The mechanism of binding of two promising anticancer agents (the cytotoxic alkylphospholipids perifosine and miltefosine) to the Akt PH domain is investigated by high-resolution field-cycling (31)P nuclear magnetic resonance (NMR) spectroscopy using a spin-labeled recombinant PH domain. These results strongly indicate that there are two discrete amphiphile binding sites on the domain: (i) the cationic site that binds phosphoinositides and some alkylphospholipids and (ii) a second site that is occupied by only the alkylphospholipids. The identification of this second site for amphiphiles on the Akt1 PH domain provides a new target for drug development as well as insights into the regulation of the activity of the intact Akt1 protein. The field-cycling NMR methodology could be used to define discrete phospholipid or amphiphile binding sites on a wide variety of peripheral membrane proteins.

Phosphatidylinositol analogs (PIAs) were originally designed to bind competitively to the Akt PH domain and prevent membrane translocation and activation. D-3-deoxydioctanoylphosphatidylinositol (D-3-deoxy-diC8PI), but not compounds with altered inositol stereochemistry (e.g., L-3-deoxy-diC8PI and L-3,5-dideoxy-diC8PI), is cytotoxic. However, high resolution NMR field cycling relaxometry shows that both cytotoxic and non-toxic PIAs bind to the Akt1 PH domain at the site occupied by the cytotoxic alkylphospholipid perifosine. This suggests another mechanism for cytotoxicity must account for the difference in efficacy of the synthetic short-chain PIAs. In MCF-7 breast cancer cells, with little constitutively active Akt, D-3-deoxy-diC8PI (but not L-compounds) decreases viability concomitant with increased cleavage of PARP and caspase 9, indicative of apoptosis. D-3-deoxy-diC8PI also induces a decrease in endogenous levels of cyclins D1 and D3 and blocks downstream retinoblastoma protein phosphorylation. siRNA-mediated depletion of cyclin D1, but not cyclin D3, reduces MCF-7 cell proliferation. Thus, growth arrest and cytotoxicity induced by the soluble D-3-deoxy-diC8PI occur by a mechanism that involves downregulation of the D-type cyclin-pRb pathway independent of its interaction with Akt. This ability to downregulate D-type cyclins contributes, at least in part, to the anti-proliferative activity of D-3-deoxy-diC8PI and may be a common feature of other cytotoxic phospholipids.

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