PI3Ks play pivotal roles in cancer and inflammation. They are recruited to cytokine and growth factor receptors via the SH2 domains of the regulatory subunit, p85. While p85 represses kinase activity in the cytosol, binding to pTyr residues on receptors or adapters activates the catalytic domain, p110, resulting in the phosphorylation of the 3'-hydroxyl group of phosphatidylinositol-4',5'-diphosphate (PIP2) to form phosphatidylinositol-3',4',5'-triphosphate (PIP3). Membrane-associated PIP3 subsequently recruits kinases such as phosphtidylinositol-dependant kinase 1 (PDK1) and Akt to the cell membrane. PDK1 phosphorylates and activates Akt, which propagates growth and survival signals. In addition to regulating PI3K kinase activity, scaffolding roles of p85, unrelated to catalytic activity, are being unraveled. In biochemical assays, doubly phosphorylated peptides enhance catalytic activity which led to the dogma that phosphopeptides will activate PI3K in cells.
We are targeting the SH2 domains of p85 with phosphatase-stable, cell-permeable prodrug analogs of Y(p)VPML, a high-affinity ligand. To allow cell entry and to protect against phosphatase degradation, we are replacing pTyr with bis-pivaloyloxymethyl esters of 4-phosphonodifluoromethylphenylalanine (F2Pmp(POM2)), which have not been reported. We synthesized Z-NMe-F2Pmp(POM2)-OPcp with the α-amino group methylated to enhance cell penetration and the carboxyl group esterified with pentachlorophenol. Z-protection was necessary for 1, phosphonate protecting group manipulation and 2, stability of the Pcp ester. This building block was coupled to VPML-amide, synthesized by SPPS. Hydrogenation in the presence of Ac2O gave the N-terminal acetyl prodrug which inhibited downstream Akt phosphorylation after EGF stimulation of breast cancer cells. In the absence of EGF, pAkt was not observed, even at 10 mM drug. These results show that in intact cells, a mono-phosphopeptide does not stimulate PI3K activity, which contradicts current dogma. Phosphopeptide mimics targeted to the SH2 domain of p85 have potential applications in cancer therapy and as immune modulators.