The Akt activation inhibitor TCN-P inhibits Akt phosphorylation by binding to the PH domain of Akt and blocking its recruitment to the plasma membrane

Persistently hyper-phosphorylated Akt contributes to human oncogenesis and resistance to therapy. TCN-P, the active metabolite of the Akt phosphorylation inhibitor triciribine (TCN), is in clinical trials, but the mechanism by which TCN-P inhibits Akt phosphorylation is unknown. Here we show that in vitro, TCN-P inhibits neither Akt activity nor the phosphorylation of Akt S473 and T308 by mTOR or PDK1, respectively. However, in intact cells, TCN inhibits EGF-stimulated Akt recruitment to the plasma membrane and phosphorylation of Akt. Surface plasmon resonance (SPR) demonstrates that TCN-P, but not TCN, binds Akt-derived pleckstrin homology (PH) domain (K(D): 690 nM). Furthermore, nuclear magnetic resonance (NMR) spectroscopy shows that TCN-P, but not TCN, binds to the PH domain in the vicinity of the PIP3 binding pocket. Finally, constitutively active Akt mutants, Akt1-T308D/S473D and myr-Akt1, but not the transforming mutant Akt1-E17K, are resistant to TCN-P and rescue from TCN inhibition of proliferation and induction of apoptosis. Thus, our studies indicate that TCN-P binds to the PH domain of Akt and blocks its recruitment to the membrane and that the subsequent inhibition of Akt phosphorylation contributes to TCN-P anti-proliferative and pro-apoptotic activity, suggesting that this drug may be beneficial to patients whose tumors express persistently phosphorylated Akt.