Abstract 3680: 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

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Persistently activated hyper-phosphorylated Akt contributes to human oncogenesis and tumor resistance. TCN-P, the active metabolite of the Akt phosphorylation inhibitor triciribine (TCN), is in clinical trials in patients whose tumors contain hyper-phosphorylated Akt, but the mechanism by which TCN-P, inhibits Akt phosphorylation is not known. In this manuscript, we show that in vitro, TCN-P inhibits neither Akt kinase activity nor the phosphorylation of Akt S473 and T308 by mTOR or PDK1, respectively. However, in intact cells, immunofluorescence and subcellular fractionation experiments demonstrate that TCN inhibits EGF-stimulated Akt recruitment to the plasma membrane and subsequent phosphorylation of Akt on Ser473 and Thr308. Surface plasmon resonance (SPR) demonstrates that TCN-P, but not TCN, binds to full-length, non-phosphorylated (inactive) Akt. However, neither TCN-P nor TCN interacts with full-length, phosphorylated (active) Akt. Furthermore, TCN-P, but not TCN, binds Akt-derived pleckstrin homology (PH) domain with KD values of 690 nM and 1.4 µM as demonstrated by SPR and isothermal calorimetry (ITC), respectively. Consistent with these data, nuclear magnetic resonance (NMR) spectroscopy shows that TCN-P, but not TCN, binds to the PH domain engaging amino acid residues in the vicinity of the PIP3 binding pocket. Thus, SPR, NMR, as well as biochemical studies indicate that the anti-cancer drug TCN-P inhibits Akt function by binding to the PH domain of Akt, blocking its recruitment to the plasma membrane and subsequent phosphorylation, suggesting that this drug may be beneficial to patients whose tumors express persistently activated Akt. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3680.