Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors

Biochem J. 2005 Jan 15;385(Pt 2):399-408. doi: 10.1042/BJ20041140.

Abstract

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 microM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 microM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 microM. 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.

MeSH terms

  • 3-Phosphoinositide-Dependent Protein Kinases
  • Adenosine Triphosphate / metabolism
  • Apoptosis Regulatory Proteins
  • Benzylamines / pharmacology
  • Binding, Competitive
  • Blood Proteins / chemistry*
  • Blood Proteins / genetics*
  • Blood Proteins / immunology
  • Carcinoma / chemistry
  • Carcinoma / metabolism
  • Carcinoma / pathology
  • Caspases / metabolism
  • Cell Line, Tumor
  • Cloning, Molecular
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / pharmacology
  • Female
  • Heterocyclic Compounds, 2-Ring / pharmacology
  • Humans
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / chemistry
  • Male
  • Membrane Glycoproteins / pharmacology
  • Molecular Structure
  • Peptides / chemistry*
  • Peptides / genetics*
  • Peptides / immunology
  • Peptides / metabolism
  • Phosphoproteins / chemistry*
  • Phosphoproteins / genetics*
  • Phosphoproteins / immunology
  • Phosphorylation / drug effects
  • Prostatic Neoplasms / chemistry
  • Prostatic Neoplasms / metabolism
  • Prostatic Neoplasms / pathology
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors*
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / antagonists & inhibitors*
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt
  • Quinoxalines / pharmacology
  • Sequence Homology, Amino Acid*
  • Signal Transduction / physiology
  • TNF-Related Apoptosis-Inducing Ligand
  • Tumor Necrosis Factor-alpha / pharmacology
  • Uterine Cervical Neoplasms / chemistry
  • Uterine Cervical Neoplasms / metabolism
  • Uterine Cervical Neoplasms / pathology

Substances

  • Akt-I-1 compound
  • Akt-I-1,2 compound
  • Apoptosis Regulatory Proteins
  • Benzylamines
  • Blood Proteins
  • Enzyme Inhibitors
  • Heterocyclic Compounds, 2-Ring
  • Isoenzymes
  • Membrane Glycoproteins
  • Peptides
  • Phosphoproteins
  • Proto-Oncogene Proteins
  • Quinoxalines
  • TNF-Related Apoptosis-Inducing Ligand
  • TNFSF10 protein, human
  • Tumor Necrosis Factor-alpha
  • platelet protein P47
  • Adenosine Triphosphate
  • 3-Phosphoinositide-Dependent Protein Kinases
  • AKT1 protein, human
  • AKT2 protein, human
  • PDPK1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Caspases