Discovery of lactoquinomycin and related ... · of AKT catalytic activation loop cysteines Lourdes Toral-Barza,1 Wei-Guo Zhang,1 Xinyi Huang,2 Leonard A. McDonald,2 Edward J. Salaski,2

Discovery of lactoquinomycin and relatedpyranonaphthoquinones as potent and allostericinhibitors of AKT/PKB: mechanistic involvementof AKT catalytic activation loop cysteines

Lourdes Toral-Barza,1 Wei-Guo Zhang,1

Xinyi Huang,2 Leonard A. McDonald,2

Edward J. Salaski,2 Laurel R. Barbieri,2

Wei-Dong Ding,2 Girija Krishnamurthy,2

Yong Bo Hu,2 Judy Lucas,1 Valerie S. Bernan,2

Ping Cai,3 Jeremy I. Levin,2 Tarek S. Mansour,2

James J. Gibbons,1 Robert T. Abraham,1

and Ker Yu1

1Oncology Research, 2Chemical and Screening Sciences, and3Preclinical Development, Wyeth Research, Pearl River, New York

AbstractThe serine/threonine kinase AKT/PKB plays a critical rolein cancer and represents a rational target for therapy.Although efforts in targeting AKT pathway have acceler-ated in recent years, relatively few small moleculeinhibitors of AKT have been reported. The developmentof selective AKT inhibitors is further challenged by theextensive conservation of the ATP-binding sites of theAGC kinase family. In this report, we have conducted ahigh-throughput screen for inhibitors of activated AKT1.We have identified lactoquinomycin as a potent inhibitorof AKT kinases (AKT1 IC50, 0.149 F 0.045 Mmol/L).Biochemical studies implicated a novel irreversible inter-action of the inhibitor and AKT involving a critical cysteineresidue(s). To examine the role of conserved cysteines inthe activation loop (T-loop), we studied mutant AKT1harboring C296A, C310A, and C296A/C310A. Whereasthe ATP-pocket inhibitor, staurosporine, indiscriminatelytargeted the wild-type and all three mutant-enzymes, theinhibition by lactoquinomycin was drastically diminishedin the single mutants C296A and C310A, and completelyabolished in the double mutant C296A/C310A. Thesedata strongly implicate the binding of lactoquinomycin tothe T-loop cysteines as critical for abrogation of catalysis,and define an unprecedented mechanism of AKT inhibition

by a small molecule. Lactoquinomycin inhibited cellularAKT substrate phosphorylation induced by growth factor,loss of PTEN, and myristoylated AKT. The inhibition wassubstantially attenuated by coexpression of C296A/C310A.Moreover, lactoquinomycin reduced cellular mammaliantarget of rapamycin signaling and cap-dependent mRNAtranslation initiation. Our results highlight T-loop targetingas a new strategy for the generation of selective AKTinhibitors. [Mol Cancer Ther 2007;6(11):3028–38]

IntroductionIn the past decade, molecular elucidation of the phosphoi-nositide-3-kinase (PI3K)/AKT (PKB) signaling pathwayand epidemiologic studies have firmly established a centralrole for AKT in human malignancy (1). The AKT family(AKT1, AKT2, and AKT3) is characterized by an NH2-terminal pleckstrin-homology domain and a COOH-terminalcatalytic domain bearing the highest sequence homologyto the AGC family founding members protein kinase A(PKA) and protein kinase C (PKC; ref. 2). AKT is thecellular homologue of the viral v-Akt encoded by theoncovirus Akt8 (3). In quiescent cells, AKT is expressed asan inactive form and becomes activated by phosphoryla-tion upon translocation to the plasma membrane inresponse to growth factor–stimulated PI3K activation (4).This process is negatively regulated by the inositol 1,4,5-trisphosphate phosphatase PTEN, a major tumor sup-pressor in human. Elevated AKT activity occurs in f50%of all human malignancies via numerous mechanisms(reviewed in refs. 5–8), including constitutive activationof cell surface growth factor receptors, loss of PTEN,activation mutation of the PI3K catalytic subunit p110a(PIK3CA), as well as overexpression of various AKT familymembers. The role of AKT in cancer is mediated through agrowing list of downstream targets that are directlyphosphorylated by AKT (5–8). AKT promotes survivalthrough several well-known apoptosis modulators suchas the forkhead transcription factors, GSK3, BAD, Bcl-XL,caspase 9, and nuclear signaling of nuclear factor-nB. AKTpromotes cell growth and proliferation through activationof the mammalian target of rapamycin (mTOR) kinase, acentral regulator of protein translation, and by influencingthe levels of D-type cyclins and cell cycle inhibitor p27/Kip1. Moreover, AKT also plays a role in tumor-inducedangiogenesis by regulating the expression of hypoxia-inducible factor 1a and vascular endothelial growth factor(9, 10).AKT represents an appealing target for anticancer

therapy. Numerous experimental approaches have further

Received 3/26/07; revised 7/22/07; accepted 9/18/07.

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.

Requests for reprints: Ker Yu, Oncology Research, Wyeth Research,401 North Middletown Road, B200/4603 Pearl River, NY 10965.Phone: 845-602-4814; Fax: 845-602-5557. E-mail: [email protected]

Copyright C 2007 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-07-0211

3028

Mol Cancer Ther 2007;6(11). November 2007

Research. on February 6, 2021. © 2007 American Association for Cancermct.aacrjournals.org Downloaded from

Published OnlineFirst November 7, 2007; DOI: 10.1158/1535-7163.MCT-07-0211

http://mct.aacrjournals.org/

corroborated clinical evidence that deregulated AKT kinaseactivity causes oncogenic transformation in a variety of celltypes and causes tumor growth in vivo . Inhibition of AKTby introducing either the PTEN tumor suppressor or adominant-negative AKT into PTEN-deficient cancer cellsleads to the inhibition of tumor growth (11, 12). Likewise,expression of antisense RNA against AKT resulted ingrowth inhibition and increased sensitivity to chemother-apeutic agents in a variety of cancer cell lines (13). Thediscovery of small molecule AKT inhibitors will furtherenhance our ability to validate the therapeutic potential oftargeting AKT in cancer. These efforts have acceleratedin recent years (reviewed in refs. 14, 15). Several groupshave reported AKT inhibitors that target the ATP-bindingpocket (16), the pleckstrin-homology domain (17), andupstream inhibitors that interfere with enzyme activation(18–20). Although significant progress has been made, thedevelopment of potent and selective AKT inhibitors isparticularly challenging due to the extensive homology inATP-binding sites of the AGC kinase family members.Consequently, a considerable interest now exists in search-ing for novel allosteric inhibitors of AKT. In this report, wedescribe our screening efforts that led to the identificationof lactoquinomycin as a potent and selective inhibitor ofAKT kinases. We show that this inhibitor class targets AKTthrough a novel allosteric mechanism that involves twocritical activation loop (T-loop) cysteines neighboring theactivating residue Thr308. Inhibition of cellular AKT bylactoquinomycin confirms the critical role of AKT ingrowth factor signaling and mRNA translation, which areessential for tumor cell growth and survival.

Materials andMethodsChemicalsAll general chemical reagents used for buffers and assays

were purchased from Sigma Corporation unless otherwisespecified. Lactoquinomycin and frenolicin B were obtainedfrom Wyeth Natural Product sample collections. Cell cycleinhibitor-779 (rapamycin ester) and wortmannin wereobtained from Wyeth Chemical & Pharmaceutical Devel-opment. Staurosporine was purchased from CalBiochem.

AKTConstructsHuman AKT1 cDNA was obtained by PCR from human

placental Quick cDNA (BD-Clontech), sequenced andconfirmed to be identical to the previous report (21). TheAKT1 cDNA was inserted into the BamHI site of themammalian expression vector pFlag-CMV5 (Sigma) inwhich AKT1 was COOH-terminally tagged with Flag-epitope. The myristoylation sequence derived from thehuman c-Src (22) was then added to the NH2 terminus ofAKT1 by PCR to generate myristoylated AKT1-Flag (myr-AKT1). This construct was then subjected to site-directedmutagenesis to generate T-loop cysteine mutants C310A,C296A, and C296A/C310A using the mutagenesis kit(Stratagene).

Expression and Purification of AKT1EnzymesAll cell culture media, supplements, and transfection

reagents were obtained from Invitrogen. HEK293 cells were

maintained in DMEM containing 10% fetal bovine serum,100 Ag/mL of penicillin, 50 Ag/mL of streptomycin, and1 mmol/L of glutamine. Plasmid DNA (50 Ag per 150 mmculture plate) of various myr-AKT1 constructs weretransiently transfected into HEK293 using LipofectAMINE2000 reagent. Cells were harvested 48 h later with all stepsdone at 4jC. Cells (1–2 � 107 per 150 mm plate) werewashed with PBS and scraped off the plate in 1.5 mL AKTlysis buffer [25 mmol/L Hepes (pH 7.5), 100 mmol/L NaCl,0.5% NP40, 1 mmol/L Na3VO4, 1 mmol/L EDTA, 1 mmol/LEGTA, 20 mmol/L h-glycerophosphate, 10 Ag/mL aproti-nin, 10 Ag/mL leupeptin, 1 mmol/L phenylmethylsulfonylfluoride, 1 Amol/L microcystin LR, 0.1% h-mercaptoetha-nol]. The cell suspension was then sonicated, incubated for30 min with gentle shaking, and cleared by centrifugationfor 30 min at 14,000 � g using a Beckman J2-HS centrifuge.The cleared lysate was collected and stored at �80jC. Forpurification of various AKT1 enzymes, frozen cell lysatewas thawed on ice and added onto an anti-Flag M2 affinitycolumn (Sigma) at 4jC following a ratio of 1 mL affinitybeads per 100 mg crude lysate. The column was washedwith 15 times the bed volume of TBS. The myr-AKT1proteins were eluted using 100 Ag/mL of Flag peptide(Sigma) in elution buffer [50 mmol/L Tris-HCl (pH 7.5),150 mmol/L NaCl, 270 mmol/L sucrose, 1 mmol/LNa3VO4, 1 mmol/L benzamidine, 0.1 mmol/L EGTA,0.2 mmol/L phenylmethylsulfonyl fluoride, 0.1% h-mer-captoethanol, 0.03% Brij-35]. Eluted proteins were quicklyfrozen in a dry-ice ethanol bath and stored at �80jC.Concentrations of all purified proteins were determinedby the Bradford method (Bio-Rad) using bovine serumalbumin as a standard.

AKTAssaysThe routine assays of AKT1 were done in low-binding

96-well plates (Corning). Myr-AKT1 (11.5 AL) or variousmutant enzymes diluted in kinase assay buffer [50 mmol/LHepes (pH 7.4), 100 mmol/L KCl, 25 mmol/L h-glycer-ophosphate, 10 mmol/L MgCl2, 1 mmol/L orthovanadateand 0.5 mmol/L EGTA] with 50 Ag/mL of bovine serumalbumin were added to each well. One microliter of DMSOvehicle or test inhibitors was then added. The kinasereactions were initiated by the addition of 12.5 AL of assaymix containing kinase assay buffer, ATP, and a biotinylatedGSK3a substrate peptide (SGRARTSSFA). The final reac-tion volume of 25 AL contained 6 ng of AKT1 (4 nmol/L),10 Amol/L of biotin-GSK3a peptide, and 50 Amol/L ofATP. The assays were incubated for 1 h at room tempera-ture and terminated with 25 AL of stop buffer [25 mmol/LTris-HCl (pH 7.5), 20 mmol/L EDTA]. Phosphorylatedbiotin-GSK3a was detected by the time-resolved fluores-cence resonance energy transfer Lance format with allreagents obtained from Perkin-Elmer. Product detectionwas done in a black low-binding plate (Dynex) in 50 AL ofdetection buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/LNaCl, and 0.5% bovine serum albumin] containing100 ng/mL of phospho-GSK3 polyconal antibody (CellSignaling Technology) labeled with Europium (Perkin-Elmer), 4 Ag/mL of streptavidin-allophycocyanin, and

Molecular Cancer Therapeutics 3029





2.5 AL of the terminated kinase reaction mix. Afterincubating for 30 min at room temperature, the plates wereread in a Victor plate reader (Wallac/Perkin-Elmer). Insome experiments, AKT activity was also measured in arate-based coupled enzyme assay in a black clear-bottomed96-well plate (half well or regular well; Corning). Inaddition to appropriate concentrations of GSK3a peptide,ATP, and thiol-free AKT, the assay mixture contained20 units/mL of pyruvate kinase, 28.5 units/mL of lacticdehydrogenase, 2 mmol/L of pyruvate enol phosphate,0.25 mmol/L of NADH in an assay buffer of 100 mmol/Lof Hepes (pH 7.5), 25 mmol/L of h-glycerophosphate,10 mmol/L of MgCl2, and 0.005% Brij-35. The assay (with afinal volume of 120 AL in the half well plate or 300 AL in theregular plate) was monitored continuously by absorbanceat 340 nm on a Gemini plate reader (Molecular Devices).Initial rates were calculated from the linear portion ofprogress curves, typically from 5 to 15 min.

Assays of Other KinasesRecombinant PKA and PKCa were obtained from

Upstate Biotechnology and assayed for phosphorylationof the myelin basic protein (23). The PKCa assay includedlipids (Upstate Biotechnology). The kinase reaction in afinal volume of 25 AL contained kinase buffer (as AKTassay), PKA (2 Ag/mL) or PKCa (0.8 Ag/mL), 100 Ag/mLof bovine serum albumin, 100 Amol/L of ATP, and myelinbasic protein (80 Ag/mL; Upstate Biotechnology). Theassays were incubated for 1 h at room temperature andterminated by adding 25 AL of stop buffer. The phosphor-ylation of myelin basic protein was measured by thedissociation-enhanced lanthanide fluorescence immunoas-say using an anti–phosphorylated (Ser/Thr)-PKA sub-strate antibody (Cell Signaling Technology) and aEuropium-labeled anti-rabbit secondary antibody (Perkin-Elmer) in 100 AL assay buffer as previously described (24).Assays for other kinases were done with recombinant

Figure 1. Development of a high-throughput assay for inhibitors of active AKT1. A, expression and purification of a C-terminally Flag-tagged myr-AKT1enzyme from HEK293. Equal amounts of the affinity-purified myr-AKT1 and a commercially purchased active AKT1 were fractionated in 10% NuPAGEgels, and were either stained with Coomassie blue (left ) or immunoblotted for total AKT and Thr308 phospho-AKT (right ). B, catalytic activity of myr-AKT1.Various amounts of myr-AKT1 (0–24 ng) were assayed in a microtiter plate format. The kinase reactions contained 10 Amol/L of biotin-GSK3a peptide and50 Amol/L of ATP, and were incubated for the indicated times. Substrate phosphorylation was detected by a homogeneous time-resolved fluorescenceresonance energy transfer Lance assay using a Europium-phosphorylated (Ser21)-GSK3 antibody as described in Materials and Methods. C, kineticparameters of myr-AKT1. To determine ATP dependence (left ), enzyme rates were measured with a constant 10 Amol/L of GSK3 peptide and varyingamounts of ATP. To determine substrate dependence (right ), enzyme rates were determined with a constant 150 Amol/L of ATP and varying amounts ofGSK3 peptide. The reactions contained 6 ng of myr-AKT1 and incubated for 1 h, terminated, and detected as in B. Km values were calculated from X -axisintercepts that coincided with the 1/2 Vmax point. Points, mean based on three independent assays; bars, SE. Similar assays were also done with thecommercial AKT1, which gave rise to similar results (data not shown).

Targeting the Activation Loop of AKT/PKB3030





enzymes produced from bacterial, insect, or human cells.Substrates used were peptides (IKKh, PDK1, S6K1, Src),proteins (CDK4, CDK2, CDC2, mTOR, Mek1), and poly(glutamic acid4-tyrosine; KDR), and autophosphorylation(epidermal growth factor receptor, HER-2, c-Met). Phos-phorylation was measured using TMB peroxidase substrate(Pierce) for CDKs and dissociation-enhanced lanthanidefluorescence immunoassay/Lance (Wallac-Perkin-Elmer)for others. The assays of a panel of 45 kinases were doneas described by Invitrogen SelectScreen profiling.4

Cell Culture,Transient Transfection, Protein Lysates,Immunoblotting and 7-methyl-GTPPull-downHEK293, Rat1, and various tumor cell lines used in this

study were obtained from American Type Culture Collec-tion. DU145-AKT was created by stable transfection ofmyr-AKT1 into DU145. MDA-MB-361 (MDA-MB-361-DYT2) was obtained from Dr. C. Discafani (Oncology,Wyeth Research, Pearl River, NY). For evaluation of cellulareffects by inhibitors, tumor cells were plated in six-wellculture plates in growth medium for 1 day. Cells were thentreated with inhibitors for 6 or 16 h in growth media, or asindicated. For insulin-like growth factor-I stimulationexperiments, serum-starved cells were treated with inhib-itors for 2 h and then stimulated with insulin-like growthfactor-I for 0.5 h. For transient expression of Flag-AKT-WTand Flag-AKT-C296A/C310A, DU145 cells were plated in

six-well culture plates for 24 h. Cells were then transfectedwithout (mock) or with 2 Ag (per well) each DNA constructusing LipofectAMINE 2000 reagent (Invitrogen). Forty-eight hours posttransfection, cells were treated withinhibitor for 12 h. Total cellular lysates were prepared withNuPAGE-LDS sample buffer (Invitrogen), sonicated, clari-fied by centrifugation, and resolved in appropriateNuPAGE gels following the protocols provided by thevendor (Invitrogen). For 7-methyl-GTP pull-down, treatedMDA-MB-468 cells in 100 mm culture plates were lysed onice for 30 min in 500 AL of AKT lysis buffer in whichNP40 was replaced with 1% Tween 20. Lysates werecollected and centrifuged for 10,000 � g for 5 min, and theprotein concentration of the cleared lysate was thendetermined. Forty microliters of a 50% slurry of 7-methyl-GTP Sepharose (Amersham) was added to 0.5 mg of clearedlysate, and incubated while rocking for 2 h at 4jC. Thecap-complexes were then collected and washed four timeswith lysis buffer, dissociated from the Sepharose by adding50 AL of NuPAGE LDS sample buffer, heated to 70jC for10 min, and resolved in NuPAGE gels. Protein blots wereprobed with antibodies: phospho-(P)-AKT (T308), P-AKT(S473), AKT, P-GSK3, GSK3, P-FKHRL1 (T32), P-ERK (T202/Y204), ERK, P-S6K1 (T389), P-S6 (S240/244), P-4EBP1 (T70),4EBP1, eIF4E, P-IKKg (S376), and InBa (Cell SignalingTechnology); P-FKHRL1 (T32), P-HER2 (Y1248), and HER2(Upstate Biotechnology); cyclin D3, eIF4A, eIF4G, and eIF3b(Santa Cruz Biotechnology); and p27/Kip1 (TransductionLaboratories).4 http://www.invitrogen.com/kinaseprofiling

Figure 2. Identification of lactoquinomycin as a potent and selective inhibitor of AKT. A, chemical structures of the pyranonaphthoquinone AKTinhibitors lactoquinomycin and frenolicin B. The specific chemical batches and molecular weights of the inhibitors are indicated. B, in vitro AKT kinaseinhibition dose-response curves of lactoquinomycin (left ) and frenolicin B (right ). Myr-AKT1 and AKT1 were assayed with DMSO vehicle and various dosesof inhibitors for 1 h, terminated and detected similarly as in Fig. 1. Points, mean IC50 values for both inhibitors based on at least four independent assays;bars, SE. C, Lactoquinomycin was assayed for inhibition dose-response against PKA, PKCa,and myr-AKT1. Assays against recombinant PKA and PKCawere done as described in Materials and Methods. D, comparison of IC50 values of lactoquinomycin in a panel of 14 tyrosine and serine/threonine kinases.Assays were done as described in Materials and Methods.






ResultsAKT1InhibitorAssayTo establish an enzyme assay for novel inhibitors of

active AKT1, we constructed an expression vector encodingmyristoylated human AKT1 with a COOH-terminal Flag-epitope tag (myr-AKT1-Flag/myr-AKT1). This approachpermitted the expression of a constitutively active form ofrecombinant AKT1 in HEK293 cells, obviating the require-ment for in vitro phosphorylation of the purified enzymeby PDK1 (4). Anti-Flag affinity chromatography of thetransfected HEK293 cell lysate yielded a relatively pureenzyme with a stoichiometry of Thr308 phosphorylationcomparable to that observed with a commercial prepara-tion of in vitro –phosphorylated AKT1 (Fig. 1A). In ahomogeneous time-resolved fluorescence resonanceenergy transfer Lance assay, the myr-AKT1 efficientlyphosphorylated a biotinylated-GSK3a peptide on Ser21 ina dose- and time-dependent manner (Fig. 1B). ApparentMichaelis constant (Km) values of the enzyme for ATPand substrate peptide were determined as 46 F 2.5 and1.35 F 0.37 Amol/L, respectively (Fig. 1C). These kineticparameters determined in the Lance assay are in goodagreement with a recent report on kinetic characterizationof the AKT family enzymes (25). Based on these data, wedeveloped and optimized the Lance assay containing 6 ng ofmyr-AKT1 (4 nmol/L), 50 Amol/L of ATP, and 10 Amol/Lof substrate peptide with a kinase reaction time of 1 h.

Discovery of Lactoquinomycin and Related Pyrano-naphthoquinones as Potent and Selective Inhibitors ofAKTKinasesScreening of the Wyeth Natural Products collection led to

the identification of lactoquinomycin from the fermentationbroth of a microbial strain Streptomyces sp. LL-AF101. Asubsequent structural similarity search also uncovered arelated inhibitor, frenolicin B. Both lactoquinomycin andfrenolicin B are antibiotics possessing a common pyrano-naphthoquinone core (Fig. 2A). Dose titration experimentswith lactoquinomycin yielded mean IC50 values of 0.133 F0.017 and 0.149 F 0.045 Amol/L, with myr-AKT1 andcommercially prepared AKT1, respectively (Fig. 2B, left),indicating an identical potency against both forms of activeAKT1. Similar results were obtained for frenolicin B, inwhich IC50 values against myr-AKT1 and AKT1 were0.157 F 0.038 and 0.313 F 0.115 Amol/L, respectively(Fig. 2B, right). Because both compounds have the samepyranonaphthoquinone core, these data imply that thepyranonaphthoquinone moiety is critical for AKT inhibi-tion. To evaluate the specificity of lactoquinomycin in AKTinhibition, we first determined the effects of this compoundon PKA and PKCa, two AGC family members the catalyticdomains of which exhibited the highest homology to AKT.Interestingly, we found that lactoquinomycin did notinhibit PKA or PKCa at concentrations up to 200 Amol/L(Fig. 2C, left), whereas staurosporine potently inhibited allthree enzymes (Fig. 2C, right). IC50 determination in a panelof 13 additional protein kinases revealed that lactoquino-mycin weakly inhibited HER2/ErbB2 (IC50, 2.42 Amol/L)and IKKh (IC50, 3.9 Amol/L), and had no effect on other

protein kinases in this panel at concentrations up to10 Amol/L (Fig. 2D). These initial interesting observationsprompted us to examine its activity against a broader rangeof kinases. Lactoquinomycin was tested at 1 Amol/L in apanel of 45 kinases in duplicate (Invitrogen SelectScreenprofiling). Mean percent inhibition values confirmed thepotent inhibition of AKT1 (92%) and AKT2 (99%; Supple-mentary Table S1).5 The compound failed to suppress theactivities of most of the protein kinases in this panel, andshowed modest (<50%) inhibitory effect on those kinasesthat were affected by lactoquinomycin (SupplementaryTable S1).5 Together, the data in Fig. 2 and SupplementaryTable S15 have identified lactoquinomycin as a potent andrelatively selective inhibitor of AKT kinases.

Kinetic Mechanism: Noncompetitive against ATPandTime DependenceTo define the kinetic mechanism of lactoquinomycin, we

first did inhibitor versus ATP matrix competition assays.

5 Supplementary material for this article is available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Figure 3. Kinetic mechanism of AKT inhibition. A, lactoquinomycin is anoncompetitive inhibitor of AKT against ATP. The kinetic mechanism oflactoquinomycin was determined in a pyruvate kinase/lactic dehydroge-nase–coupled enzyme assay. The assay mixture contained 100 Amol/L ofGSK3a peptide, 100 to 1,000 Amol/L of ATP, and various amounts oflactoquinomycin. AKT (400 nmol/L) was added to start the reactions. Thedata were best-fit in SigmaPlot to noncompetitive inhibition. The doublereciprocal plot was graphed using GraphPad Prism 4.0. B, inhibition ofAKT by lactoquinomycin was time-dependent. AKT (183 nmol/L) wasincubated at room temperature with 0 to 10 Amol/L of lactoquinomycin for2, 15, 30, 45, and 60 min. The reactions were started by the addition ofsubstrate/coupling reaction mix. The assay contained final concentrationsof 300 Amol/L of ATP and 40 Amol/L of GSK3a peptide.






AKT activity was measured in a rate-based pyruvatekinase/lactic dehydrogenase–coupled enzyme assay inthe presence of constant substrate peptide and varyingamounts of ATP and lactoquinomycin. Double-reciprocalplots of the initial enzyme rate versus ATP concentrationwere best-fit to noncompetitive inhibition (Fig. 3A). Toassess whether AKT might be irreversibly inhibited,lactoquinomycin dose-response was assayed at varioustime points after initiation of the kinase reaction. IC50

values determined at 2, 15, 30, 45, and 60 min were 1.7, 0.66,0.31, 0.26, and 0.18 Amol/L, respectively (Fig. 3B), indicat-ing a progressive, time-dependent decrease in IC50 values,a characteristic of irreversible inhibitors that interactcovalently with their target enzyme. Similar assays with

a known reversible ATP-pocket inhibitor staurosporineyielded nearly identical IC50 values in these assays (datanot shown). Thus, lactoquinomycin seemed to act via adistinct kinetic mechanism that may involve its irreversiblebinding to AKT at a site outside of the ATP-pocket.

Mechanistic Involvement of AKT T-Loop CysteinesTo further define the lactoquinomycin-AKT interaction,

we examined the effect of this compound on an AKT1mutant lacking the pleckstrin-homology domain. Thisdeletion mutant was equally sensitive to lactoquinomycinand frenolicin B (data not shown), implying that theseinhibitors bind within the catalytic domain. Because thepyranonaphthoquinone core of lactoquinomycin andfrenolicin B could potentially react with thiols, we

Figure 4. AKT1 T-loop cysteines are required for enzyme inhibition by lactoquinomycin. A, AKT inhibitory activity of pyranonaphthoquinone inhibitorswas attenuated by the presence of cysteine. AKT enzyme was premixed with or without 1 mmol/L of cysteine-HCl for 10 min prior to the initiation of kinasereaction. B, expression, purification, and activity of AKT1 T-loop Cys-mutants. A Coomassie blue staining of the purified enzymes corresponding to thewild-type and various mutant myr-AKT1 (left ), and assay of enzymatic activity with various amounts of each of the purified AKT (right ). C, comparison ofenzyme inhibition sensitivity of Cys-mutants versus wild-type AKT in response to inhibitors. Equivalent amounts of active AKT1 wild-type(2 ng), C296A (2 ng), C310A (10 ng), and C296A/C310A (10 ng) mutants were assayed for inhibition by various doses of lactoquinomycin (top ),frenolicin B (middle ), and staurosporine (bottom ). Assays were done and analyzed similarly as in Fig. 2 except that the kinase reactions contained0.1 mmol/L of DTT. The graphs in C are representative of three independent assays.






speculated that these inhibitors might interfere withcatalytic activity through the T-loop cysteine(s) surround-ing the critical activating phosphorylation residue Thr308.This model was particularly attractive in light of theinactive AKT2 crystal structure which shows that theT-loop is disordered and contains a redox-sensitivecysteine-disulfide bond (26, 27). To test this hypothesis,we did cysteine-competition experiments. The IC50 valuesfor lactoquinomycin and frenolicin B were increased by66- and 55-fold, respectively, when AKT was assayed after

preincubation with 1 mmol/L of cysteine (Fig. 4A),consistent with a potential involvement of cysteine(s) inthe AKT-inhibitor interaction. To definitively test thismodel, we created T-loop cysteine mutants in myr-AKT1for assessing inhibitor effects. AKT enzymes harboringeither C310A or C296A and the double mutant C296A/C310A were expressed in HEK293 cells and affinity-purified (Fig. 4B, left). The purified AKT Cys-mutants wereall active in the in vitro kinase assays; however, we notedthat the activities of the C310A and C296A/C310A mutants

Figure 5. Lactoquinomycin inhibits cellular AKT signaling in cell models. A, lactoquinomycin inhibits the phosphorylation of AKT substrates in cells.Cells of U87MG glioma (left ) and DU-AKT (middle ) were plated in six-well plates for 24 h in growth medium and were treated with the indicated doses oflactoquinomycin and wortmannin (0.5 Ag/mL) for 6 h. Rat1 cells (right ) were plated, serum-starved for 24 h, and treated with inhibitor for 2 h prior toinsulin-like growth factor-1 stimulation for 30 min. Total cell lysates were prepared and subject to immunoblotting with antibodies against P-GSK3, totalGSK3, P-AKT (T308), P-AKT (S473), P-FKHRL1 (T32), P-ERK (T202/Y204), and total ERK. B, transiently expressed AKT C296A/C310A attenuates theAKT substrate inhibition by lactoquinomycin. DU145 cells were transfected with DNA vectors encoding the Flag-tagged myr-AKT-WT or myr-AKT-C296A/C310A, or mock-transfected for 48 h, and treated with the indicated doses of lactoquinomycin for 12 h. Lysates were similarly analyzed as in A, andprobed with anti-Flag. All blots were stained with Ponceau S for total protein loading control. The blotting data in B were quantified and graphed.






were moderately reduced relative to the wild-type myr-AKT1 (Fig. 4B, right). In subsequent experiments, thewild-type and Cys-mutants of AKT were assayed forsensitivity to lactoquinomycin and frenolicin B (Fig. 4C).We found that although lactoquinomycin (Fig. 4C, top)efficiently inhibited the wild-type AKT (IC50, 0.36 Amol/L),it only weakly inhibited C310A (IC50, 38.5 Amol/L) andC296A (IC50, 30.4 Amol/L), and was inactive against thedouble mutant C296A/C310A at concentrations up to40 Amol/L. Similar results were obtained for frenolicin B(Fig. 4C, middle) with IC50 values of 0.46, 39.6, and27.4 Amol/L against the wild-type, C310A, and C296A,respectively. In contrast to the results with the pyrano-naphthoquinone inhibitors, the ATP-competitive inhibitorstaurosporine (Fig. 4C, bottom) as well as a chemically

unrelated compound (data not shown) indiscriminatelyinhibited all four enzymes with nearly identical potencies.These observations suggest that the active site conforma-tion in the Cys-mutants likely remained comparable to thatof the wild-type AKT because no significant difference inthe inhibitory activity was detected for these ATP inhib-itors. We conclude from these data and additional experi-ments (see below) that the pyranonaphthoquinones inhibitAKT kinase activity through a binding mechanism thatrequires the T-loop Cys296 and Cys310 residues in thecatalytic domain.

Inhibition of AKTDownstream Substrate Phosphory-lation inTumor CellsLactoquinomycin was examined for inhibition of AKT

substrate phosphorylation in tumor cells. Treatment of

Figure 6. Cellular inhibition of AKT down-regulates mTOR activity and cap-dependent translation initiation. A, DU-AKT cells were plated in six-wellplates and grown for 24 h in full growth medium, switched to medium containing 0.5% serum and treated with DMSO vehicle, wortmannin (0.5 Ag/mL),and the indicated doses of lactoquinomycin for 16 h. B,MDA-MB-468 cells were similarly plated and treated in growth medium with the indicated doses oflactoquinomycin for 16 h. Total cell lysates of cells from A and B were subject to immunoblotting with antibodies against P-AKT (S473), P-GSK3, P-S6K(T389), P-FKHRL1 (T32), P-S6 (S240/244), 4EBP1, cyclin D3, and p27/Kip1. All blots were stained with Ponceau S for total protein loading control.C,MDA-MB-468 cells were grown in 100-mm dishes for 24 h and treated with DMSO vehicle, an mTOR inhibitor cell cycle inhibitor-779 (50 nmol/L), andthe indicated doses of lactoquinomycin for 16 h. Total lysates were prepared and subject to 7-methyl-GTP pull-down as described in Materials andMethods. D, total lysates as in C were immunoprecipitated with an anti-eIF3b antibody. Total lysates and protein complexes obtained in C and D weresubject to immunoblotting with antibodies against eIF4E, 4EBP1, eIF4G, eIF4A, and eIF3b.






PTEN-negative U87MG cells with 10, 5, 2.5, and 1.25 Amol/Lof lactoquinomycin for 6 h resulted in a dose-dependentinhibition of GSK3 phosphorylation (Fig. 5A, left). To verifythat the inhibition was not a result of targeting upstreamkinases, we studied a stable DU145-myr-AKT1 (DU-AKT)cell line in which AKT is activated constitutively, in aPI3K-independent fashion. Phosphorylation of FKHRL1was completely suppressed in cells treated for 6 h withlactoquinomycin, but not with the PI3K inhibitor wort-mannin (Fig. 5A, middle). Lactoquinomycin at 2 Amol/Lalso blocked insulin-like growth factor-I – stimulatedphosphorylation of FKHRL1 in serum-starved Rat1 cells(Fig. 5A, right). Notably, Thr308 and Ser473 phosphoryla-tions of AKT were minimally inhibited under these acutetreatment conditions (Fig. 5A, middle and right), furthersupporting a direct targeting of cellular AKT rather thanupstream kinases. The lack of suppression of AKTphosphorylation in cells is consistent with the in vitroenzyme inhibition data in which lactoquinomycin didnot inhibit PDK1 and mTOR, two known kinases ofThr308 and Ser473, respectively (Fig. 2D; data not shown).It was noted that lactoquinomycin dose-dependentlyincreased phospho-ERK in these cells (Fig. 5A), whichmight be a consequence of AKT suppression and/orcellular stress (28, 29). To further address the role ofT-loop cysteines in mediating AKT inhibition in cells,DU145 cells were transiently transfected with the Flag-tagged wild-type and C296A/C310A myr-AKT. Bothconstructs expressed comparable myr-AKT and inducedphospho-FKHRL1 and phospho-GSK3 (Fig. 5B). Treatmentwith lactoquinomycin (1–6 Amol/L) resulted in a dose-dependent suppression of phospho-FKHRL1 and phospho-GSK3 in the wild-type AKT-transfected cells, and theinhibition of both markers were substantially reduced inthe C296A/C310A AKT cells (Fig. 5B). These data areconsistent with the biochemical data in Fig. 4 in confirmingthe critical involvement of T-loop cysteines in the inhibitionmechanism of lactoquinomycin.

Suppression of Cellular AKT Down-regulates mTORActivity and Abrogates Cap-Dependent TranslationInitiationAKT is an upstream regulator of the mTOR in the

canonical growth factor–stimulated cellular mRNA trans-lation (reviewed in ref. 30). We found that both mTORsubstrates S6K1 and 4EBP1 were constitutively phosphor-ylated in serum-starved DU-AKT cells as compared withthe DU145 parent (Fig. 6A, left ), indicating mTORactivation by AKT. We therefore determined whetherlactoquinomycin would interfere with mTOR signalingand mRNA translation. Treatment of DU-AKT cells for16 h with 2, 1, and 0.5 Amol/L of lactoquinomycin resultedin a dose-dependent decrease in phospho-S6K andphospho-4EBP1, whereas the inhibition was not observedin wortmannin-treated cells (Fig. 6A, right). Notably, Ser473

phosphorylation of AKT was also partially reduced with2 Amol/L of lactoquinomycin (Fig. 6A, right), which maybe a result of blocking the signaling of mTOR complex2 after a relatively prolonged treatment. In the PTEN-

negative MDA-MB-468 cells, lactoquinomycin at concen-trations as low as 0.25 Amol/L substantially inhibitedphosphorylation of S6K, S6, 4EBP1, and FKHRL1 (Fig. 6B).Notably, these alterations in mTOR substrate phosphory-lation were accompanied by a marked reduction in cyclinD3- and an increase in p27/Kip1-expression. Both of thecell cycle regulatory proteins are indirectly regulated bymTOR signaling and were shown to be modulated by themTOR inhibitor cell cycle inhibitor-779 in MDA-MB-468cells (31). In additional studies, we examined the effectsof lactoquinomycin on the structure of the cap-bindingtranslation initiation complex, which is known to bemodulated by AKT/mTOR signaling. 7-Methyl-GTP pull-down assays showed a profound dose-dependent increasein the binding of 4EBP1 to the translation initiation factoreIF4E (Fig. 6C), consistent with the known inhibitoryeffects of 4EBP1 on the assembly of an active, cap-bindingtranslation initiation complex (32, 33). The increase ineIF4E-bound 4EBP1 was accompanied by a decrease in thecoprecipitation of eIF4G and eIF4A with eIF4E (Fig. 6C).Treatment with the mTOR inhibitor cell cycle inhibitor-779(50 nmol/L) caused a similar but relatively modestincrease in eIF4E-bound 4EBP1 and loss of eIF4G and 4A(Fig. 6C). Similar results were obtained when we exam-ined the levels of eIF3b-bound eIF4E. Although the controlsample contained abundant eIF4E, samples from treatedcells exhibited a concentration-dependent loss of eIF4E(Fig. 6D). As expected, a similar but partial loss of eIF4Ewas also observed in the cell cycle inhibitor-779–treatedcells (Fig. 6D). Thus, it is clear from the data in Fig. 6 thatinhibition of AKT by lactoquinomycin in these cellsresulted in a substantial suppression of mTOR signalingand disruption of cap-initiation complexes eIF4F and eIF3,thereby inhibiting cap-dependent translation initiation, arate-limiting step of cellular protein synthesis.

DiscussionDespite widespread screening efforts, few small moleculeinhibitors of AKT have been reported (14, 15). Moreover,the development of selective AKT inhibitors is hindered bythe extensive conservation of the ATP-binding pockets ofthe AGC kinase family. Members of this family participatein a wide array of critical cellular functions, and broadtargeting of this family might lead to adverse side effects.Consequently, AKT inhibitors that avoid the conservedATP binding site have attracted considerable interest. Inthis regard, a class of AKT pleckstrin-homology domaininhibitors was reported to possess remarkable specificityfor AKT over other AGC family members and to inhibit thephosphorylation of AKT in cells (17). It remains to be seenwhether these inhibitors potently block the phosphoryla-tion of AKT downstream substrates in tumor cells withconstitutive PI3K/AKT signaling pathways (17).In the present study, we have identified two pyrano-

naphthoquinones that potently inhibited AKT throughtargeting of the catalytic T-loop of the enzyme. Binding ofthese inhibitors to AKT did not require the pleckstrin-homology domain and did not discriminate appreciably






between AKT1 and AKT2 isoenzymes. A novel mechanisminvolving an irreversible inhibitor–T-loop interaction wasstrongly supported by mutational analysis that highlightedthe T-loop Cys296 and Cys310 residues to be critical forsensitivity to the pyranonaphthoquinone inhibitors. Thisconclusion was further supported by the observation thattransiently expressed C296A/C310A myr-AKT substantial-ly attenuated the cellular inhibition of AKT substratephosphorylation in response to lactoquinomycin. Massspectrometry studies have been used to elucidate thespecific covalent interaction of the same T-loop cysteinesin AKT2 with lactoquinomycin and related pyranonaph-thoquinones. These results and the proposed mode ofchemical inactivation of AKT will be described elsewhere.In various tumor cell models, lactoquinomycin potentlyand acutely inhibited the phosphorylation of AKT down-stream substrates GSK3a/h, FKHRL1, as well as mTOR(data not shown). In serum-starved Rat1 cells, this com-pound blocked the insulin-like growth factor-I–stimulatedphosphorylation of known AKT substrates. These data thusindicate that lactoquinomycin could inhibit both thegrowth factor–stimulated as well as the constitutivelyactive AKT signaling pathways in exponentially proliferat-ing tumor cells. The observation that lactoquinomycin didnot inhibit Thr308 phosphorylation of AKT itself in thesecells indicates that binding of the inhibitor to the AKTT-loop does not interfere with the PDK1-dependent Thr308

phosphorylation. Alternatively, lactoquinomycin mightpreferentially target the phosphorylated form of AKT.Lactoquinomycin caused an increase of phospho-ERK incells. Although the mechanism for ERK phosphorylationwas not investigated in this study, previous reports haveindicated a negative regulation of the Raf/Mek/ERKpathway by AKT as well as ERK activation in stressresponse (28, 29). Thus, an enhanced phospho-ERKinduced by lactoquinomycin may reflect a consequence ofAKT suppression in these cells and/or stress response.Nevertheless, despite its induction of phospho-ERK,lactoquinomycin inhibited the proliferation of a broadpanel of cancer cells in the culture (data not shown).The selective targeting of AKT over those tested AGC

kinases by these pyranonaphthoquinones indicates thatT-loop inhibition may be a viable strategy for developingnovel inhibitors of AKT. This selectivity likely reflects thefact that, in contrast to the high degree of ATP binding siteconservation among the AGC kinases, the T-loops of theseprotein kinases are relatively polymorphic (27). Althoughone or both of the equivalent T-loop cysteines areconserved in all AGC family members, lactoquinomycinwas inactive against these highly related enzymes. Ittherefore seems that the T-loop region of the AKT familymay confer a distinct conformational feature(s) that rendersthe two target cysteines susceptible to modification by thepyranonaphthoquinones. The AKT2 crystal structuresreveal that the inactive AKT2 has an unstructured T-loop,which becomes structured upon phosphorylation of Thr309

by PDK1 (26, 27). This structuring of the T-loop isaccompanied by the realignment of the rest of the catalytic

domain, resulting in compatibility with both ATP andsubstrate binding. It is conceivable that binding oflactoquinomycin in the T-loop may either directly interferewith interaction of AKT with substrate or cause aninhibitor-induced conformational misalignment that abro-gates catalysis. Interestingly, although the pyranonaphtho-quinones represent the first class of T-loop inhibitorstargeting AKT, the antiinflammatory natural productparthenolide was previously shown to bind the T-loopcysteine of IKKh and block its kinase activity (34). Atpresent, it is not known whether the T-loop cysteines ofactive AKT generally play a role in physiologic regulationof AKT activity in cells, an earlier report implicated redoxregulation of T-loop cysteines of AKT2 in response tohydrogen peroxide–induced apoptosis, which involvesdisulfide bond formation between Cys297 and Cys311 anddephosphorylation by phosphatase 2A (35). We observedthat inhibition of AKT by lactoquinomycin did not involvedisulfide formation (data not shown) and did not requiredephosphorylation of AKT in cells. It is noteworthy thatlactoquinomycin also inhibited HER2 and IKKh in vitro andin select cell models at higher doses (data not shown).Interestingly, the active site Cys805 of HER2 was previouslyimplicated in the covalent inhibition by HKI-272 (36).However, the mechanism of action by lactoquinomycin intargeting HER2 remains to be elucidated.The PI3K/AKT/mTOR pathway positively regulates the

cap-dependent mRNA translation through dynamic phos-phorylation of the mTOR substrates 4EBP1 and S6K1,which are crucial regulatory events required for thefunctional assembly of key translation initiation proteincomplexes (30, 32, 33, 37, 38). In particular, mTOR-dependent phosphorylation of 4EBP1 promotes its releasefrom eIF4E bound to the mRNA 5¶ cap structure (7-methyl-GTP; refs. 32, 33), allowing for the recruitment of the RNAhelicase eIF4A and the scaffolding protein eIF4G. Thisprocess is essential for the recruitment of the 40S ribosomalsubunit to the mRNA and is abrogated by the mTORinhibitor rapamycin (32). Constitutive phosphorylation of4EBP1 and/or S6K1 is believed to contribute to oncogenictransformation in tumors harboring deregulated PI3K/AKT/mTOR signaling (30, 39, 40). In DU-AKT cells,constitutive phosphorylation of S6K1 and 4EBP1 was nor-malized by lactoquinomycin. In exponentially proliferatingMDA-MB-468 cells, lactoquinomycin-induced dephosphor-ylation of 4EBP1 correlated with a drastic increase inbinding of 4EBP1 to eIF4E and disruption of cap-initiationcomplexes eIF4F and eIF3. Thus, our data, obtained with aselective chemical inhibitor of AKT, corroborated previousgenetic and biochemical data in further establishing AKT asa critical positive regulator of cap-dependent mRNAtranslation.Given its critical role in tumor growth and survival, AKT

is an attractive target for the development of new therapies.Our results highlight T-loop targeting as a new strategy forthe development of potent and selective AKT inhibitors forthe treatment of cancer and other proliferative diseases.Nevertheless, lactoquinomycin itself is not suitable for






therapy because the general redox properties of thenaphthoquinones confer nonspecific cytotoxicity and mightlimit the therapeutic window (41). Interestingly, the AKTinhibition by the pyranonaphthoquinones does not seem touse the general redox mechanism, and AKT inhibition isnot a common property of benzoquinones (data notshown). The specific structural features of the pyranonaph-thoquinone scaffold are critical for AKT inactivation.Hence, new chemical analogues with further improvedAKT inhibition potency, selectivity, and reduced redoxproperties might offer higher therapeutic potential. Ourstudies on the elucidation of the chemical structure andactivity relationship of the pyranonaphthoquinones will bedescribed elsewhere.

Acknowledgments

We thank Frank Erardi, Frank Ritaco, Jason Lotvin, Mark Tischler, MaireadYoung, and Celine Shi for technical assistance; and Drs. Philip Frost, GuyCarter, and Jerauld Skotnicki for helpful discussions and generous support.

References

1. Luo J, Manning BD, Cantley LC. Targeting the PI3K-Akt pathway inhuman cancer: rationale and promise. Cancer Cell 2003;4:257–62.

2. Coffer PJ, Woodgett JR. Molecular cloning and characterisation of anovel putative protein-serine kinase related to the cAMP-dependent andprotein kinase C families. Eur J Biochem 1991;201:475–81.

3. Bellacosa A, Testa JR, Staal SP, Tsichlis PN. A retroviral oncogene,akt, encoding a serine-threonine kinase containing an SH2-like region.Science 1991;254:274–7.

4. Alessi DR, Andjelkovic M, Caudwell B, et al. Mechanism of activationof protein kinase B by insulin and IGF-1. EMBO J 1996;15:6541–51.

5. Cantley LC, Neel BG. New insights into tumor suppression: PTENsuppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc Natl Acad Sci U S A 1999;96:4240–5.

6. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKTpathway in human cancer. Nat Rev Cancer 2002;2:489–501.

7. Altomare DA, Testa JR. Perturbations of the AKT signaling pathway inhuman cancer. Oncogene 2005;24:7455–64.

8. Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov 2005;4:988–1004.

9. Mazure NM, Chen EY, Laderoute KR, Giaccia AJ. Induction of vascularendothelial growth factor by hypoxia is modulated by a phosphatidylinositol3-kinase/Akt signaling pathway inHa-ras-transformed cells through a hypoxiainducible factor-1 transcriptional element. Blood 1997;90:3322–31.

10. Zundel W, Schindler C, Haas-Kogan D, et al. Loss of PTEN facilitatesHIF-1-mediated gene expression. Genes Dev 2000;14:391–6.

11. Davies MA, Lu Y, Sano T, et al. Adenoviral transgene expression ofMMAC/PTEN in human glioma cells inhibits Akt activation and inducesanoikis. Cancer Res 1998;58:5285–90.

12. Jetzt A, Howe JA, Horn MT, et al. Adenoviral-mediated expression ofa kinase-dead mutant of Akt induces apoptosis selectively in tumor cellsand suppresses tumor growth in mice. Cancer Res 2003;63:6697–706.

13. Liu X, Shi Y, Han EK, et al. Downregulation of Akt1 inhibitsanchorage-independent cell growth and induces apoptosis in cancer cells.Neoplasia 2001;3:278–86.

14. Barnett SF, Bilodeau MT, Lindsley CW. The Akt/PKB family of proteinkinases: a review of small molecule inhibitors and progress towards targetvalidation. Curr Top Med Chem 2005;5:109–25.

15. Chen YL, Law PY, Loh HH. Inhibition of PI3K/Akt signaling: anemerging paradigm for targeted cancer therapy. Curr Med ChemAnticancer Agents 2005;5:575–89.

16. Luo Y, Shoemaker AR, Liu X, et al. Potent and selective inhibitors ofAkt kinases slow the progress of tumors in vivo. Mol Cancer Ther 2005;4:977–86.

17. Barnett SF, Defeo-Jones D, Fu S, et al. Identification and character-

ization of pleckstrin-homology-domain-dependent and isoenzyme-specificAkt inhibitors. Biochem J 2005;385:399–408.

18. Kondapaka SB, Singh SS, Dasmahapatra GP, Sausville EA, Roy KK.Perifosine, a novel alkylphospholipid, inhibits protein kinase B activation.Mol Cancer Ther 2003;2:1093–103.

19. Meuillet EJ, Mahadevan D, Vankayalapati H, et al. Specific inhibitionof the Akt1 pleckstrin homology domain by D-3-deoxy-phosphatidyl-myo-inositol analogues. Mol Cancer Ther 2003;2:389–99.

20. Martelli AM, Tazzari PL, Tabellini G, et al. A new selective AKTpharmacological inhibitor reduces resistance to chemotherapeutic drugs,TRAIL, all-trans-retinoic acid, and ionizing radiation of human leukemiacells. Leukemia 2003;17:1794–805.

21. Jones PF, Jakubowicz T, Pitossi FJ,Maurer F, Hemmings BA.Molecularcloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci U S A 1991;88:4171–5.

22. Klippel A, Reinhard C, Kavanaugh WM, Apell G, Escobedo MA,Williams LT. Membrane localization of phosphatidylinositol 3-kinase issufficient to activate multiple signal-transducing kinase pathways. MolCell Biol 1996;16:4117–27.

23. Kishimoto A, Nishiyama K, Nakanishi H, et al. Studies on thephosphorylation of myelin basic protein by protein kinase C and adenosine3¶:5¶-monophosphate-dependent protein kinase. J Biol Chem 1985;260:12492–9.

24. Toral-Barza L, Zhang WG, Lamison C, Larocque J, Gibbons J, Yu K.Characterization of the cloned full-length and a truncated human target ofrapamycin: activity, specificity, and enzyme inhibition as studied by a highcapacity assay. Biochem Biophys Res Commun 2005;332:304–10.

25. Zhang X, Zhang S, Yamane H, et al. Kinetic mechanism of AKT/PKBenzyme family. J Biol Chem 2006;281:13949–56.

26. Huang X, Begley M, Morgenstern KA, et al. Crystal structure of aninactive Akt2 kinase domain. Structure 2003;11:21–30.

27. Yang J, Cron P, Good VM, Thompson V, Hemmings BA, Barford D.Crystal structure of an activated Akt/protein kinase B ternary complex withGSK3-peptide and AMP-PNP. Nat Struct Biol 2002;9:940–4.

28. Reusch HP, Zimmermann S, Schaefer M, Paul M, Moelling K.Regulation of Raf by Akt controls growth and differentiation in vascularsmooth muscle cells. J Biol Chem 2001;276:33630–7.

29. Cuevas BD, Abell AN, Johnson GL. Role of mitogen-activated proteinkinase kinase kinases in signal integration. Oncogene 2007;26:3159–71.

30. Ruggero D, Sonenberg N. The Akt of translational control. Oncogene2005;24:7426–34.

31. Yu K, Toral-Barza L, Discafani C, et al. mTOR, a novel target in breastcancer: the effect of CCI-779, an mTOR inhibitor, in preclinical models ofbreast cancer. Endocr Relat Cancer 2001;8:249–58.

32. Brunn GJ, Hudson CC, Sekulic A, et al. Phosphorylation of thetranslational repressor PHAS-I by the mammalian target of rapamycin.Science 1997;277:99–101.

33. Lawrence JC, Jr., Abraham RT. PHAS/4E-BPs as regulators of mRNAtranslation and cell proliferation. Trends Biochem Sci 1997;22:345–9.

34. Kwok BH, Koh B, Ndubuisi MI, Elofsson M, Crews CM. The anti-inflammatory natural product parthenolide from themedicinal herb feverfewdirectly binds to and inhibits InB kinase. Chem Biol 2001;8:759–66.

35. Murata H, Ihara Y, Nakamura H, Yodoi J, Sumikawa K, Kondo T.Glutaredoxin exerts an antiapoptotic effect by regulating the redox state ofAkt. J Biol Chem 2003;278:50226–33.

36. Rabindran SK, Discafani CM, Rosfjord EC, et al. Antitumor activity ofHKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosinekinase. Cancer Res 2004;64:3958–65.

37. Holz MK, Ballif BA, Gygi SP, Blenis J. mTOR and S6K1 mediateassembly of the translation preinitiation complex through dynamic proteininterchange and ordered phosphorylation events. Cell 2005;123:569–80.

38. Harris TE, Chi A, Shabanowitz J, Hunt DF, Rhoads RE, Lawrence JC,Jr. mTOR-dependent stimulation of the association of eIF4G and eIF3 byinsulin. EMBO J 2006;25:1659–68.

39. Clemens MJ. Targets and mechanisms for the regulation oftranslation in malignant transformation. Oncogene 2004;23:3180–8.

40. Bjornsti MA, Houghton PJ. Lost in translation: dysregulation of cap-dependent translation and cancer. Cancer Cell 2004;5:519–23.

41. Nomoto K, Okabe T, Suzuki H, Tanaka N. Mechanism of action oflactoquinomycin A with special reference to the radical formation. JAntibiot (Tokyo) 1988;41:1124–9.






2007;6:3028-3038. Published OnlineFirst November 7, 2007.Mol Cancer Ther Lourdes Toral-Barza, Wei-Guo Zhang, Xinyi Huang, et al. activation loop cysteinesof AKT/PKB: mechanistic involvement of AKT catalyticpyranonaphthoquinones as potent and allosteric inhibitors Discovery of lactoquinomycin and related

Updated version

10.1158/1535-7163.MCT-07-0211doi:

Access the most recent version of this article at:

Cited articles

http://mct.aacrjournals.org/content/6/11/3028.full#ref-list-1

This article cites 41 articles, 18 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/6/11/3028.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://mct.aacrjournals.org/content/6/11/3028To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-07-0211

http://mct.aacrjournals.org/content/6/11/3028.full#ref-list-1

http://mct.aacrjournals.org/content/6/11/3028.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/6/11/3028


Documents

Discovery of lactoquinomycin and related ... · of AKT catalytic activation loop cysteines Lourdes Toral-Barza,1 Wei-Guo Zhang,1 Xinyi Huang,2 Leonard A. McDonald,2 Edward J. Salaski,2