Abstract
A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 mum, 17 compounds inhibited insulin-like growth factor 1 (IGF-I)-stimulated phosphorylation of Akt (Ser-473) by at least 50% but did not inhibit IGF-I-stimulated phosphorylation of Erk-1/2 (Thr-202/Tyr-204). Substitutions at the 2-position (Cl or CF3) did not alter inhibitory activity, whereas N10-substitutions with derivatives having acetyl (20B) or morpholino (12B) side chain lost activity compared with propyl or butyl substituents (7B and 14B). Inhibition of Akt phosphorylation was associated with the inhibition of IGF-I stimulation of the mammalian target of rapamycin phosphorylation (Ser-2448 and Ser-2481), phosphorylation of p70 S6 kinase (Thr-389), and ribosomal protein S6 (Ser-235/236) in Rh1, Rh18, and Rh30 cell lines. The two most potent compounds 10-[4'-(N-diethylamino)butyl]-2-chlorophenoxazine (10B) and 10-[4'-[(beta-hydroxyethyl)piperazino]butyl]-2-chlorophenoxazine (15B) (in vitro, IC50 approximately 1-2 microM) were studied further. Inhibition of Akt phosphorylation correlated with inhibition of its kinase activity as determined in vitro after immunoprecipitation. Akt inhibitory phenoxazines did not inhibit the activity of recombinant phosphatidylinositol 3'-kinase, PDK1, or SGK1 but potently inhibited the kinase activity of recombinant Akt and Akt deltaPH, a mutant lacking the pleckstrin homology domain. Akt inhibitory phenoxazines blocked IGF-I-stimulated nuclear translocation of Akt in Rh1 cells and suppressed growth of Rh1, Rh18, and Rh30 cells (IC50 2-5 microM), whereas "inactive" derivatives were > or = 10-fold less potent inhibitors of cell growth. In contrast to rapamycin analogs, Akt inhibitory phenoxazines induced significant levels of apoptosis under serum-containing culture conditions at concentrations of agent consistent with Akt inhibition. Thus, the cellular responses to phenoxazine inhibitors of Akt appear qualitatively different from the rapamycin analogs. Modeling studies suggest inhibitory phenoxazines may bind in the ATP-binding site, although ATP competition studies were unable to distinguish between competitive and noncompetitive inhibition.
Highlights
A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt
As it is established that Akt signaling protects against cellular stress, including cytotoxic agents, we have investigated whether phenoxazine derivatives inhibit Akt and induce apoptosis
Phenoxazine Derivatives Inhibit Akt Phosphorylation in Cells—In order to identify novel inhibitors of Akt, phenoxazines shown in Table I, were investigated to determine whether they would inhibit the phosphorylation of Akt (Ser473 or Thr-308) in cancer cells
Summary
A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. We screened a number of phenoxazine derivatives for their effects on Akt activation in cells derived from pediatric cancers From among these compounds, we report the identification of a small group of novel lead compounds, which at low micromolar concentrations block Akt activation and downstream signaling to substrates such as mTOR, p70 S6 kinase, and ribosomal protein S6 (S6). We report the identification of a small group of novel lead compounds, which at low micromolar concentrations block Akt activation and downstream signaling to substrates such as mTOR, p70 S6 kinase, and ribosomal protein S6 (S6) These agents do not affect activation of the upstream kinases, PDK-1, PI 3-kinase, or other kinases downstream of ras such as Erk-1/2. At low micromolar concentrations, under normal growth conditions, these small molecule inhibitors induce apoptosis in rhabdomyosarcoma cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
