Identification of N10-substituted phenoxazines as potent and specific inhibitors of Akt signaling.
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.
Thimmaiah
Kuntebommanahalli N
KN
Department of Chemistry, Western Illinois University, Macomb 61455, USA.
Easton
John B
JB
Germain
Glen S
GS
Morton
Christopher L
CL
Kamath
Shantaram
S
Buolamwini
John K
JK
Houghton
Peter J
PJ
D064413:3-Phosphoinositide-Dependent Protein Kinases; D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology; D001386:Azides / Q000378:metabolism; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D007700:Kinetics; D010078:Oxazines / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000502:physiology*; D000099184:Serum-Glucocorticoid Regulated Kinases
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000255:Adenosine Triphosphate; D001386:Azides; D017874:Immediate-Early Proteins; D010078:Oxazines; D019869:Phosphatidylinositol 3-Kinases; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D000099184:Serum-Glucocorticoid Regulated Kinases; C017040:8-azidoadenosine 5'-triphosphate; C039203:phenoxazine; C494918:AKT1 protein, human; C578562:PDPK1 protein, human