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Molecular Cancer Therapeutics
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Research Articles: Therapeutics, Targets, and Development

Discovery of a novel class of AKT pleckstrin homology domain inhibitors

Daruka Mahadevan, Garth Powis, Eugene A. Mash, Benjamin George, Vijay M. Gokhale, Shuxing Zhang, Kishore Shakalya, Lei Du-Cuny, Margareta Berggren, M. Ahad Ali, Umasish Jana, Nathan Ihle, Sylvestor Moses, Chloe Franklin, Satya Narayan, Nikhil Shirahatti and Emmanuelle J. Meuillet
Daruka Mahadevan
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Garth Powis
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Eugene A. Mash
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Benjamin George
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Vijay M. Gokhale
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Shuxing Zhang
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Kishore Shakalya
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Lei Du-Cuny
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Margareta Berggren
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M. Ahad Ali
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Umasish Jana
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Nathan Ihle
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Sylvestor Moses
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Chloe Franklin
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Satya Narayan
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Nikhil Shirahatti
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Emmanuelle J. Meuillet
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DOI: 10.1158/1535-7163.MCT-07-2276 Published September 2008
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  • Figure 1.
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    Figure 1.

    Interactions of compound 1 with the human AKT1 PH domain. A, schematic representation of the interaction of compound 1 (NSC 348900, 4-[1-(4-chlorobenzoyl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-4-ylazo]-N-pyrimidine-2-yl-benzenesulfonamide, C21H16ClN7O4S) with amino acid residues of the AKT1 PH domain (Arg86, Asn53, Arg23, and Ile19). B, the docking pose of compound 1 with the AKT1 PH domain. The AKT1 PH domain (red); residues Arg23, Arg25, and Arg86 are colored according to atom type. Compound 1 is colored according to atom type (capped stick). Hydrogen bonding interactions (dotted lines). The sulfamido group interacts with Arg86 through a hydrogen bonding interaction whereas similar hydrogen bonds are involved in the interaction of the diazopyrazolyl group with Arg23. These two arginines are intimately involved in the interaction with the phosphate head groups of the substrate phosphoinositol-1,3,4,5-tetrakisphosphate. C and D, the binding mode of compound 1 in the binding pocket of the PH domain of PDK1 with the amino acids involved in the binding pocket (D). Note that compound 1 exhibits the reverse binding pose in the PDK PH domain similar to compound 2 in the PH domain of AKT1. Supplementary Fig. 1A and B show the binding mode of compound 1 in the binding pocket of the PH domain of IRS1.

  • Figure 2.
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    Figure 2.

    Synthesis of compounds 1 to 6.

  • Figure 3.
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    Figure 3.

    Binding of the compounds (1–2) to the PH domain of AKT1 and IRS1. The proteins were immobilized on a CM5 Sensorchip as described in Materials and Methods. The drugs were injected over the surface at the indicated concentrations and binding to the proteins was measured by surface plasmon resonance. A, the binding curves of compound 1 (left) and compound 2 (right) to the PH domain of AKT. B, the binding curves of compound 1 (left) and compound 2 (right) binding to the PH domain of IRS1. Overlay plot of typical sensorgrams obtained with increasing concentrations of compound 1 or 2 (arrows; A).

  • Figure 4.
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    Figure 4.

    ELISA competitive binding assay. Competition binding curves for compounds 1 (A) and 2 (B), respectively, to the (□) AKT PH domain and (•) IRS1 PH domain. Points, mean of three determinations; bars, SD (**, P < 0.05). Compound 1 binds the PH domains of AKT1 and IRS1 in a similar manner. Compound 2 binds only the PH domain of AKT1.

  • Figure 5.
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    Figure 5.

    Inhibition of AKT in cancer cells. A, HT-29 colon cancer cells were treated with compounds 1 to 6, at 20 μmol/L for 2 h and stimulated with 50 ng/nL of epidermal growth factor for 30 min. Akt activity was measured by Western blotting using anti–phospho-Ser437 AKT antibody. All other downstream targets of AKT were also detected by Western blotting using specific anti–phospho-antibodies. β-Actin was used as a loading control. Note that compounds 2 and 6 inhibit AKT phosphorylation and downstream GSK3 phosphorylation. B, apoptosis measured as described in Materials and Methods. Note that both compounds 2 and 6 at 20 μmol/L induce significant apoptosis significantly as compared with controls. Columns, mean of three determinations; bars, SD (**, P < 0.05 and ***, P < 0.001). Compound 1 (C) or its analogue compound 2 (D) were also tested at the concentrations shown for 2 h, and in HT-29 cells, stimulated with 50 ng/nL of epidermal growth factor for 30 min. Akt activity was measured by Western blotting using anti–phospho-Ser437 AKT antibody, PDK activity by anti–phospho-Ser241 PDK antibody as well as downstream target PKC using pan–phospho-PKC antibodies. β-Actin was used as a loading control.

  • Figure 6.
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    Figure 6.

    In vivo activity of compound 1. A, pharmacokinetics of compound 1 in mice. Female scid mice were administered compound 1 at a dose of 250 mg/kg either (•) i.p. or (○) p.o. by oral gavage and plasma concentrations measured. Points, means of three mice; bars, SE. B, antitumor activity in female scid mice with HT-29 colon cancer xenografts treated p.o. daily for 5 days (arrows) with (•) vehicle alone or (□) compound 1 at 250 mg/kg daily. Points, means of 10 mice; bars, SE. C. Effect on tumor phospho-AKT in female scid mice with HT-29 colon cancer xenografts treated p.o. with (open columns) vehicle alone or (filled columns) compound 1 at 250 mg/kg. Tumors were removed at various times for Western blotting. Columns, mean of four mice; bars, SE (*, P < 0.05; **, P < 0.01).

Tables

  • Figures
  • Table 1.

    Structures and calculated properties of lead compound 1 (NSC 348900) and analogues (2–6)

    CompoundsAKT1 FlexX scoreAKT1 G scoreAKT1 cKD(μmol/L)PDK1 FlexX scorePDK1 G scorePDK1 cKD(μmol/L)IRS1 FlexX scoreIRS1 G scoreIRS1 cKD(μmol/L)
    Embedded ImageNSNS4.0NSNSNSNSNSNS
    1Embedded Image−31.0−96.91.2−17.4−109.01.74−16.0−128.01.99
    2Embedded Image−29.6−31.92.4−17.0−40.02.60−17.1−96.22.40
    3Embedded Image−28.2−99.51.2−17.1−103.41.70−14.8−79.710.70
    4Embedded Image−29.1−71.93.0−17.5−88.62.20−17.9−145.51.80
    5Embedded Image−33.0−120.61.3−20.1−137.12.40−14.6−90.110.70
    6Embedded Image−24.3−132.00.85−21.0−109.11.45−14.5−140.60.52
    • Abbreviation: NS, not shown.

  • Table 2.

    Sequence alignments of the PH domain of AKT

    Alignment of human AKT PH domain isoforms
    AA numbers14 20 23 25 27 52 55
    AKT1 PHMSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQREAPLNNFSVA58
    AKT2 PHMSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQREAPLNNFSVA58
    AKT3 PHMSDVTIVKEGWVQKRGEYIKNWRPRYFLLKTDGSFIGYKEKPQDVDLP-YPLNNFSVA57
    AA numbers71 74 76
    AKT1 PHQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGL110
    AKT2 PHQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGL110
    AKT3 PHKCQLMKTERPKPNTFIIRCLQWTTVIERTFHVDTPEEREEWTEAIQAVADRL109
    Alignment of human (h) and mouse (m) AKT PH domains
    AA numbers14 20 23 25 27 52 55
    hAKT PHMSDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQREAPLNNFSVAQC
    mAKT PHMNDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDVDQRESPLNNFSVAQC
    AA numbers71 74 76
    hAKT PHQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWTTAIQTVADGL110
    mAKT PHQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWATAIQTVADGL110
    • The PH domain binding residues are shown underlined in the sequence alignment above obtained using CLUSTAL W (1.82) multiple sequence alignment.

  • Table 3.

    Selectivity for other PH domains

    CompoundsAKT1 PH mKD (μmol/L)IRS1 PH mKD (μmol/L)PDK1 PH mKD (μmol/L)
    PtdIns(3,4,5)P33.08 ± 0.49NDND
    DPIEL5.04 ± 0.4831.56 ± 8.49NB
    10.37 ± 0.040.39 ± 0.0131.28 ± 9.54
    23.66 ± 0.03NB0.17 ± 0.10
    31.37 ± 0.25NB3.57 ± 0.96
    40.51 ± 0.060.14 ± 0.02NB
    51.35 ± 0.021.74 ± 0.410.42 ± 0.17
    61.62 ± 0.02NB0.98 ± 0.48
    • NOTE: Binding affinities were measured by surface plasmon resonance spectroscopy as described in Materials and Methods and are referred to as mKD (μmol/L) for measured.

    • Abbreviations: NB, no measurable binding; ND, not determined.

  • Table 4.

    Biological properties of compound 1 and analogues

    CompoundAKT inhibition (IC50 μmol/L)
    Cytotoxicity (IC50 μmol/L)Log PMetabolic half-life (min)Solubility (μmol/L)Permeability (nm/s)
    NIH3T3HT-29Caco2MDCK
    1413242.16217.99091
    21120141.96228.38334
    3>20>20253.29128.69539
    4ND>20NI1.2>48012.9238
    55>20NI1.513813.1185200
    635ND1.9ND<0.1145
    • Abbreviations: NI, not inhibitory for IC50 >100 μmol/L; ND, not determined.

    • NOTE: For each of the analogues, phospho-Ser473 AKT inhibition was measured in either mouse NIH3T3 or human HT-29 colon cancer cells. Cytotoxicity was measured in HT-29 cells. Metabolic stability was measured by incubating with HT-29 cells at the maximum concentration in DMEM at 37°C. The apparent permeability (nm/s) in Caco-2 and MDCK cells was obtained using the QikProp software (Schrodinger Inc.). Values ranged between <25 for poor and >500 for great permeability.

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Molecular Cancer Therapeutics: 7 (9)
September 2008
Volume 7, Issue 9
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Discovery of a novel class of AKT pleckstrin homology domain inhibitors
Daruka Mahadevan, Garth Powis, Eugene A. Mash, Benjamin George, Vijay M. Gokhale, Shuxing Zhang, Kishore Shakalya, Lei Du-Cuny, Margareta Berggren, M. Ahad Ali, Umasish Jana, Nathan Ihle, Sylvestor Moses, Chloe Franklin, Satya Narayan, Nikhil Shirahatti and Emmanuelle J. Meuillet
Mol Cancer Ther September 1 2008 (7) (9) 2621-2632; DOI: 10.1158/1535-7163.MCT-07-2276

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Discovery of a novel class of AKT pleckstrin homology domain inhibitors
Daruka Mahadevan, Garth Powis, Eugene A. Mash, Benjamin George, Vijay M. Gokhale, Shuxing Zhang, Kishore Shakalya, Lei Du-Cuny, Margareta Berggren, M. Ahad Ali, Umasish Jana, Nathan Ihle, Sylvestor Moses, Chloe Franklin, Satya Narayan, Nikhil Shirahatti and Emmanuelle J. Meuillet
Mol Cancer Ther September 1 2008 (7) (9) 2621-2632; DOI: 10.1158/1535-7163.MCT-07-2276
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