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J. Biol. Chem., Vol. 280, Issue 36, 31924-31935, September 9, 2005

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Identification of N¹⁰-Substituted Phenoxazines as Potent and Specific Inhibitors of Akt Signaling^*

Kuntebommanahalli N. Thimmaiah, John B. Easton¶, Glen S. Germain¶, Christopher L. Morton¶, Shantaram Kamath||, John K. Buolamwini||, and Peter J. Houghton¶**

From the ^¶Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, the Department of Chemistry, Western Illinois University, Macomb, Illinois 61455, and the ^||Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163

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'-[(-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 AktPH, 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.

Received for publication, June 28, 2005 , and in revised form, July 11, 2005.

^* This work was supported by United States Public Health Service Awards CA23099 (to P. J. H.), CA96996 (to P. J. H.), CA77776 (to P. J. H.), and CA100202 (to J. K. B.), NCI Cancer Center Support Grant CA21675 from the National Institutes of Health, and by American, Lebanese, Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^** To whom correspondence should be addressed: Dept. of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 North Lauderdale St., Memphis, TN 38105. Tel.: 901-495-3440; Fax: 901-495-4290; E-mail: peter.houghton{at}stjude.org.

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