obtained from the American Tis-sue Type Culture Collection (Rockville, MD). All cell lineswere maintained in bulk culture in DMEM supplemented with10% FBS and passaged using 0.25% trypsin and 0.02%EDTA.

Measurement of Phospho-Akt1 in NIH3T3, MCF-7, DU-145, and LNCaP Cells. Cells were grown in 35-mm culturedishes in DMEM with 10% heat-inactivated FBS, 4.5 grams/liter glucose, 100 units/ml penicillin, and 100 �g/ml strepto-mycin in a 5% CO2 atmosphere at 37°C to 75% confluence.Sixteen hours before the study, the medium was replaced byDMEM without FBS. The cells were incubated with the DPIsin DMEM for 4 h and stimulated with 50 ng/ml PDGF for 1 h(for NIH3T3 cells), with 100 ng/ml epidermal growth factor for30 min (for MCF-7, DU-145, and LNCaP). Control cells wereincubated with DMEM without growth factor. The culturemedia was aspirated, and the cells were lysed in 50 mM

HEPES (pH 7.5), 50 mM NaCl, 1% NP40, 0.25% sodiumdeoxycholate, 1 mM EDTA, and 1 mM sodium orthovanadate.A measurement of 20 �g of total cell lysates was boiled for5 min, and the samples were loaded on a 12% acrylamide/bisacrylamide gel and separated by electrophoresis at 160 Vfor 40 min (34). Proteins were electrophoretically transferredto polyvinylidene fluoride membranes, preincubated inblocking buffer (137 mM NaCl, 2.7 mM KCl, 897 �M CaCl2,491 �M MgCl2, 3.4 mM Na2HPO4, 593 mM KH2PO4, and 5%BSA), and incubated with anti-phospho-Ser473-Akt poly-clonal antibody. Immunoreactive bands were detected usingdonkey antirabbit IgG peroxidase-coupled secondary anti-body and detected using the Renaissance chemilumines-

Fig. 1. Structures of PtdIns, DPI, DPIEL, and DCIEL. Note the absence ofthe hydroxyl group on the 3-position of the myo-inositol head group andthe 1-carbonyl group in DCIEL replacing the phosphate in DPIEL and DPI.

390 Novel Akt Inhibitors



cence kit on Kodak X-Omat Blue XB films. Bands werequantified using Eagle Eye software (Stratagene). The detec-tion of pPKCs, pPDK-1, and pERK was performed as de-scribed for pAkt using specific anti-phospho antibodies.

Akt1 Kinase Assay. The kinase assay was performedaccording to the manufacturer’s instructions (UBI). Briefly,NIH3T3 cells were grown in T75-cm2 flasks (�5.16cells/flasks), stimulated with 50 ng/ml PDGF for 10 min, and thenlysed with 50 mM Tris-HCl (pH 7.4), 1% NP40, 0.25% sodiumdeoxycholate, 150 mM NaCl, 1 mM ethylene glycol bis(�-aminoethylether)N,N,N�,N�-tetraacetic acid, 1 mM p-toluene-sulfonyl fluoride, 1 �g/ml aprotinin, 1 �g/ml leupeptin, 1 mM

Na3VO4, 1 mM NaF, and 1 �M microcystin. The lysate wasmixed with 4 �g of rabbit antihuman Akt1 polyclonal anti-body coupled to protein G agarose beads (UBI) for 2 h at 4°C.After several washes in 20 mM 3-(N-morpholino) propanesulfonic acid (pH 7.2), 25 mM glycerol phosphate, 1 mM DTT,and 1 mM sodium orthovanadate, the immunoprecipitatedAkt1 was preincubated for 1 h with the DPI analogue con-centrations ranging from 10 to 20 �M at room temperature.An in vitro kinase assay was performed for 10 min at 30°Cusing as substrate 10 �l of a 0.4 mM solution of Akt/GSK and10 �Ci/sample of [�-32P]-ATP (1 mCi/100 �l, 3000 Ci/mmol;NEN Life Science Products). To each sample, 20 �l of 40%trichloroacetic acid were added, and after 5 min, 40 �l of thesupernatant fraction were transferred to a P81 phosphocel-lulose paper square (Whatman International, Ltd., Kent,United Kingdom). The assay squares were washed threetimes with 0.75% phosphoric acid and once with acetone,and the radioactivity was determined by liquid scintillationcounting.

Cell Growth Inhibition. Mouse NIH3T3 cells were seededat a density of 30,000 cells/24-mm diameter dish in DMEMwith 10% FBS. After 16 h, the DPIs were added to the culturemedium at concentrations ranging from 0.5 to 20 �M. Threedays later, cells were counted using a hemocytometer andtrypan blue exclusion to discriminate viable from dead cells.All measurements were made in triplicate.

Measurement of Apoptosis. The annexin V apoptosis kit(Roche, Indianapolis, IN) was used according to manufacturerinstructions with minor modifications. Briefly, prostate cancercells were incubated with DPIEL for 24 h, collected, andwashed in 137 mM NaCl, 2.7 mM KCl, 897 �M CaCl2, 491 �M

MgCl2, 3.44 mM Na2HPO4, and 593 mM KH2PO4. Cells wereresuspended in 20 �l of diluted FITC-annexin V/propidium io-dide stain. Cells were incubated at 22°C for 20 min and thendiluted with 500 �l of buffer supplied with the kit. The fluores-cence emission of 10,000 cells was counted by flow cytometryfor each condition using a FACScan (Becton Dickinson, SanJose, CA), and the results were analyzed using CELL-Questsoftware.

Myristoylated HA-Akt1 and GST-Akt1 Transfections inNIH3T3 Cells. NIH3T3 cells were seeded into 100-mm di-ameter dishes, grown to 50% confluency in DMEM with 10%FBS, and transiently transfected using LipoTAXI (StratageneCloning Systems) with pCMV6 plasmid containing the cDNAinsert for HA-tagged myr-Akt1 and pCMV5 plasmid contain-ing the cDNA for GST-Akt1. After 24 h, the cells were re-seeded into 24- and 40-mm diameter dishes for cell growth

and Akt activity measurements, respectively. Expression wasconfirmed by Western blotting using anti-HA antibodies forthe myr-Akt1 and monoclonal anti-GST antibodies for Akt(data not shown).

PI-3 Kinase Assay. In vitro PI-3 kinase activity was meas-ured as described previously (35). Briefly, NIH3T3 cells werestimulated with 100 ng/ml PDGF for 10 min and lysed, andPI-3 kinase was immunoprecipitated using antiphosphoty-rosine antibody (clone 4G10) coupled to agarose beads. PI-3kinase was eluted from the beads using 2 mM phenylphos-phate, and PI-3 kinase activity was measured in the presenceof 10 �M [�-32P]-ATP (1 mCi/100 �l, 3000 Ci/mmol; NEN LifeScience Products) and 10 �l of a 10 mg/ml solution ofL-�-PtdIns for 45 min at 37°C. Samples were quenched bythe addition of 100 �l of 1N HCl, extracted with 400 �l of 1:1chloroform/methanol, and centrifuged at 2000 � g for 1 min.For each sample, 25 �l of the lower organic phase werespotted onto the preabsorbent strip of an individual lane in amultichannel thin-layer chromatography plate (Whatman,Hillsboro, OR). Plates were developed in 65% n-propylalcohol/35% 2 M acetic acid. [32P]-labeled PtdIns productwas quantitated by phosphorimager analysis (Molecular Dy-namics, Sunnyvale, CA).

Translocation of Akt1 in NIH3T3 Cells. NIH3T3 cellswere grown to 50% confluency on glass coverslips in DMEMwith 10% FBS. The cells were then incubated in DMEMwithout FBS for 2 h. DPIEL was added for 3 h, Wortmanninwas added for 1 h, and cells were stimulated with 100 ng/mlPDGF for 60 min. After the stimulation, cells were fixed with4% paraformaldehyde for 45 min and incubated for 1 h in aPBS buffer containing 137 mM NaCl, 2.7 mM KCl, 897 �M

CaCl2, 491 �M MgCl2, 3.44 mM Na2HPO4, 593 mM KH2PO4

supplemented with 1% BSA, 0.1% Triton X-100, and 1%goat serum. Antibody against Akt was added overnight in thesame buffer, and after several washes, slides were incubatedwith 10 �g/ml antigoat secondary antibody coupled to AlexaFluor 568. Nuclear staining was performed using a solution of1.5 ng/ml YOYO-1, according to the instructions from themanufacturer. Slides were examined under a Leica-TCS con-focal microscope equipped with an Ar/Kr laser.

Lipid Binding to Akt-PH Domain. Lipids, dissolved inchloroform at 1 mg/ml, were added to the wells of a poly-carbonate 96-well plate at 100 �g/well, and the chloroformwas allowed to evaporate. The wells were then blocked with3% BSA fatty acid free for 1 h at room temperature. Ameasurement of 100 �l of a solution containing 0.5 �g/ml PHdomain of Akt1 was added to each well overnight at 4°C. Theplate was washed twice with PBS, and a sheep polyclonalantibody against the PH domain of Akt1 was added for 1 h atroom temperature. The wells were washed twice in PBS, andbound PH domain was detected using a peroxidase-coupledsecondary antisheep antibody, followed by a developing so-lution containing 2% 2,2�-azino-di-(3-ethylbenzethiazolinesulfonate) in 1 M sodium acetate and 0.03% H2O2. Plateswere read at 405 nm using a SpectraMAX Plus spectropho-tomer-ELISA reader (Molecular Devices).

Molecular Modeling and Docking Studies. A homologymodel of the PH domain of human Akt 1 was built based onthe crystal structure of Btk PH domain as described previ-

391Molecular Cancer Therapeutics



ously by Rong et al. (36). The model was used for dockingstudies of DPIs. To explore the interactions of DPIEL with thePH domain, a molecular model of the compound was built,energy was minimized performing the necessary replace-ments, hydrogen atoms were added using INSIGHT II (Ver-sion 2000; ACCELRYS, Inc., San Diego, CA)4, and refiningwas done by energy minimization (1000 cycles of steepestdescent and 2 � 1000 cycles of conjugate gradient), whileconstraining the positions of the heavy atoms. The entirestructure was then subjected to conjugate gradient minimi-zation, and constraints were gradually removed until conver-gence was reached. After 100-ps molecular dynamic simu-lations at 300 K, energy minimization (2 � 1000 cycles ofconjugate gradient minimization) was performed usingDISCOVER-3 with Class II Force Fields.5 This structure sub-sequently served as the model structure for further energyrefinement, docking, molecular dynamics, and complex forma-tion. The low energy model was manually docked into the activesite, and energies were computed using AFFINITY (37, 38).

To clarify the orientation of the ligand in the binding site,the electrostatic potentials at the Van der Waals surface ofthe active site pocket of the PH domain were determinedusing solvent surface calculations. Simulated annealingdocking with 100 fs per stage duration (50 simulated anneal-ing stages) was then performed to find the most favorableorientation. The orientation with the lowest intermolecularpotential energy was obtained while moving the ligand andhighly amended ligand-binding amino acid residues. Theresulting ligand-PH domain complex trajectories were en-ergy minimized using 1000 cycles of conjugate gradient min-imizer, and the binding energies were calculated for eachcomplex (39).

ResultsDPIs Bind Specifically to the PH Domain of Akt1. DPIswere synthesized based on the fact that these compoundsare not able to be phosphorylated by PI-3 kinase on the3-position of the myo-inositol ring (Fig. 1) but may act ascompetitors for Akt activation at the plasma membrane and,thus, behave as downstream inhibitors of the effects ofPtdIns-3-phosphates. First, the ability of the DPIs to directlybind the PH domain of Akt was tested using an in vitrobinding assay (Fig. 2). DAG and PtdIns used as control lipidsbound only weakly to the PH domain of Akt. DPI and DPIELbound to the PH domain of Akt to the same extent toPtdIns(3,4)P2, whereas DCIEL showed a lesser binding.

To better understand these binding differences, molecularmodeling and docking studies were used to calculate therelative binding affinity of DPIs for the PH domain of Akt(Table 1 and Fig. 3). The myo-inositol ring is involved in manyinteractions with Arg25, Tyr38, Arg48, and Arg86 (36). Dockinganalysis of the DPIs into the PH domain of Akt revealed thatthe myo-inositol moiety of DPIEL binds to the positively

charged binding pocket of the PH domain with high energy(�109.3 kcal/mol) as compared with DPI or DCIEL (�59.4and �56.6 kcal/mol, respectively). The 3-deoxy-myo-inositolring of DPIEL is stabilized by hydrogen bond interactionswith the terminal nitrogen atom of Arg25, and the 1-phos-phate group exhibits a strong interaction with the positivelycharged pocket using a network of hydrogen bonds: twobonds with 1-P�O. . . HN-Lys14 and the carbonyl oxygenatom of the side chain from the Arg23 residue with a distanceof 2.48 and 3.34 Å, respectively (Fig. 3). Two other hydrogenbonds are between the ether oxygen atoms of 1-phosphateand amine hydrogen atoms of Arg23 and Arg25 residues. Thefunctional methoxy group exhibits a strong steric interactionbetween Lys14 and Thr21.

The comparison of the binding mode of DPIEL with DPI andDCIEL revealed that the 3-deoxy-myo-inositol ring of all threecompounds exhibits a similar position. All hydroxyl groups ofthe DPI’s ring retained their interactions with Tyr38 and Arg48. Incontrast, the interaction between the 1-phosphate group of DPIand Lys14 is lost. The 1-phosphate group position is shifted 1.2Å toward Arg25, and the hydroxyl group of the phosphate func-tion displays a hydrogen bond interaction with the functionalamine group of Arg25 with a distance of 2.04 Å. For DCIEL, nohydrogen bond interactions were formed because of the ab-sence of the 1-phosphate group. The position and orientation of3-deoxy-myo-inositol ring is stabilized by participation of the4,5-OH groups in hydrogen bond interactions with Arg25 andArg48. DPIEL was calculated to bind much more strongly to thePH domain of Akt as compared with DPI and DCIEL. DPIELdocks into the PH domain of Akt and exhibits strong interac-tions with the amino acids thought to be involved into thebinding of PtdIns-phosphates.

DPIs Inhibit Akt Kinase Activity in Vitro. PtdIns phos-phates are known to modulate Akt kinase activity (15). Thus,we studied the effects of DPIs and Wortmannin, a PI-3 kinaseinhibitor (40, 41) on Akt1 kinase activity in an in vitro assay(Fig. 4). Akt1 kinase activity was increased in PDGF-stimu-

4 INSIGHT II, Molecular Modeling Software. San Diego: Accelrys, Inc.,2000.5 Discover-3 (Version 2.9.5) Molecular Mechanism force field INSIGHT II2000, Molecular Modeling Software. San Diego: Accelrys, Inc., 2000.

Fig. 2. DPIs bind to the PH domain of Akt. A measurement of 100 �g ofDAG, PtdIns, PtdIns(3,4)P2, DPI, DCIEL, or DPIEL was used to coat thewells of a 96-well plate. Recombinant PH domain of Akt (amino acids1–149) was added, and afterward, equilibration-bound PH domain wasdetected using specific anti-PH domain antibody. The results are theaverage of three separate experiments. Bars, SD. �, P � 0.1; ��, P � 0.05compared with DAG used as the control.




lated cells. Akt activity was not inhibited by 5 �M Wortmanninbut was inhibited by the DPIs with approximate IC50s for DPIand DPIEL of 8 and 17.5 �M, respectively.

DPIs Inhibit Akt Activity in NIH3T3 Mouse Fibroblasts.Akt1 is the major Akt isoform expressed in mouse NIH3T3fibroblast cells where it represents 59% of the total Aktpresent. Akt2 and Akt3 are 23 and 18%, respectively (Fig.5A). Akt1 activation measured by phosphorylation on Ser473,was increased �2-fold on PDGF stimulation of NIH3T3 cells(Fig. 5B, Lane 2 as compared with Lane 1 and Fig. 5C). In thepresence of 1 �M Wortmannin, Akt1 phosphorylation wascompletely inhibited (Fig. 5B, Lane 3). DPIEL (Lane 6) and, toa lesser extent, DPI and DCIEL decreased Akt1 phosphoryl-ation (Fig. 5B, Lanes 5 and 4, respectively). PtdIns was usedas a control and did not affect the phosphorylation of Akt(Lane 7). Fig. 5C represents the quantification of phospho-Ser473-Akt from at least five different Western blots.

DPIs Inhibit Cell Growth and Induce Apoptosis in aDose-dependent Manner. Increasing concentrations ofDPI, DPIEL, and DCIEL inhibited Akt1 phosphorylation inNIH3T3 cells in a dose-dependent manner (Table 2) withIC50s (�SD, n � 4) of 27.4 � 4.4 �M, 1.5 � 0.3 �M, and12.5 � 2 �M, respectively. The DPIs also inhibited NIH3T3cell growth with IC50s for the DPI, DPIEL, and DCIEL of 17.6,4.3, and 14.1 �M, respectively. Human cancer cell lines werealso tested (36), and results are summarized in Table 2.Overall, DPIEL appeared to better inhibit the proliferation ofNIH3T3 and MCF-7 cells. Interestingly, DPIEL and DCIELhad similar effects on HT29 cell proliferation. DPIEL inhibited

Table 1 Computed energies (kcal/mol) for the DPIEL, DPI, and DCIEL (ligand) with the PH domain of Akt

Compounds [PL]a Van derWaals

[PL]Electrostatic

[PL] Totalenergy

[L]b Van derWaals

[L]Electrostatic

[L]Total

energy

Bindingenergy

DPIEL �65.2 �254.1 �319.3 �89.1 �339.5 �428.6 �109.3DPI �89.7 �177.7 �267.4 �61.4 �265.4 �326.8 �59.4DCIEL �87.3 �180.4 �267.7 �53.2 �271.1 �324.3 �56.6

a [PL], protein-ligand complex.b [L], ligand.

Fig. 3. Binding sites and specificity of PH domains. A, interactions be-tween the Akt PH domain and DPIEL; B, interactions between the Akt PHdomain and DCIEL; C, interactions between the Akt PH domain and DPI.The ribbon of the PH domains is colored in green. The amino acidsthought to be involved in PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2 binding inthe PH domain are represented in ball and stick model and numbered inwhite. White dotted lines, hydrogen bonds occurring between the com-pound and amino acids of the protein.

Fig. 4. DPIs inhibit Akt1 kinase activity in vitro. Akt1 was immunopre-cipitated from NIH3T3 cells using an anti-Akt1 isoform-specific antibody.DPIs and Wortmannin were added for 1 h, and kinase activity was meas-ured as described in “Material and Methods.” The results are the averageof three separate experiments. Bars, SD. �, P � 0.1; ��, P � 0.05compared with the PDGF-stimulated control.




Akt in DU-145 and LNCaP prostate cancer cells (Fig. 6A) andcaused a dose-dependent increase in apoptosis (Fig. 6B).

DPIs Inhibit Bad Phosphorylation in a Dose-dependentManner. Akt phosphorylates the antiapoptotic protein Badon Ser136 (42, 43). Fig. 7, A and B shows the effects of theDPIs on Bad phosphorylation measured in cell lysates usinga specific anti-phospho-Ser136-Bad antibody (Panel A). BothDPI and DPIEL significantly inhibited Bad phosphorylation inPDGF-stimulated NIH3T3 cells. Panel B represents the quan-tification of phospho-Ser136-Bad from at least five differentWestern blots.

DPIs Inhibit PI-3 Kinase Activity in Vitro. The ability ofthe DPIs to inhibit PI-3 kinase was measured in an in vitrokinase assay (Fig. 8, A and B). DPI, DPIEL, and DCIEL (data

not shown for DCIEL) inhibited PI-3 kinase activity in a dose-dependent manner with IC50s (�SD) of 38.4 � 4.1, 16.4 �2.9, and 15.5 � 1.8 �M, respectively. A representative chro-matogram is shown in Panel A.

DPIs Do Not Inhibit Other PH Domain-containing Pro-teins Involved in the Pathway. PDK-1 is a PH domaincontaining serine kinase that phosphorylates Akt on Ser473

(18). It also phosphorylates conventional PKCs (44). To in-vestigate the effects of DPIEL on PDK-1 activity, we havemeasured the phosphorylation of PKCs in MCF-7 breastcancer cells. DPIEL had no effect on PKC phosphorylation inMCF-7 cells (Fig. 9). The phosphorylation on Ser241 of PDK-1is necessary for its activity (45). We tested whether DPIELcan affect PDK-1 activation by using specific anti-Phosho-Ser241-PDK-1 antibodies. No change in PDK-1 phosphoryl-ation was observed in the presence of DPIEL in MCF-7breast cancer cells (Fig. 9). Erk (p42/44) phosphorylation wasalso not affected by DPIEL. As shown in other cell lines(NIH3T3, Fig. 5B; DU145 and LNCaP; Fig. 6A), increasingconcentrations of DPIEL inhibit Akt phosphorylation onSer473 in MCF-7 cells.

Constitutive myr-Akt Activity Is Not Affected by DPIs.To test the possibility that DPIs interact directly with the PHdomain of Akt as suggested by the immunohistochemistrystudies, NIH3T3 cells were transiently transfected with amyristylated (myr) Akt1 construct, which permanently local-izes Akt at the plasma membrane where it is constitutivelyactivated (46). The cells were incubated with DPI, DPIEL, orWortmannin for 2 days and counted (Fig. 10A). The growthinhibitory effects of DPIEL were abolished in myr-Akt-trans-fected cells, and the growth inhibitory effects of DPI wereconsiderably reduced. We also measured Akt1 phosphoryl-

Fig. 5. Inhibition of Akt activity by DPIs in NIH3T3 mouse fibroblast cells.A, Akt isoforms present in NIH3T3 cells after immunoprecipitation withspecific antibodies (duplicate samples). Left panel, a total cell lysate ofNIH3T3 cells. In B, NIH3T3 cells were treated with 1 �M Wortmannin for 1 hor with 20 �M DPI, DPIEL, DCIEL, or PtdIns for 2 h and then stimulatedwith 50 ng/ml PGDF for 1 h. After stimulation, cells were lysed, andsamples were run on a 7.5% SDS-PAGE. Akt activation was measured byWestern blotting using specific anti-phospho-Ser473Akt antibodies (toppart, B). Arrow, the position of phospho-Ser473Akt. Total Akt is shown onthe bottom part of B. C, densitometric analysis of the bands correspond-ing to phosphorylated Akt. The values are reported as a percentage, with100% corresponding to phosphorylated Akt in the presence of PDGF.Mean of five observations with SD.

Table 2 Effects of DPIs on cell proliferation of cancer cells and Aktactivity in NIH3T3 cells

CompoundNIH3T3 cellproliferation(IC50, �M)

HT29 cellproliferation(IC50, �M)

MCF-7 cellproliferation(IC50, �M)

Akt inhibitionin NIH-3T3(IC50, �M)

DPI 17.6 ND ND 27.4 � 4.4DPIEL 4.3 2.1 7.2 1.5 � 0.3DCIEL 14.1 2.5 12.0 12.5 � 2.0

Fig. 6. DPIEL inhibits Akt and induces apoptosis in a dose-dependentmanner in human prostate cancer cells. A, Akt phosphorylation and ex-pression (top and bottom panels, respectively) in human DU-145 andLNCaP prostate cancer cells in the presence of 5 and 20 �M DPIEL. In B,apoptosis was measured in prostate cancer cells after 24-h exposure toincreasing concentrations of DPIEL. Values are the means of triplicatesamples; bars, SD.




ation in Akt1-transfected- and myr-Akt1-transfected NIH3T3cells in the presence and absence of the DPIs (Fig. 10, B andC). Myr-Akt1 was phosphorylated in the absence of PDGF(46), and this phosphorylation was not affected by DPIEL(Fig. 10, B and C). DPI caused a small inhibition of myr-Aktphosphorylation. Unexpectedly, Wortmannin completely in-hibited myr-Akt phosphorylation.

DiscussionOur work shows that the DPIs are inhibitors of the activationof Akt1 in NIH3T3 cells and that this inhibition is associatedwith increased apoptosis and the inhibition of the phospho-rylation of at least one of the downstream targets of Akt, thecell survival protein, Bad (42, 43). The most active DPI wasthe ether lipid analogue DPIEL with an IC50 for Akt1 inhibitionin NIH3T3 cells of 1.5 �M. The DPIs were effective inhibitorsof cell growth with IC50s for DPI, DPIEL, and DCIEL of 18, 4,and 14 �M, respectively. The cell growth inhibitory effects ofDPIEL on NIH3T3 cells could be prevented by expressingmyristoylated-Akt1, which is a membrane-bound and con-stitutively active Akt (46). DPIEL had no effect on myristoy-lated-Akt1 phosphorylation. DPIs also inhibited Akt in mouseNIH3T3 cells, human MCF-7 breast cancer cells, and humanLNCaP and DU-145 prostate cancer cells. Thus, the growthinhibitory effects of the DPIs appear to be associated withthe inhibition of Akt.

The mechanism by which the DPIs could enter cells toinhibit Akt is not known, but related compounds with ether

lipid groups, such as alkyl-lysophospholipids, i.e., edelfosine(or ET-18-OCH3), miltefosine (HePC) rapidly enter cells andinhibit cell growth (47, 48). Edelfosine is able to inhibit Akt athigher concentrations than DPIEL (data not shown and Ref.49). DPIEL appears to be a specific inhibitor of the PH do-main of Akt. DPIEL had no effect on the activity of another PHdomain containing protein in the Akt pathway, PDK-1. PDK-1is constitutively located at the plasma membrane and re-quires phosphorylation on Ser241 to be active (45). DPIEL didnot affect the phosphorylation on Ser241 and did not affect

Fig. 7. DPIs inhibit Bad phosphorylation in mouse NIH3T3 fibroblasts.NIH3T3 cells were stimulated with 50 ng/ml PDGF in the presence or ab-sence of DPI, DPIEL, or Wortmannin, as described in Fig. 5. Anti-phospho-Ser136-Bad antibody was used to detect phosphorylated Bad (A). The resultsare the average of three separate experiments; bars, SD. �, P � 0.1; ��, P �0.05 as compared with the PDGF-stimulated control (B).

Fig. 8. Effects of DPIs on PI-3 kinase activity. NIH3T3 cells were stim-ulated for 10 min with 50 ng/ml PDGF, and PI-3 kinase was immunopre-cipitated with antiphosphotyrosine antibody. PI-3 kinase activity wasmeasured by an in vitro kinase assay in the presence or absence of theDPIs or Wortmannin. A, radiolabeled PtdIns(3,4,5)P3 (arrow) detected afterthin-layer chromatography. B, inhibition of PI-3 kinase activity by DPI (E)and DPIEL (F).

Fig. 9. pPKCs, pERKs, and pPDK-1 are not altered by DPIEL. HumanMCF-7 breast cancer cells were treated with increasing concentrations ofDPIEL for 2 h and stimulated with 100 ng/ml epidermal growth factor for30 min. After stimulation, cells were lysed, and samples were run on a7.5% SDS-PAGE. Membranes were probed with anti-phospho-Ser473-Akt antibody, anti-phospho-Thr308-Akt antibody, a Pan phospho-PKCantibody, an anti-phospho-Erk1/2, and an anti-phospho-Ser241-PDK-1antibody.




PDK-1 activity as measured by phosphorylation of PKCs,which are also substrates for PDK-1. DPIEL also did notsignificantly alter p42/44 Erk phosphorylation. DPIEL is ableto inhibit PI-3 kinase (IC50 16 �M) and is a weak directinhibitor of Akt1 kinase activity (IC50 17 �M). However, theinhibition of Akt1 activation and cell growth by DPIEL oc-curred at 10-fold lower concentrations. Thus, the predomi-nant activity of DPIEL in cells at concentration caused celldeath and apoptosis.

We studied two other DPI analogues, DPI itself and thecarbonate analogue DCIEL that was synthesized to preventpossible breakdown by PtdIns-specific phospholipase C (50,51). Both analogues were less potent than DPIEL at inhibitingAkt1 in cells and also less specific. DPI and DCIEL wereinhibitors of PI-3 kinase at concentrations that inhibited Akt1in cells. Although DPI is able to inhibit Akt kinase activity invitro at a lower concentration than DPIEL (8 versus 17.5 �M,respectively), it was a weak inhibitor of Akt in cells, probablybecause DPI may be broken down by phospholipases. DPI,unlike DPIEL, inhibited myristoylated-Akt1 phosphorylation.In this respect, it was similar to the PI-3 kinase inhibitorWortmannin (40, 41), which, surprisingly, almost completelyblocked myristoylated-Akt1 phosphorylation in NIH3T3 cells.This observation suggests that PI-3 kinase, which has serine/threonine kinase activity (52), could be phosphorylatingmembrane-bound Akt1 directly. However, the Wortmanninconcentration used in the cells was much higher than re-quired to inhibit purified PI-3 kinase so that other mecha-nisms could be responsible for Wortmannin’s effects onmyristoylated-Akt1 phosphorylation.

The PH domain is a phosphoinositide-binding motif foundin a number of signal-transducing proteins, including Akt,that gives membrane-binding properties to the host proteins.The three-dimensional organization of individual PH domainsgives different binding specificities (53) with the Group 1 PHdomain (containing, e.g., IRS-1’s or Btk’s PH domains) rec-ognizing PtdIns(3,4,5)P3, the Group 2 PH domain recognizingPtdIns(4,5)P2 (containing, e.g., the PH domains of mSos1 or�ARK), and the Group 3 PH domain recognizingPtdIns(3,4)P2 and PtdIns(3,4,5)P (containing, e.g., Akt; Ref.54). We found that DPIs bind directly to the PH domain of Aktin an in vitro binding assay. However, the assay only showsthat the DPIs bind to the PH domain of Akt and does notallow the calculation of relative binding affinities. Molecularmodeling and docking analysis of DPIEL into the PtdIns-3-phosphate binding pocket of a variety of PH domains al-lowed us to demonstrate a high affinity for the binding ofDPIEL to Group 3 PH domain of Akt (see Fig. 4 and data notshown). DPIEL bound strongly to the PH domain of Akt ascompared with DPI and DCIEL. These results are consistentwith the respective effects of each compound on Akt activityin cells. DPIEL appears to be the most potent Akt inhibitor inthe cells because it binds the best to the PH domain of Akt.Moreover, we found that DPIEL bound with relatively lowaffinity to the Group 1 PH domains of the Group Btks andIRS-1, whereas no significant binding of DPIEL to the Group2 PH domains of �ARK and mSos1 PH was observed (datanot shown). As noted previously we also found that DPIELdid not block the activity of the Group 3 PH domain proteinPDK-1 in cells. Thus, the docking and activity studies sug-gest that DPIEL has also specificity for Akt among the PHdomain containing proteins investigated.

There are a number of cellular mechanisms by which Aktinhibition by the DPIEL may occur (Fig. 11). We have foundusing immunohistochemistry that DPIEL blocks the translo-cation of Akt1 from the cytoplasm to the plasma membranein NIH3T3 cells where it normally binds to PtdIns-3-phos-phates in the inner leaflet of the membrane (data not shown).

Fig. 10. Myr-Akt1-transfected cells are not affected by DPIs. NIH3T3cells were transiently transfected with myr-Akt1-HA or Akt1-GST for 2days. Cells were then treated as described in Fig. 2. In A, cell proliferationwas measured after 3 days of exposure to DPI and DPIEL. Values are themean of three determinations; bars, SD. B, Akt activation measured byWestern blotting using a specific anti-phospho-Ser473Akt antibody. Val-ues are the mean of at least three Western blots from three separateexperiments. Bars, SD. �, P � 0.1; ��, P � 0.05 as compared with thePDGF-stimulated control. C, a representative Western Blot of phospho-Ser473Akt in NIH3T3 cells and myr-Akt-HA NIH3T3-transfected cells in thepresence or absence of PDGF and presence of 10 and 20 �M DPIEL, 20�M DPI, and 1 �M Wortmannin.




Taken together, a first mechanism that can be suggested isthat DPIEL could block the formation of PtdIns-3-phos-phates. However, inhibition of PI-3 kinase by DPIEL onlyoccurs at concentrations of an order of magnitude higherthan required to inhibit Akt. We have also not seen a de-crease in the levels of tumor PtdIns(3,4,5)P3 by DPIEL meas-ured by immunohistochemistry.6 A second mechanism isthat DPIEL, or a metabolite, in the cytoplasm binds the PHdomain of Akt, thus trapping Akt in the cytoplasm. We foundthat DPIEL binds to the PH domain of Akt1 to the sameextent as PtdIns(3,4)P2. We do not know the localization ofDPIEL in the cell or whether it is metabolized. We haveobserved that DPIEL treatment gives a more diffuse immu-nohistochemical staining of Akt1 in the cytoplasm than theone seen in nontreated cells (data not shown). Akt has beenreported to oligomerize through a PH domain interaction,which could contribute to the activation of Akt (55). By bind-ing to the PH domain, DPIEL, or a metabolite, may preventthe oligomerization of Akt. A third mechanism is that DPIELis in the plasma membrane as described similarly for alkyl-lysophospholipids (48). Akt binds to DPIEL at the membraneand by preventing Akt phosphorylation, induces the releaseof Akt back into the cytosol. Such a mechanism could ex-plain the diffuse immunohistochemical staining observed asAkt is shuttling from the cytosol to the plasma membranewithout complete activation (data not shown). A fourth mech-anism is a reorganization of the plasma membrane by DPIEL,which prevents the binding of Akt to PtdIns(3,4,5)P3 withoutthere being direct binding of DPIEL to Akt. Although we haveno evidence to support or disprove such a mechanism, ourfinding that DPIEL binds to the PH domain of Akt and thedocking studies that show a high affinity specific interactionwith Akt’s PH domain compared with the PH domain of otherproteins and compared with the other DPIs suggest that amechanism with direct binding to the PH domain of Akt ismost likely to occur in the cells.

In summary, we have shown that the DPIs bind to the PHdomain of Akt and inhibit Akt activation in cells. DPIEL wasthe most active compound, and it prevented Akt transloca-tion to the plasma membrane. DPIEL had no effect on myr-Akt phosphorylation and was much less effective at inhibitingthe growth of these cells. The results suggest that the growthinhibitory effects of DPIEL are caused by inhibition of Aktactivation probably after a block of Akt’s translocation fromthe cytoplasm to the plasma membrane. This is attributable,at least in part, to the binding of DPIEL or a metabolite to thePH domain of Akt. Thus, the DPIs may represent a new classof potential anticancer drugs with a novel mechanism ofaction causing specific inhibition of the PH domain-depen-dent translocation of Akt.

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2003;2:389-399. Mol Cancer Ther Emmanuelle J. Meuillet, Daruka Mahadevan, Hariprasad Vankayalapati, et al.

1-Inositol Analoguesmyoby D-3-Deoxy-Phosphatidyl-Specific Inhibition of the Akt1 Pleckstrin Homology Domain

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