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Akt Inhibitors

A-674563 is a potent selective protein kinase B/Akt inhibitor with an IC50 of 14 nM. A-674563 also shows inhibitory activity against PKA and CDK2 with IC50 of 16 and 46 nM, respectively. While promising efficacy was observed in vivo, A-674563 indicated significantly effects on depolarization of purkinje fiber in an in vitro assay and severe CV toxicity (e.g. hypotension) in vivo. An X-ray structure of A-674563 bound to protein kinase A, which has 80% homology with Akt in the kinase domain, suggested the phenyl group is not tightly bound in the ligand-protein complex. The Akt inhibitor A-674563 displayed proliferation of tumor cells with an EC50 of 0.4 μM. When given in combination, A-674563 enhanced the efficacy of paclitaxel in a PC-3 xenograft model.[1][2]

References on A-674563:

[1] Luo Y et al. Mol Cancer Ther. 2005 Jun;4(6):977-86.

[2] http://oasys2.confex.com/acs/229nm/techprogram/P850964.HTM

AT7867 is a potent and oral AKT and p70 S6 kinase inhibitor with an IC50 of 17 nM. AT7867 potently prevents both AKT and p70S6K activity at the cellular level, as measured by prevention of GSK3β and S6 ribosomal protein phosphorylation, and also gives rise to the suppression of growth in a range of human cancer cell lines as a single agent. Induction of apoptosis was detected by multiple methods in tumor cells following AT7867 treatment. Administration of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) to athymic mice implanted with the PTEN-deficient U87MG human glioblastoma xenograft model gave rise to inhibition of phosphorylation of downstream substrates of both AKT and p70S6K and induction of apoptosis, confirming the observations made in vitro. These doses of AT7867 also led to inhibition of human tumor growth in PTEN-deficient xenograft models. [1] 

Protocol:

Biochemical assay:
Cells were plated in 96-well microplates at 5,000 per well in medium supplemented with 10% fetal bovine serum and grown for 24 hours before treatment with AT7867. Inhibitor or vehicle control was added to the cells for 72 hours. Following this, Alamar Blue solution was added as stated in the manufacturer’s instructions. The IC50 value for AT7867 was calculated in GraphPad Prism using nonlinear regression analysis and a sigmoidal dose-response equation. [1]

Cell assay:
Cells were plated in 96-well microplates at 16,000 per well in medium supplemented with 10% fetal bovine serum and grown for 24 hours before treatment with AT7867. AT7867 or vehicle control was added to the cells for 1 hour. Following this, cells were fixed with 3% paraformaldehyde, 0.25% glutaraldehyde, 0.25% Triton-X100, washed, and blocked with 5% milk in TBS with 0.1% Tween 20 before overnight incubation with a phosphorylated GSK3β (Ser9) antibody. The plates were then washed, secondary antibody was added, and enhancement of the signal was done using DELFIA reagents as stated in the manufacturer’s instructions. Europium counts were normalized to the protein concentration, and the IC50 value for each inhibitor was calculated in GraphPad Prism using nonlinear regression analysis and a sigmoidal dose-response (variable slope) equation. [1]

Animal study:
A single dose of AT7867 (90 mg/kg p.o. or 20 mg/kg i.p.) was administrated into mice bearing MES-SA tumor xenografts to assess the pharmacokinetic and pharmacodynamic profile. AT7867 was formulated in vehicle containing 0.1% Tween 20. Plasma and tumor samples were harvested at 2, 6, and 24 hours after dosing. Mice were bled by cardiac puncture, and plasma samples were collected and frozen at −20°C until analysis. Tumors were dissected and divided into two approximately equal pieces. One piece was frozen at −20°C until pharmacokinetic analysis, whereas the other was snap frozen in liquid nitrogen until pharmacodynamic analysis. For pharmacokinetic bioanalysis, all tissue samples were first homogenized in five volumes (w/v) of acetonitrile/water (50:50). AT7867 was extracted from plasma and tissue homogenates and quantified as described above. For pharmacodynamic studies, tumors were ground to a powder under liquid nitrogen, lysed, and centrifuged to remove debris. Protein content was analyzed using bicinchoninic acid reagent, and samples run on Western blots as described above. [1]

References on AT7867:

[1] Grimshaw KM et al. Mol Cancer Ther. 2010 May;9(5):1100-10.

CCT128930 is a novel potent ATP-competitive, AKT inhibitor with an IC50 of 6 nM. CCT128930 prevents AKT activity in vitro and in vivo and induces marked antitumor responses. CCT128930 showed significantly antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G1 arrest in PTEN-null U87MG human glioblastoma cells, in  good agreement with AKT pathway blockade.[1]

References on CCT128930:

[1] Yap TA et al. Mol Cancer Ther. 2011 Feb;10(2):360-71. 

GSK690693 is an aminofurazan-derived，novel ATP-competitive, low-nanomolar pan-Akt kinase inhibitor. It is selective for the Akt isoforms(inhibits Akt kinases 1, 2, and 3 with IC50 values of 2, 13, and 9 nM respectively)versus the majority of kinases in other families; however, it does inhibit additional members of the AGC kinase family. [1,2]

GSK690693 inhibited proliferation and induced apoptosis in a subset of tumor cells with potency consistent with intracellular inhibition of Akt kinase activity. [1]

Daily administration of GSK690693 produced significant antitumor activity in mice bearing established human SKOV-3 ovarian, LNCaP prostate, and BT474 and HCC-1954 breast carcinoma xenografts. [1]

References on GSK690693:

[1] Nelson Rhodes et al. Cancer Res.2008 April 1;68:2366-2374

[2] Heerding DA et al. J Med Chem. 2008 Sep 25;51(18):5663-79

Honokiol is a biphenolic compound present in the cones, bark, and leaves of Magnolia grandifloris. It inhibits phosphorylation of Akt, p44/42 mitogen-activated protein kinase (MAPK), and src. Additionally, honokiol modulates the nuclear factor kappa B (NF-κB) activation pathway, an upstream effector of vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX-2), and MCL1, all significant pro-angiogenic and survival factors. Honokiol induces caspase-dependent apoptosis in a TRAIL-mediated manner, and potentiates the pro-apoptotic effects of doxorubicin and other etoposides. Honokiol has been shown to promote neurite outgrowth and have neuroprotective effects in rat cortical neurons. [1]

References on Honokiol:

[1] http://en.wikipedia.org/wiki/Honokiol

MK-2206 is a highly selective, potent non-ATP competitive allosteric Akt inhibitor with IC50 of 5.3 nM,12 nM, 65 nM for Akt1, Akt2 and Akt3 respectively. MK-2206 possesses extensively preclinical antitumor activity. In vitro, MK-2206 synergistically inhibited cell proliferation of human cancer cell lines in combination with molecular targeted agents such as erlotinib or lapatinib. Complementary prevention of erlotinib-insensitive Akt phosphorylation by MK-2206 was one mechanism of synergism, and a synergistic effect was found even in erlotinib-insensitive cell lines. 1 hr treatment of 1 μM MK-2206 abolished Akt phosphorylation in U87MG cells. MK-2206 treatment abolished IR-induced Akt phosphorylation. Moreover, treatment with MK-2206 also increased the radiosensitivity of U87MG cells. [1] MK-2206 also revealed synergistic responses in combination with cytotoxic agents such as topoisomerase inhibitors (doxorubicin, camptothecin), antimetabolites (gemcitabine, 5-fluorouracil), anti-microtubule agents (docetaxel), and DNA cross-linkers (carboplatin) in lung NCI-H460 or ovarian A2780 tumor cells. The synergy with docetaxel depended on the treatment sequence; a schedule of MK-2206 dosed before docetaxel was not effective. MK-2206 inhibited the Akt phosphorylation that is induced by carboplatin and gemcitabine. [1]
In vivo, MK-2206 in combination with these agents exerted significantly more potent tumor inhibitory activities than each agent in the monotherapy setting. MK-2206 is generally well tolerated at doses up to 60 mg QOD with plasma concentrations that portend activity in preclinical models. PK/PD results suggest a substantial and maintained target inhibition should at 60 mg. [2] The phase II clinical trials of MK-2206 against the treatment of endometrial cancer are recruiting participant Dana-Farber Cancer Institute.

References on MK-2206:

[1] Hirai H et al. Mol Cancer Ther. 2010 Jul;9(7):1956-67.

[2] A. W. Tolcher . Meeting: 2009 ASCO Annual Meeting

Palomid 529 (P529) is a novel potent antitumour PI3K/Akt/mTOR inhibitor with a GI50 of <35 μM in the NCI-60 cell lines panel. Palomid 529 (P529) inhibits the TORC1 and TORC2 complexes and shows both inhibition of Akt signaling and mTOR signaling similarly in tumor and vasculature. Palomid 529 (P529) inhibits tumor growth, angiogenesis, and vascular permeability. However, Palomid 529 (P529) has the additional benefit of blocking pAktS473 signaling consistent with blocking TORC2 in all cells and thus bypassing feedback loops that lead to increased Akt signaling in some tumor cells. Palomid 529 (P529) inhibited both VEGF-driven (IC50 = 20 nM) and bFGF-driven (IC50 = 30 nM) endothelial cell proliferation and retained the ability to induce endothelial cell apoptosis. In addition, Palomid 529 (P529) significantly enhanced the antiproliferative effect of radiation in prostate cancer cells (PC-3). [1][2][3]

References on Palomid 529 (P529):

[1] Diaz R et al. Br J Cancer. 2009 Mar 24;100(6):932-40.

[2] Gravina GL et al. Endocr Relat Cancer. 2011 May 6.

[3] Xue Q et al. Cancer Res. 2008 Nov 15;68(22):9551-7.

Perifosine is a novel Akt and PI3K inhibitor with IC50 of median 4.7 µM. Perifosine possesses antiproliferative properties. Perifosine is a p.o.-bioavailable ALK which prevents Akt activation. [1] Targeting cellular membranes, perifosine modulates membrane permeability, membrane lipid composition, phospholipid metabolism, and mitogenic signal transduction, resulting in cell differentiation and inhibition of cell growth.  Perifosine also prevents the anti-apoptotic mitogen-activated protein kinase (MAPK) pathway and regulates the balance between the MAPK and pro-apoptotic stress-activated protein kinase (SAPK/JNK) pathways, thereby inducing apoptosis. Perifosine has a lower gastrointestinal toxicity profile than the related agent miltefosine. Immortalized keratinocytes (HaCaT) as well as head and neck squamous carcinoma cells were sensitive to the antiproliferative properties of perifosine. [2] Perifosine is originally developed by Asta Medica and Zentaris.

Protocol:

Cell assay:
Cell proliferation studies by measuring the uptake of [3H]thymidine was performed as described. Briefly, HNSCC and HaCaT cells (1-2 × 104/well) were grown overnight in 24-well plates and exposed to either perifosine (0.1-30 μm) or PBS (control). After treatment (24-48 h), cells were pulsed with [3H]thymidine (1 μCi/well) for 4-6 h, fixed (5% trichloroacetic acid), and solubilized (0.5 m NaOH) before scintillation counting. Experiments were performed in triplicates. [2]

Animal study:
Mice were given subcutaneous injections of 5 × 106 cells in 100 μl HBSS into the right hind limb and tumor size was measured using calipers at least two times per week. Tumor volume was calculated as π/6 × length × width × height, where tumor volume at the start of treatment was normalized to 100%. When tumors had grown to an average volume of 100 mm3, mice were separated into 4 groups: control, perifosine, radiotherapy, and combined therapy. Perifosine and combined groups were given perifosine in a loading dose of 300 mg/kg (2 × 150 mg/kg separated by 12 hours) followed by daily maintenance doses of 35 mg/kg for 5 days. Two fractions of 5 Gy radiation were delivered the next day and 4 days after the start of perifosine treatment. [3]

References on Perifosine:

[1] http://en.wikipedia.org/wiki/Perifosine

[2] Patel V et al. Cancer Res. 2002 Mar 1;62(5):1401-9.

[3] Gao Y et al. Radiat Oncol. 2011 Apr 15;6:39.

PHT-427 is a Novel Akt/phosphatidylinositide-dependent protein kinase 1 pleckstrin homology domain inhibitor with an IC50 of 6.3 μM for AKT inhibition in Panc-1 cells. Phosphatidylinositol 3-kinase (PIK3)/ PtdIns dependent protein kinase-1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. PHT-427 caused apoptosis and inhibited AKT phosphorylation. [1] PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 prevented Akt and PDKP1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When administrated orally PHT-427 suppressed the growth of human tumor xenografts in immunodeficient mice with up to 80% inhibition in the most sensitive tumors and revealed greater activity than analogs with C4, C6 or C8 alkyl chains. Suppression of PDKP1 was more closely correlated to antitumor activity than Akt prevention. Tumors with PIK3CA mutation were the most sensitive and K-Ras mutant tumors the least sensitive. Combination studies revealed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer, and with erlotinib in NSC lung cancer. When given over 5 days PHT-427 caused no weight loss or change in blood chemistry. [2]

Protocol:

Cell assay:
Panc-1 cells stably transfected with green fluorescent protein (GFP) tagged Akt or PDKP1 PH domains were serum starved in phenol red free growth medium on glass-bottom 96-well imaging plates for 16 hours. They were then treated with PHT-427 at 1, 5, and 10µM or PI-103 for 4 hr, and stimulated with 50ng/mL IGF-1 for 10 min. Images were taken before and after IGF-1 treatment using an IN Cell Analyzer 1000 instrument with a Nikon Plan Fluor ELWD 20X/0.45 objective loaded and using a 300msec exposure time. [2]

Animal study:
1×107 BxPC-3 or MiaPaCa-2 pancreatic cancer cells were injected s.c. into the flanks of female scid mice and allowed to grow to approximately 300 mm3. Mice received a single oral dose of PHT-427 of 200 mg/kg in 0.1 ml vehicle. Mice were killed after various times, the tumors removed and immediately frozen in liquid N2. The tumors were homogenized in 50 mM HEPES buffer, pH 7.5, 50 mM NaCl, 1% Nonidet® P40 and 0.25 % sodium deoxycholate. Western blotting was performed. [2]

References on PHT-427:

[1] Moses SA et al. Cancer Res. 2009 Jun 15;69(12):5073-81. 
  
[2] Meuillet EJ et al. Mol Cancer Ther. 2010 Mar;9(3):706-17. 

Triciribine is a synthetic tricyclic nucleoside which acts as a specific inhibitor of the Akt signaling pathway. It selectively inhibits the phosphorylation and activation of Akt1, -2 and -3 but does not inhibit Akt kinase activity nor known upstream Akt activators such as PI 3-Kinase and PDK1. It inhibits cell growth and induces apoptosis preferentially in cells that express aberrant Akt. This agent is shown≥ 80% inhibition in tumor growth in mice at 1 mg/kg/day, i.p. [1,2]

References on Triciribine:

[1] Porcari AR et al. J Med Chem. 2000 Jun 15;43(12):2438-48.

[2] Porcari AR, Leroy B. Townsend. Nucleosides Nucleotides Nucleic Acids. 2004, 23(1-2):31-9.

Triciribine phosphate (NSC-280594) is a novel potent, small-molecule Akt inhibitor. Triciribine phosphate (NSC-280594) prevents the phosphorylation of Akt. Triciribine phosphate (NSC-280594) disrupts a specific signaling pathway associated with chemoresistance and cancer cell survival in ovarian cancer. Triciribine phosphate (NSC-280594) in combination with trastuzumab was shown to inhibit cell growth and induce cell death in breast cancer cells that were previously trastuzumab resistant. Triciribine phosphate (NSC-280594) has been demonstrated anti-tumor activity against a wide spectrum of cancers in preclinical and clinical studies. [1] Triciribine phosphate (NSC-280594) is a nucleotide derivative first synthesized by Schram and Townsend.

References on Triciribine phosphate (NSC-280594):

[1] Garrett CR et al. Invest New Drugs. 2011 Dec;29(6):1381-9.