Potent and selective inhibitors of Akt kinases slow the ... · Potent and selective inhibitors of...

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Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo Yan Luo, 1 Alexander R. Shoemaker, 1 Xuesong Liu, 1 Keith W. Woods, 1 Sheela A. Thomas, 1 Ron de Jong, 4 Edward K. Han, 1 Tongmei Li, 1 Vincent S. Stoll, 2 Jessica A. Powlas, 1 Anatol Oleksijew, 1 Michael J. Mitten, 1 Yan Shi, 1 Ran Guan, 1 Thomas P. McGonigal, 1 Vered Klinghofer, 1 Eric F. Johnson, 1 Joel D. Leverson, 1 Jennifer J. Bouska, 1 Mulugeta Mamo, 2 Richard A. Smith, 2 Emily E. Gramling-Evans, 2 Bradley A. Zinker, 3 Amanda K. Mika, 3 Phong T. Nguyen, 3 Tilman Oltersdorf, 2 Saul H. Rosenberg, 1 Qun Li, 1 and Vincent L. Giranda 1 1 Cancer Research, 2 Structural Biology, and 3 Metabolic Disease Research, Abbott Laboratories, Abbott Park, Illinois and 4 Idun Pharmaceuticals, San Diego, California Abstract The Akt kinases are central nodes in signal transduction pathways that are important for cellular transformation and tumor progression. We report the development of a series of potent and selective indazole-pyridine based Akt inhibitors. These compounds, exemplified by A-443654 (K i = 160 pmol/L versus Akt1), inhibit Akt-dependent signal transduction in cells and in vivo in a dose- responsive manner. In vivo , the Akt inhibitors slow the progression of tumors when used as monotherapy or in combination with paclitaxel or rapamycin. Tumor growth inhibition was observed during the dosing interval, and the tumors regrew when compound administration was ceased. The therapeutic window for these compounds is narrow. Efficacy is achieved at doses f2-fold lower than the maximally tolerated doses. Consistent with data from knockout animals, the Akt inhibitors induce an increase in insulin secretion. They also induce a reactive increase in Akt phosphorylation. Other toxicities observed, including malaise and weight loss, are consistent with abnormalities in glucose metabolism. These data show that direct Akt inhibition may be useful in cancer therapy, but significant metabolic toxicities are likely dose limiting. [Mol Cancer Ther 2005;4(6):977 – 86] Introduction Akt activity is elevated in a large proportion of human malignancies, where it plays a central role in inducing a malignant phenotype by both promoting cell growth and decreasing apoptosis (1, 2). Akt1 is a serine/threonine protein kinase that was first discovered as the human homologue of the transforming gene in the AKT-8 onco- genic virus, which was isolated from a spontaneous thymoma in the AKR mouse (3, 4). Since the discovery of human Akt1 (also called protein kinase B), two additional mammalian Akt isoforms, Akt2 and Akt3, have been identified (5 – 8). Akt is downstream of phosphatidylinositol 3-kinase (PI3K) and is a critical node in this signal transduction pathway. The activation of Akt by PI3K is antagonized by the tumor suppressor PTEN (9). Thus, the increased Akt activity that is observed in most human malignancies could be the result of (a) an increased Akt expression, (b) increased PI3K activity, or (c) decreased PTEN activity (10). Correlative evidence for all three of these mechanisms has been found in human tumors. Akts are overexpressed in a variety of human tumors (7, 11–13), and at the genomic level, AKT1 and AKT2 have been shown to be amplified in a number of cancer types (7). PI3K activity is increased by numerous growth factors, many of which are themselves targets for cancer therapy (e.g., KDR and HER2; refs. 14–16). PTEN mutations that result in increased Akt activity have likewise been described in a wide variety of malignancies (17 – 19). Alterations that increase Akt activity (e.g., PTEN loss, PI3K up-regulation, and increased Akt expression) rival alterations in the p53/ p16 pathway as the most common changes found in malignant tumor cells (10, 20). In addition to the correlative data, there is also consid- erable experimental evidence for the importance of PI3K, Akt, and PTEN in neoplasia. The constitutive expression of active Akts promotes tumorigenesis when these Akt- expressing clones are inoculated into nude mice (21 – 23). Mice heterozygous for Pten deletions, where Akt activity is dramatically increased, develop neoplasms in colon, testis, thyroid, prostate, endometrium, and liver. These animals also have an increased incidence of lymphoma and leukemia (24, 25). In humans, there are three closely related and inherited cancer syndromes caused by mutations in the PTEN locus: Cowden disease (26), Lhermitte-Duclos disease (26), and Bannayan-Zonana syndrome (27). Patients with these disorders have multiple benign tumors and greatly increased incidence of carcinomas. Received 1/6/05; revised 3/16/05; accepted 4/11/05. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: R. de Jong is currently at Syrrx, Inc., 10410 Science Center Drive, San Diego, CA 92121. Requests for reprints: Vincent L. Giranda, Cancer Research, Abbott Laboratories, 100 Abbott Park Road, IL 60064-6117. Phone: 847-937- 0268; Fax: 847-938-2365. E-mail: [email protected] Copyright C 2005 American Association for Cancer Research. 977 Mol Cancer Ther 2005;4(6). June 2005 on April 18, 2021. © 2005 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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Page 1: Potent and selective inhibitors of Akt kinases slow the ... · Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo YanLuo,1 AlexanderR.Shoemaker,1 XuesongLiu,1

Potent and selective inhibitors of Akt kinases slow theprogress of tumors in vivo

Yan Luo,1 Alexander R. Shoemaker,1 Xuesong Liu,1

Keith W. Woods,1 Sheela A. Thomas,1

Ron de Jong,4 Edward K. Han,1 Tongmei Li,1

Vincent S. Stoll,2 Jessica A. Powlas,1

Anatol Oleksijew,1 Michael J. Mitten,1 Yan Shi,1

Ran Guan,1 Thomas P. McGonigal,1

Vered Klinghofer,1 Eric F. Johnson,1

Joel D. Leverson,1 Jennifer J. Bouska,1

Mulugeta Mamo,2 Richard A. Smith,2

Emily E. Gramling-Evans,2 Bradley A. Zinker,3

Amanda K. Mika,3 Phong T. Nguyen,3

Tilman Oltersdorf,2 Saul H. Rosenberg,1 Qun Li,1

and Vincent L. Giranda1

1Cancer Research, 2Structural Biology, and 3Metabolic DiseaseResearch, Abbott Laboratories, Abbott Park, Illinois and4Idun Pharmaceuticals, San Diego, California

AbstractThe Akt kinases are central nodes in signal transductionpathways that are important for cellular transformationand tumor progression. We report the development of aseries of potent and selective indazole-pyridine based Aktinhibitors. These compounds, exemplified by A-443654(K i = 160 pmol/L versus Akt1), inhibit Akt-dependentsignal transduction in cells and in vivo in a dose-responsive manner. In vivo, the Akt inhibitors slow theprogression of tumors when used as monotherapy or incombination with paclitaxel or rapamycin. Tumor growthinhibition was observed during the dosing interval, and thetumors regrew when compound administration wasceased. The therapeutic window for these compounds isnarrow. Efficacy is achieved at doses f2-fold lower thanthe maximally tolerated doses. Consistent with data fromknockout animals, the Akt inhibitors induce an increase ininsulin secretion. They also induce a reactive increase inAkt phosphorylation. Other toxicities observed, includingmalaise and weight loss, are consistent with abnormalitiesin glucose metabolism. These data show that direct Akt

inhibition may be useful in cancer therapy, but significantmetabolic toxicities are likely dose limiting. [Mol CancerTher 2005;4(6):977–86]

IntroductionAkt activity is elevated in a large proportion of humanmalignancies, where it plays a central role in inducing amalignant phenotype by both promoting cell growth anddecreasing apoptosis (1, 2). Akt1 is a serine/threonineprotein kinase that was first discovered as the humanhomologue of the transforming gene in the AKT-8 onco-genic virus, which was isolated from a spontaneousthymoma in the AKR mouse (3, 4). Since the discovery ofhuman Akt1 (also called protein kinase B), two additionalmammalian Akt isoforms, Akt2 and Akt3, have beenidentified (5–8).

Akt is downstream of phosphatidylinositol 3-kinase(PI3K) and is a critical node in this signal transductionpathway. The activation of Akt by PI3K is antagonized bythe tumor suppressor PTEN (9). Thus, the increased Aktactivity that is observed in most human malignanciescould be the result of (a) an increased Akt expression, (b)increased PI3K activity, or (c) decreased PTEN activity(10). Correlative evidence for all three of these mechanismshas been found in human tumors. Akts are overexpressedin a variety of human tumors (7, 11–13), and at thegenomic level, AKT1 and AKT2 have been shown to beamplified in a number of cancer types (7). PI3K activity isincreased by numerous growth factors, many of which arethemselves targets for cancer therapy (e.g., KDR andHER2; refs. 14 – 16). PTEN mutations that result inincreased Akt activity have likewise been described in awide variety of malignancies (17–19). Alterations thatincrease Akt activity (e.g., PTEN loss, PI3K up-regulation,and increased Akt expression) rival alterations in the p53/p16 pathway as the most common changes found inmalignant tumor cells (10, 20).

In addition to the correlative data, there is also consid-erable experimental evidence for the importance of PI3K,Akt, and PTEN in neoplasia. The constitutive expression ofactive Akts promotes tumorigenesis when these Akt-expressing clones are inoculated into nude mice (21–23).Mice heterozygous for Pten deletions, where Akt activity isdramatically increased, develop neoplasms in colon, testis,thyroid, prostate, endometrium, and liver. These animalsalso have an increased incidence of lymphoma andleukemia (24, 25). In humans, there are three closely relatedand inherited cancer syndromes caused by mutations in thePTEN locus: Cowden disease (26), Lhermitte-Duclosdisease (26), and Bannayan-Zonana syndrome (27). Patientswith these disorders have multiple benign tumors andgreatly increased incidence of carcinomas.

Received 1/6/05; revised 3/16/05; accepted 4/11/05.

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 toindicate this fact.

Note: R. de Jong is currently at Syrrx, Inc., 10410 Science Center Drive,San Diego, CA 92121.

Requests for reprints: Vincent L. Giranda, Cancer Research, AbbottLaboratories, 100 Abbott Park Road, IL 60064-6117. Phone: 847-937-0268; Fax: 847-938-2365. E-mail: [email protected]

Copyright C 2005 American Association for Cancer Research.

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In Drosophila , a germ line deletion of dAkt reverses thelarge cell phenotype in dPten�/� flies (28). More recently,deletion of Akt1 has been shown to reverse the aggressivegrowth phenotype of Pten�/� mouse embryonic stem cells(29). These data suggest the tumorigenic phenotypecaused by loss of PTEN can be largely reversed byinhibition of Akt.

The prevalence of Akt activation in human tumors,coupled with the genetic experiments showing that PTENdeletions or active Akt can induce tumor formation,suggests that inhibition of Akt may be useful in thetreatment of neoplastic diseases. Indeed, there have beenattempts to modulate the activity of this pathway.Inhibition of PI3K directly has been reported to decreasethe progression of tumors in rodents (30, 31). Likewise,using rapamycin or one of its analogues to inhibit the PI3Kpathway downstream at mammalian target of rapamycin(mTOR) has been shown to reduce tumor growth in mousemodels (several compounds are currently in human clinicaltrials; refs. 32, 33). There have also been reports ofcompounds that inhibit the activation of Akt [e.g., heatshock protein 90 inhibitors (34), pleckstrin homologydomain inhibitors (35, 36), and others (37)]. Here we reporton the discovery of potent compounds that directly inhibitthe kinase activity of Akt. We also report the effects of thesecompounds on transformed cells in vitro as well as ontumor progression in vivo .

Materials andMethodsCrystallization and X-rayAnalysisProtein kinase A (PKA) was purified (38), concentrated to

20 mg/mL, and complexed with peptide inhibitor of PKAfor 1 hour and complexed with A-443654 (39). Crystalswere transferred to cryosolutions that contained wellsolution plus increasing amounts of glycerol, soaking for1 minute in 5%, 15%, and 25% glycerol. Crystals were thenfrozen in a stream of 100jK nitrogen. Diffraction data wererecorded using a MAR-165 CCD detector system on aRigaku RU-2000 rotating anode X-ray generator (Danvers,MA) operating at 100 mA and 50 kV. Diffraction data werereduced using HKL2000 (HKL Research, Inc., Charlottes-ville, VA) and the protein model (accession number 1YDT)with the inhibitor (H89) and the phosphorylation sitesomitted for initial phasing. Generation of initial electrondensity maps and structure refinement was achieved usingCNX program package. Electron density maps weregenerated using the program QUANTA 97/2001 (Accelrys,San Diego, CA), and compound A-44654 was fit to theelectron density. The structure was determined to aresolution of 2.7 A and refined to Rwork = 24.41% andRFree = 30.18 %.

In vitro KinaseAssaysRecombinant CK2, PKCg, and PKCy (Calbiochem, San

Diego, CA); PKA (Panvera, Madison, WI); cyclin-dependentkinase 2/CyclinA, GSK3h, MAPK-AP2, Src, and RSK2 (UpstateBiotechnology, Charlottesville, VA); extracellular signal–regulated kinase 2 (New England Biolabs, Beverly, MA),

cKit (544-976; ProQinase, Freiburg, Germany) were com-mercially obtained. His-tagged Akt1 (S378A, S381A, T450D,S473D; 139-480), Chk1 (1-269), KDR (789-1354), Flt-1 (786-1338), and phosphatidylinositide-dependent kinase 1 (1-396), were expressed using the FastBac bacculovirusexpression system (Life Technologies, Gaithersburg, MD)and purified using either nickel (his-tag) or glutathioneS-transferase affinity chromatography. Peptides substrateshad the general structure biotin-Ahx-peptide with thefollowing sequences: Akt, EELSPFRGRSRSAPPNLWA-AQR; PKA, LRRASLG; PKCg and PKCy, ERMRPRK-RQGSVRRRV; CK2, RRADDSDDDDD; cyclin-dependentkinase 2, LPPCSPPKQGKKENGPPHSHTLKGRRAAFD-NQL; GSK3h, YRRAAVPPSPSLSRHSSPHQS(p)EDEEE;MAPK-AP2, KKLNRTLSVA; RSK2, KKKNRTLSVA; extra-cellular signal– regulated kinase 2, KRELVEPLTPSGE-APNQALLR; Chk1, AKVSRSGLYRSPSMPENLNRPR;phosphatidylinositide-dependent kinase 1, KTFCGTPEY-LAPEVRREPRILSEEEQEMFRDFDYIADWC; KDR, Flt-1,and cKit, AEEEYFFLFA-amide. For Src assays, the bio-tinylated substrate PTK-2 (Promega, Madison, WI) wasused. Inhibition of kinase activity was assessed using aradioactive FlashPlate-based assay platform as previouslydescribed (40).

Alamar Blue CellViabilityAssayThe cells on 96-well plates were gently washed with

200 AL of PBS. Alamar Blue reagent (Biosource Interna-tional, Carmarillo, CA) was diluted 1:10 in normalgrowth media. The diluted Alamar Blue reagent (100 AL)was added to each well on the 96-well plates andincubated until the reaction was complete as permanufacturer’s instructions. Analysis was done using anfmax Fluorescence Microplate Reader (Molecular Devices,Sunnyvalle, CA), set at the excitation wavelength of544 nm and emission wavelength of 595 nm. Data wereanalyzed using SOFTmax PRO software provided by themanufacturer.

Protein Extraction andWestern Blot AnalysisCells were treated with 0 to 30 Amol/L of Akt inhibitors

for 2 hours. All drug samples were adjusted to containequal volumes of the vehicle (DMSO). Cells were har-vested, sonicated for 5 minutes in ice-cold insect cell lysisbuffer [BD PharMingen, San Diego, CA; 10 mmol/L Tris-HCl (pH 7.5), 130 mmol/l NaCl, 1% Triton X-100, 10mmol/L NaF, 10 mmol/L NaPi, 10 mmol/L NaPPi and1 Ag/mL microcyctin LR plus the protease inhibitorsaprotinin (10 Ag/mL), leupeptin (10 Ag/mL), and phenyl-methylsulfonyl fluoride (1 mmol/L)], and centrifuged at15,300 rpm for 10 minutes. The concentrations of the totallysate protein were determined using a standard Bradfordassay (Bio-Rad, San Diego, CA).

MiaPaCa-2 pancreatic tumor–bearing mice were treatedwith control vehicle or A-443654 at 7.5 or 75 mpk for2 hours. Tumors were isolated and immediately frozen inliquid nitrogen. Frozen tumor tissues were sliced in thinsections and resuspended in 500 AL of lysis buffer.This suspension was homogenized with a PolytronPT 1200C (Kinematica AG, Luzern, Switzerland) for

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2 minutes. The homogenized tissues were centrifuged at15,300 rpm for 10 minutes, supernatants were collected,and the protein concentrations were determined with theBradford method.

For Western blot analysis, 40 Ag of protein from the totalcell lysate were electrophoresed by SDS-PAGE. Theproteins were then electrotransfered onto immobilon-Pmembranes (Millipore, Bedford, MA), using transfer buffer(25 mmol/L Tris, 190 mmol/L glycine, and 10% methanol).Membranes were treated with blocking buffer (50 mmol/LTris, 200 mmol/L NaCl, 0.2% Tween 20, 5% nonfat drymilk) for 1 hour at room temperature and incubated with1:1,000 dilution of antibodies (phospho-GSK3 a/h Ser21/9,phospho-TSC2 Thr1462, phospho-FOXO1A/3A Thr24/32,phospho-ribosomal protein S6 Ser235/236 phospho-Akt1Ser473, and phospho-mTOR Ser2448; all from Cell SignalingTechnology, Beverly, MA) for 16 hours at 4jC. Afterwashing with blocking buffer without 5% nonfat dry milk(washing buffer) for 30 minutes, the membranes wereincubated with horseradish peroxidase–linked anti-rabbitdonkey serum (1:1,000; Amersham, Arlington Heights, IL)for 1 hour. Membranes were then washed with washingbuffer and immune detection was done using theenhanced chemiluminescence Western blotting detectionsystem (Amersham). Quantitation of Western blots wasdone using a phospho-imager (Amersham Biosciences,Piscataway, NJ).

FL5.12Akt1-,Akt2-,orAkt3-OverexpressingCellLinesStable transfectants of FL5.12 cells expressing full-length

human Akt1, Akt2, or Akt3 were generated by electro-poration with a pCIneo plasmid (Promega) containing AktcDNAs with an NH2-terminal HA-tag (41). Single cloneswere isolated by limiting dilution and selection for G418resistance (Invitrogen, Carlsbad, CA). Expression wasconfirmed by immunoblotting with Akt isoform-specificantibodies. FL5.12/Akt cell lines were maintained in RPMI1640 supplemented with 10% fetal bovine serum, 10%WEHI-3B conditioned medium as a source of IL-3,nonessential amino acids, 1 mmol/L sodium pyruvate,2 mmol/L glutamine, 50 Amol/L h-mercaptoethanol, 50Ag/mL G418, and 2.5 mmol/L HEPES (pH 7.5) at 37jC in ahumidified atmosphere containing 5% CO2.

FOXO3ATranslocation AssayThe pFOXO3A-hrGFP construct was engineered by

inserting a FOXO3A fragment encoding NH2-terminalamino acids 1 to 400 into plasmid phrGFP (Stratagene, LaJolla, CA) using standard PCR and DNA recombinanttechniques. This sequence includes the nuclear import andexport signals and Akt phoshorylation sites (42). HeLacells were plated into 6-well plates at a density of 0.3 �106 cells per well and incubated at 37jC at 5% CO2 overnight. Cells were transiently transfected with pFOXO3A-hrGFP plasmid using Effectene (Qiagen, Valencia, CA). At40 hours post-transfection, cells were treated with com-pounds added to culture medium lacking phenol red.Equal amounts of DMSO were added to cells to a finalconcentration of 0.5%. After 6 hours, images of fluorescentlive cells were captured by microscopy.

Tumor Efficacy StudiesAnimal studies were conducted following the guide-

lines of the internal Institutional Animal Care and UseCommittee. Immunocompromised male scid mice (C.B-17-Prkdcscid) were obtained from Charles River Laboratories(Wilmington, MA) at 6 to 8 weeks of age. MiaPaCa-2 andPC-3 cells were obtained from the American Type CultureCollection (Manassas, VA). The 3T3-Akt1 cell line wasdeveloped and characterized in our laboratory (23). The 1� 106 3T3-Akt1 or 2 � 106 MiaPaCa-2 and PC-3 cells in50% Matrigel (BD Biosciences, Bedford, MA) were inocu-lated s.c. into the flank. For early treatment studies, micewere randomly assigned to treatment groups and therapywas initiated the day after inoculation. Ten animals wereassigned to each group, including controls. For establishedtumor studies, tumors were allowed to reach a designatedsize and mice were assigned to treatment groups of equaltumor size (n = 10 mice per group). Tumor size wasevaluated by twice weekly measurements with digitalcalipers. Tumor volume was estimated using the formula:V = L � W2 / 2. A-443654 was given s.c. in a vehicle of0.2% HPMC. A-674563 was given orally in a vehicle of 5%dextrose. Gemcitabine and paclitaxel were obtained fromEli Lilly and Company (Indianapolis, IN) and Bristol-Myers Squibb Co. (Princeton, NJ), respectively, and givenaccording to the manufacturers’ guidelines.

For the determination of plasma drug concentrations,proteins were precipitated with two volumes of acidifiedmethanol. Tissue samples were taken from the sameanimals and homogenized with 2 volumes of saline, thenprecipitated with 2 volumes of acetonitrile. Precipitatedproteins were removed by centrifugation and super-natants were stored at �20jC until analysis by UV-liquidchromatography or liquid chromatography-mass spec-trometry. Plasma and tissue extracts were injecteddirectly on a C18 reversed phase column (YMC-AQ 5A; Waters Co., Milford, MA) and eluted using combina-tions of acetonitrile, methanol and 8 mmol/L triethyl-amine acetate buffer (pH 4) with UV detection(Shimadzu 10A/VP, Shimadzu Scientific, Columbia,MD) or with acetonitrile and 0.1% acetic acid in waterfor mass spectrometer analysis (LCQ Duo, Thermoquest,San Jose, CA). Pharmacokinetic variables were calculatedusing WinNonlin software.(Pharsight Co., MountainView, CA).

Immunohistochemistry3T3-Akt1 and MiaPaCa-2 tumors were fixed for 48

hours in Streck Tissue Fixative (Streck Laboratories,Omaha, NE), processed, and embedded in paraffin; 5-Amtissue sections were blocked with streptavidin/biotincomplex followed by 0.3% bovine serum albumin beforeincubation with primary antibody (caspase-3: rabbit anti-active Caspase-3 at 1:400, BD PharMingen; phospho-Akt:rabbit anti-phospho-Akt at 1:200, Cell Signaling Technol-ogy) for 1 hour at room temperature. Secondary antibodywas used at 1:250 to 300 followed by streptavidin-biotinhorseradish peroxidase and 3,3V-diaminobenzidine colordevelopment.

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ResultsDiscoveryof Indazole-PyridineSeriesofAkt InhibitorsThe initial lead in this series was obtained from a high-

throughput screen (Fig. 1A, compound 1). This compoundweakly inhibited Akt (K i = 5 Amol/L). The potency wasimproved by addition of an indole ring to the aliphatic sidechain (Fig. 1A, compound 2), and still further improved byconstraining rotatable bonds in the linker between thecentral and distal pyridine rings (Fig. 1A, compound 3).Addition of a second hydrogen bonding interaction to thehinge-binding region resulted in A-443654, that exhibited aK i of 160 pmol/L, a 30,000-fold improvement in potencyversus the initial lead molecule. Finally, a compound withoral activity was achieved by replacing the indole with aphenyl moiety (Fig. 1A, A-674563).

The series of indazole-pyridine compounds bind to theATP site of Akt. These compounds are potent, ATPcompetitive, and reversible inhibitors of the Akt catalyzedphosphorylation activity. The three-dimensional structuresof these compounds complexed with a closely relatedprotein kinase, PKA (Fig. 1B-C), were determined by X-raycrystallography. Three hydrogen bond sets are critical tomaintain potency. The first is a canonical hydrogen bond tothe hinge region backbone found with most ATP-compet-itive kinase inhibitors. The second is formed between theinhibitor’s central pyridine ring and a conserved lysine(Lys72 in PKA and Lys181 in Akt1). The third set ofimportant hydrogen bonding interactions form between theprimary amino group of the inhibitor’s aliphatic side chainand the PKA residues Asn171 and Asp184 (Asn279 andAsp292 in Akt1). This position is normally occupied by adivalent cation of Mg2+ ATP.

In vitro ActivityA-443654 is a pan Akt inhibitor and has equal potency

against Akt1, Akt2, or Akt3 within cells (Fig. 2). A-443654is, however, very selective for Akt versus other kinases(Table 1). It is the least selective towards several closelyrelated kinases in the AGC family, including PKA andprotein kinase C (PKC). Even so, A-443654 is 40-foldselective for Akt over PKA. A-674563 is somewhat lessselective, particularly against the cyclin-dependent kinasesin the CMGC family. However, in spite of the selectivitydifferences between these two classes of Akt inhibitors,their behavior is similar in cells and in vivo (see below).

The phosphorylation of Akt downstream targets suchas GSK3a/h, FOXO3, TSC2, and mTOR were measuredas an indication of Akt activity within cells. The Aktinhibitors reduced the phosphorylation of all of theseproteins in a dose-dependent fashion (Fig. 3A). Asexpected, we also observed that FOXO3 translocates intothe nucleus upon the reversal of its phosphorylationinduced by Akt inhibition (Fig. 3B). Phosphorylation ofsignaling molecules further downstream in the Aktpathway, such as the S6 protein, was also reduced by theAkt inhibitors.

Together with the decrease in phosphorylation of Akttargets, we observed a concomitant increase in the Thr308

and Ser473 phosphorylation of Akt. This increase has been

observed with all of the Akt inhibitors tested and seems asensitive marker of Akt inhibition. However, in spite of theincreased Akt activation, the ability of Akt to phosphory-late its downstream targets was markedly decreased inthe presence of the inhibitors (Fig. 3A). The addition

Figure 1. A, a schematic representation of the discovery of the indazole-pyridine series of Akt inhibitors. B, the X-ray structure of A-443654bound to the ATP binding site of PKA. The structures of both Akt andPKA contain very similar ATP binding sites, with only four amino aciddifferences within the inhibitor binding sites and minimal differencesbetween the protein backbones. Dotted lines , hydrogen bonds betweenA-443654 and PKA. C, the line drawing also illustrates the A-443654mode of binding, with the indazole binding to the hinge region, thepyridine N binding to a conserved lysine, and the indole ring bindingunder the glycine-rich loop.

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of 20 Amol/L of the PI3K inhibitor Ly294002 together withA-443654 eliminates the Akt inhibitor induced increase inAkt phosphorylation (data not shown), suggesting in-creased phosphatidylinositol 3,4,5-triphosphate is requiredfor the A-443654 induced increase in Akt phosphorylation.

To investigate the effects of Akt inhibition on cellproliferation, we did an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The Akt inhibitorsslowed proliferation of tumor cells with an EC50 of 0.1Amol/L for A-443654 and 0.4 Amol/L for A-674563 (Fig. 3C).

In vivo AntitumorActivityWe tested both A-443654 and A-674563 for their ability to

inhibit tumor growth in vivo (Fig. 4). A-443654 is morepotent and more selective than A-674563 but is notavailable orally. Furthermore, the length of time A-443654

can be given is limited to 14 days due to severe injectionsite irritation. Nevertheless, in spite of the differencesbetween these compounds, both compounds behavesimilarly in vivo.

The effect of the inhibitors was examined in several Akt-dependent models. We tested a genetically engineeredmodel, where 3T3 murine fibroblasts stably express aconstitutively active form of Akt1. In contrast to theparental 3T3 cell line, the 3T3-Akt1 cells have a dramat-ically increased ability to form colonies in soft agar as wellas tumors in immunocompromised mice, suggesting theactive Akt1 is responsible for the transformed phenotype(23). In this model, A-443654 inhibited the growth of thetumors (Fig. 4A); whereas the 2-methyl analogue of A-443654, which is 5,000-fold less active versus Akt1,showed no effect in this model. We also tested thecompounds in a MiaPaCa-2 human pancreatic cellxenograft model, where we compared the efficacy withgemcitabine. MiaPaCa-2 is a human pancreatic carcinomaline with constitutively active Akt (43). Here again, theAkt inhibitor significantly slowed the growth of thetumors (Fig. 4B). A-443654 also showed antitumor activityin two rat orthotopic syngeneic models of tumor growth, aMatLyLu prostate carcinoma and a 9L glioblastoma (datanot shown).

In all models tested, the tumors rapidly regrew aftercompound administration was stopped. The cytostaticbehavior of the inhibitors suggests that continuous dosingmay lead to increased efficacy. The number of consecutivedays the orally available compounds could be dosed,unlike the parenteral compounds, was not limited byinjection site toxicity. Nevertheless, the oral compoundscould only be given for 15 to 25 days, after which timethe animals became moribund. In all cases, the com-pounds were given at their respective maximally tolerateddoses.

Activation of the Akt pathway is thought to inhibitapoptosis induced by a variety of cytotoxic agents and hasbeen reported to be an effective survival mechanism inmany tumor cells (44–47). Therefore, we attempted tocombine the Akt inhibitors with the antimitotic agentpaclitaxel in a PC-3 xenograft model (48, 49). PC-3 is a

Table 1. Selectivity of Akt inhibitors for selected kinases

Family Kinase A-443654 fold(K i, Amol/L)

A-674563 fold(K i, Amol/L)

AGC Akt1 1 (0.00016) 1 (0.011)PKA 40 (0.0063) 1.4 (0.016)PKCg 150 (0.024) 110 (1.2)PKCy 200 (0.033) 32 (0.36)PDK1 >120,000 (>20) >1,800 (>20)

TK KDR 19,000 (3.1) >330 (>3.7)cKIT 7,300 (1.2) >530 (>5.8)SRC 16,000 (2.6) 1,200 (13)Flt1 22,000 (3.6) >200 (>2.2)

CMGC CDK2 150 (0.024) 4.2 (0.046)ERK2 2,100 (0.34) 24 (0.26)

GSK3h 260 (0.041) 10 (0.11)CK2 15,000 (2.4) 490 (5.4)

CAMK Chk1 15,000 (2.3) 235 (2.6)RSK2 68 (0.011) 53 (0.58)

MAPK-AP2 21,000 (3.3) 100 (1.1)

NOTE: The fold selectivity versus Akt1 is shown for each kinase and the K i

is in parentheses.Abbreviations: PDK1, phosphatidylinositide-dependent kinase 1; MAPK,mitogen-activated protein kinase; GSK3, glycogen synthase kinase 3; ERK2,extracellular signal – regulated kinase 2.

Figure 2. A-443654 inhibits Akt1, Akt2,or Akt3 equally within cells. MurineFL5.12 cells were stably transfected withconstitutively active myristoylated humanAkt1 (.), Akt2 (E), or Akt3 (n). There isno appreciable phosphorylation of GSK3 inthe parental cell lines, whereas there arehigh levels of phospho-GSK3 (P-GSK3 )observed in all three Akt overexpressinglines. A-443654 reduces the P-GSK3 in adose-responsive manner in all three celllines. The broad-spectrum kinase inhibitorstaurosporine was added at 1 Amol/L as apositive control. The P-GSK3 was quanti-tated and normalized to the total GSK3.Plot of the resulting inhibition for eachoverexpressing cell line (right ).

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PTEN-deficient human prostate carcinoma cell line, whereresistance to chemotherapeutic agents is at least partiallydependent on Akt activity (50). When given in combina-tion, A-674563 increased the efficacy of paclitaxel in a PC-3xenograft model (Fig. 4C).

We tested the pharmacokinetics and pharmacodynamicsto insure that we were inhibiting Akt in vivo (Fig. 5).When the inhibitors were given at their maximallytolerated doses on a twice daily (bid) schedule, theirconcentration in plasma remained above the cellular EC50

for f5 hours (Fig. 5A). Concentrations in tumors weresignificantly higher than those in plasma, and drugconcentrations in tumor remained above the cellularEC50 for the entire 12-hour dosing interval (Fig. 5B). Inthe tumors, as we have seen in the cellular assays, theinhibition of GSK3 and S6 phosphorylation are inhibitedin a dose responsive manner by the Akt inhibitors (Fig.5C). As also seen in the cellular studies, the increase in Aktphosphorylation seems the most sensitive marker for Aktinhibition. This suggests that the cells, in vivo , are alsosensitive to Akt inhibition, and attempt to compensate forthe loss of the activity.

We also examined caspase-3 activation (a measure ofapoptosis) and Akt phosphorylation by immunohistochem-istry. As expected, at 8 hours after a single dose ofA-443654, we observed an increase in apoptotic cells(Fig. 5D), suggesting that at least part of the antitumoractivity of the Akt inhibitors is due to apoptosis induction.Normal liver and colon tissues showed no increase inapoptosis following treatment (data not shown). Corrobo-rating the Western blot analysis, we also observed that thephosphorylation of Akt increased when inhibitors aregiven (Fig. 5E).

As seen in Fig. 5C, doses near the MTD do notmaximally inhibit Akt within tumors. Furthermore, drugwashout studies in cells suggested that compounds weremore effective at inducing apoptosis when maintainedabove their cellular EC50 for at least 12 hours (data notshown). As stated above, the plasma concentrations ofA-443654 fall below this EC50 after about 5 hours. In anattempt to increase the efficacy of A-443654 by intensi-fying the dosing regime, the compound was given thricein a single day, 3 hours apart, for a total of 50 mg/kg/d.The animals could not tolerate 50 mg/kg/d every day;thus, compound was given every fourth day. Thisregime maintains the plasma concentrations of A-443654 above its cellular EC50 for >12 hours. This thricedaily (tid)/q4d schedule proved to be more efficaciousthan the bid/daily schedule (Fig. 4D). Note that for thetid schedule, dosing was started on established tumors

Figure 3. Akt inhibitors affect the phosphorylation and localizationof cellular Akt substrates. A, MiaPaCa-2 cells were treated for 2h with various concentrations of Akt inhibitors A-443654 and A-674563. Western blot analyses were done to assess the phosphor-ylation of Akt, GSK3 a/h, TSC2, mTOR, and ribosomal protein S6.B, HeLa cells transiently transfected with pFOXO3A-hrGFP weretreated with A-443654 for 6 h. Images of live cells taken byfluorescence microscopy. Transcription factor FOXO3A is retained inthe cytoplasm when phosphorylated by Akt. DMSO vehicle and 20mmol/L LY 294002 (an inhibitor of PI3K) were used as negativeand positive controls, respectively. C, MiaPaCa-2 cells were treatedwith A-443654 or A-674563 for 48 h. Alamar blue cell viabilityassays were done to measure the cell growth inhibition. Points,average of three determinations; bars, SD.

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whereas for the bid schedule dosing was startedimmediately after tumor cell inoculation. Similar to whatwas observed with the bid/daily schedule, when treatedon the tid/q4d schedule, the tumors regrew soon afterdosing was stopped.

Akt inhibition was compared with inhibition furtherdownstream in the PI3K pathway at mTOR, usingrapamycin (Fig. 4D). When dosed, both the Aktinhibitors and rapamycin are efficacious in the Mia-PaCa-2 model. However, rapamycin is significantly lesstoxic and has a much larger therapeutic window thanthe Akt inhibitors. Furthermore, rapamycin therapy canbe maintained longer, leading to more durableresponses. The function of mTOR in tumor progressiondoes not seem completely epistatic to that of Akt, as theefficacy observed for the combination of the Akt andmTOR inhibitors was better than that observed for eitheragent alone.

Metabolic Consequences of Akt InhibitionAkt is downstream of the insulin receptor and

transduces the insulin response in vivo . We thereforemeasured blood sugar and insulin responses related tothe inhibition of Akt. We examined random bloodsugars during multiple tumor studies and have neverobserved any differences between the treated andcontrol groups. We also measured both blood sugarand plasma insulin levels in glucose challenge tests.Blood sugars were not changed upon administration ofAkt inhibitors, even when supertherapeutic doses weregiven (data not shown). We did, however, observesignificant increases in plasma insulin following admin-istration of a single dose of Akt inhibitors at therapeu-tic doses (Fig. 5F). This data is consistent with ahomeostatic response, where the animals increaseinsulin secretion to maintain blood glucose concentra-tions. This is also what has been observed in Akt2

Figure 4. Akt inhibitors slow tumor growth in vivo. A, A-443654 inhibits tumor growth in the 3T3-Akt1 flank tumor model. The scid micebearing established 3T3-Akt1 tumors were treated s.c. bid for 14 d with vehicle (4), A-443654 at 7.5mg/kg/d (n), or the inactive analogue, 2-methyl A-443654 at 7.5 mg/kg/d (5). Therapy was initiated 21 d after tumor inoculation when the mean tumor volume of each group was f245mm3. A-443654 was statistically different from vehicle from day 26 onward (P < 0.02). B, A-443654 inhibits tumor growth in the MiaPaCa-2xenograft tumor model. scid mice were inoculated with cells on day 0 and therapy was begun on day 1. A-443654 at 7.5 mg/kg/d (E) or vehicle (4)was given s.c., bid for 14 d. Gemcitabine at 120 mg/kg/d (n) or vehicle (5) was given i.p., qd on days 3, 6, 9, and 12. A-443654 and gemcitabine bothsignificantly inhibited tumor growth (P < 0.03). C, A-674563/paclitaxel combination therapy in the PC-3 prostate cancer xenograft model. scid micebearing established PC-3 tumors were treated with A-674563 (4) p.o., bid at 40 mg/kg/d for 21 d; paclitaxel (E) i.p., qd at 15 mg/kg/d on days 20, 24,and 28; the combination (n); or the combination vehicle (5). Therapy was initiated 20 d after tumor inoculation when the mean tumor volume for eachgroup was f270 mm3. Although A-674563 showed no significant monotherapy activity, the efficacy of the combination therapy was significantlyimproved compared to paclitaxel monotherapy (P < 0.002). D, A-443654 and rapamycin efficacy in the MiaPaCa-2 pancreatic cancer xenograft model.scid mice bearing established tumors were treated with A-443654 (n) s.c., tid at 50 mg/kg/d on days 16, 20, and 24; rapamycin (5) i.p., qd at 20 mg/kg/d for 15 d; the combination of rapamycin plus A-443654 (y); s.c. vehicle (E); or combination vehicle (4). Therapy was initiated 16 d after tumorinoculation when the mean tumor volume for each group was f255 mm3. Each monotherapy showed significant activity throughout the study (P <0.01) and the combination was statistically better than either monotherapy from day 23 onward (P < 0.01).

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Figure 5. The effects of Akt inhibitors after in vivo administration. Plasma (A) and tumor (B) concentrations of A-443654 are plotted versusthe time after a final dose of A-443654 was given. Animals were treated at (.) 3.8, (n) 7.5, or (E) 15 mg/kg bid for 3 d before measurement.C, 2 h after A-443654 administration, the phosphorylation of Akt (solid columns ), S6 Protein (open columns ), and GSK3 (cross-hatchedcolumns ) were measured via Western blotting (bottom ). Akt phosphorylation increases in response to the Akt inhibitor. In spite of this increasein P-Akt, phosphorylation of targets downstream from Akt is inhibited in a dose-responsive manner. D, A-443654 induces apoptosis in 3T3-Akt1 flank tumors. The scid mice bearing established 3T3-Akt1 tumors were given a single s.c. dose of A-443654 at 50 mg/kg or vehicle.Tumors were removed 8 h after treatment and apoptosis was examined by immunohistochemical staining with an antibody specific for theactivated form of caspase-3. E, A-443654 treatment leads to increased levels of phosphorylated Akt1 in MiaPaCa-2 tumors. The scid micebearing established MiaPaCa-2 flank tumors were given a single s.c. 30 mg/kg dose of A-443654. One hour after treatment, tumors wereremoved and the level of phospho-Akt was examined by immunohistochemical staining. F, A-674563 and A-443654 increase plasma insulin inan oral glucose tolerance test. Fasted animals administered vehicle (n), 20 mg/kg A-674563 (.), 100 mg/kg A-674563 (o), or 20 mg/kg A-443654 (E) 30 min before the 1 g/kg glucose challenge (time 0). Insulin levels were elevated in all A-674563- and A-443654-treated animals.Similar increases in insulin responses were seen in the sham-challenged (no glucose challenge) animals after treating with Akt inhibitors (data notshown).

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knockout animals (51, 52). When young, these animalshave normal blood sugars but increased plasma insulin.Only when older, after islet cell failure, do the animalslose their ability to maintain normal blood glucoseconcentrations.

While determining the maximum tolerated dose, wefound that the Akt inhibitors induce a dose- and time-dependent weight loss in treated animals. This weight lossis typically >10% of total body mass when compounds aregiven at thrice their maximum tolerated dose (data notshown). Whereas weight loss is a general toxicity, it isconsistent with the expectation that the compounds mayinterfere with the utilization of glucose by interfering withthe insulin pathway.

DiscussionWe have shown that inhibitors of Akt, when dosed tolevels that inhibit Akt in vivo , can slow the tumor growthin laboratory models of cancer. Several compounds havebeen tested and have shown efficacy in a number oftumor models. In all of these models, the dosing period islimited due to toxicity of the therapy. In addition, in allmodels, the tumors regrew rapidly when dosing isstopped. The Akt inhibitors show efficacy in the samemodels where activity is observed with rapamycin.However, the Akt inhibitors have a narrower therapeuticwindow than does rapamycin.

The dose-limiting toxicities for the oral compounds,malaise and weight loss, are consistent with a mechanism-based toxicity, where the inhibitors interfere with themetabolism of glucose (the limiting toxicity for parenteralcompounds is an injection site irritation). However, weightloss is a general toxicity seen with many anticancer agentsthat do not inhibit Akt. Also consistent with mechanism-based toxicity is the observation that compounds withsubstantially different selectivity profiles, but similar Aktinhibitory activity, elicit the same types of toxicity. Theincrease in plasma insulin attendant with Akt inhibition alsosuggests metabolic toxicities may be dose limiting. Furtherstudies with more widely divergent Akt inhibitors will berequired to ascertain if Akt inhibition as cancer therapy willbe limited by mechanism-based metabolic toxicities.

Whereas the efficacy observed in the tumor models ispromising, it is clear that inhibition of Akt as a therapy forcancer would benefit from a widening of the therapeuticwindow. A number of active investigations are under wayto further improve the therapeutic window. The inhibitorsdescribed here are pan-Akt inhibitors, and in cells haveequal potency inhibiting Akt1, Akt2, or Akt3 within cells.Akt2 loss seems to predominate in toxicity related toinsulin signaling (51, 52), whereas Akt1 has been reportedto be more important in cancer cell survival. Thus,generation of inhibitors more selective for Akt1 over Akt2might improve therapeutic window. Furthermore, it is clearfrom the paclitaxel study that Akt inhibitors may collabo-rate with chemotherapeutics that induce apoptosis. Akt hasrecently been shown to collaborate with Bcl2/BclXL in

several cell lines to inhibit apoptosis in cancer cells (50).Thus, using these Akt inhibitors with other agents mayproduce the desired improvement in therapeutic windowby improving efficacy without increasing toxicity.

Finally, the Akt inhibitors, along with rapamycin, illus-trate that different nodes in the PI3K signal transductionpathway can be inhibited to slow tumor progression. Theefficacy of the Akt and mTOR inhibitors suggests that, asmonotherapy, other inhibitors of the PI3K pathway will alsobe efficacious. However, the differences in toxicity suggestthat inhibiting at different nodes in the PI3K pathway mightyield very different therapeutic windows. There are anumber of potential targets in this pathway, includingPI3K, phosphatidylinositide-dependent kinase 1, ILK, Akt1,Akt2, Akt3, mTOR, p70S6K, and 4E-BP1, all of which areunder investigation for utility in human cancer therapy. Theresults of these investigations will ultimately determinewhere to target the PI3K pathway to provide the bestefficacy and therapeutic window for the treatment of cancer.

Acknowledgments

We thank Lisa Li and Jennifer Hensel for their technical assistance withmany of the cellular assays and Stephen Fesik for his assistance in editingthis article.

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