We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 μM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 μM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 μM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.
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Research Article|
January 07 2005
Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors
Stanley F. BARNETT;
Stanley F. BARNETT
1
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
1To whom correspondence should be addressed (email stan_barnett@merck.com).
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Deborah DEFEO-JONES;
Deborah DEFEO-JONES
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Sheng FU;
Sheng FU
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Paula J. HANCOCK;
Paula J. HANCOCK
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Kathleen M. HASKELL;
Kathleen M. HASKELL
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Raymond E. JONES;
Raymond E. JONES
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Jason A. KAHANA;
Jason A. KAHANA
†Department of Biological Chemistry, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Astrid M. KRAL;
Astrid M. KRAL
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Karen LEANDER;
Karen LEANDER
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Ling L. LEE;
Ling L. LEE
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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John MALINOWSKI;
John MALINOWSKI
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Elizabeth M. McAVOY;
Elizabeth M. McAVOY
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Debbie D. NAHAS;
Debbie D. NAHAS
‡Department of Bioprocess Engineering, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Ronald G. ROBINSON;
Ronald G. ROBINSON
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Hans E. HUBER
Hans E. HUBER
*Department of Cancer Research, Merck and Company, Sumneytown Pike, West Point, PA 19454, U.S.A.
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Publisher: Portland Press Ltd
Received:
July 02 2004
Revision Received:
September 22 2004
Accepted:
September 29 2004
Accepted Manuscript online:
September 29 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 385 (2): 399–408.
Article history
Received:
July 02 2004
Revision Received:
September 22 2004
Accepted:
September 29 2004
Accepted Manuscript online:
September 29 2004
Citation
Stanley F. BARNETT, Deborah DEFEO-JONES, Sheng FU, Paula J. HANCOCK, Kathleen M. HASKELL, Raymond E. JONES, Jason A. KAHANA, Astrid M. KRAL, Karen LEANDER, Ling L. LEE, John MALINOWSKI, Elizabeth M. McAVOY, Debbie D. NAHAS, Ronald G. ROBINSON, Hans E. HUBER; Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors. Biochem J 15 January 2005; 385 (2): 399–408. doi: https://doi.org/10.1042/BJ20041140
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