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A series of 30 N10-substituted phenoxazines were synthesized and screened as potential inhibitors of Akt. In cellular assays at 5 μM, 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′-[(β-hydroxyethyl)piperazino]butyl]-2-chlorophenoxazine (15B) (in vitro, IC50 ∼1–2 μM) 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 AktΔPH, 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 μM), whereas ‘inactive’ derivatives were ≥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. [ABSTRACT FROM AUTHOR]

The cyclin-dependent kinase inhibitor p21Cip1 regulates multiple cellular functions and protects cells from genotoxic and other cellular stresses. Activation of apoptosis signal-regulating kinase 1 (ASK1) induced by inhibition of mTOR signaling leads to sustained phospho-c-Jun that is suppressed in cells with functional p53 or by forced expression of p21Cip1. Here we show that small deletions of p21Cip1 around S98 abrogate its association with ASK1 but do not affect binding to Cdk1, hence distinguishing between the cell cycle-regulating functions of p21Cip1 and its ability to suppress activation of the ASK1/Jun N-terminal protein kinase (JNK) pathway. p21Cip1 is phosphorylated in vitro by both ASK1 and JNK1 at S98. In vivo phosphorylation of p21Cip1, predominantly carried out by ASK1, is associated with binding to ASK1 and inactivation of ASK1 kinase function. Binding of p21Cip1 to ASK1 requires ASK1 kinase function and may involve phosphorylation of S98. [ABSTRACT FROM AUTHOR]

The cyclin-dependent kinase inhibitor p21(Cip1) regulates multiple cellular functions and protects cells from genotoxic and other cellular stresses. Activation of apoptosis signal-regulating kinase 1 (ASK1) induced by inhibition of mTOR signaling leads to sustained phospho-c-Jun that is suppressed in cells with functional p53 or by forced expression of p21(Cip1). Here we show that small deletions of p21(Cip1) around S98 abrogate its association with ASK1 but do not affect binding to Cdk1, hence distinguishing between the cell cycle-regulating functions of p21(Cip1) and its ability to suppress activation of the ASK1/Jun N-terminal protein kinase (JNK) pathway. p21(Cip1) is phosphorylated in vitro by both ASK1 and JNK1 at S98. In vivo phosphorylation of p21(Cip1), predominantly carried out by ASK1, is associated with binding to ASK1 and inactivation of ASK1 kinase function. Binding of p21(Cip1) to ASK1 requires ASK1 kinase function and may involve phosphorylation of S98.

We examined some in silico approaches to identify Akt (protein kinase B) inhibitors. Experimental validation of selected compounds was achieved using a fluorescence-based enzymatic assay and a substrate phosphorylation assay involving the protein GSK-3. We report on success and failure obtained by using several strategies including FlexX, GOLD, and CSCORE, where the 100-200 top-scoring compounds from a 50000-compound library were experimentally tested. This study led to the identification of low micromolar Akt1 inhibitors.