Abstract 4838: Peptide prodrugs targeting the SH2 domains of p85 block PI3K signaling
Authors: Mandal Pijus K. , Yao Yanhua , Bresnick Anne R. , Backer Jonathan M. , McMurray John S. 
Published: July 2016

Class IA phosphoinositol-3-kinases (PI3Ks) play pivotal roles in cancer and inflammation and are targets for anti-cancer therapies. p85 was first described as a regulatory subunit of Class I PI3Ks which complexes with the p110 catalytic domain to form the heterodimeric enzyme. PI3Ks are recruited to cytokine and growth factor receptors via the SH2 domains of p85. While p85 represses the kinase activity of p110 subunits in the cytosol, binding to pTyr residues on receptors or adapters activates 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 PDK1 and Akt to the cell membrane. PDK1 phosphorylates and activates Akt, which propagates growth and survival signals. In biochemical assays, doubly phosphorylated peptides enhance catalytic activity of p85/p110 dimers, which led to the dogma that phosphopeptides should activate PI3K in cells.

Due to the challenges of cellular entry of negatively charged phosphopeptides, blocking protein-protein interactions mediated by p85 has been largely unexplored in intact cells. We targeted the SH2 domains of p85 with phosphatase-stable, cell-permeable prodrug analogs of Y(p)VPML, a high-affinity ligand derived from Tyr751 of PDGF. To allow cell entry and to protect against phosphatase degradation, we replaced pTyr with bis-pivaloyloxymethyl esters of 4-phosphonodifluoromethylphenylalanine (F2Pmp(POM2)), which have not been reported. We synthesized Z-NMe-F2Pmp(POM2)-OPcp with the á-amino group methylated to enhance cell penetration and the carboxyl group esterified with pentachlorophenol. Z-protection was necessary for phosphonate protecting group manipulation and for stability of the Pcp ester. A series of inhibitors was prepared by coupling Z-NMe-F2Pmp(POM2)-OPcp to analogs of VPML. Hydrogenation in the presence of Ac2O gave an N-terminal acetyl prodrug. Screening for inhibition of EGF-stimulation of pAkt in serum starved MDA-MB-468 cells led to the identification of the lead inhibitor, PM-190I. In serum starved MDB-MB-231 cells, PM-190I inhibited EGF-stimulated phosphorylation of Akt with an IC50 of 5 ìM. At 30 ìM Akt phosphorylation was negligible. These results suggest that the phosphopeptide mimic blocks recruitment to p85/p110 dimers to EGFR or its substrates, thereby preventing PI3K pathway signaling. Further, in intact cells, a mono-phosphopeptide does not stimulate PI3K activity, which contradicts current dogma.

In addition to its role as a regulatory subunit, non-catalytic roles of p85 include complexing Rab4 and Rab5, stabilizing PTEN, invadopodium formation, cytokinesis, and nuclear trafficking of proteins such as XBP-1 and OPNi. Phosphopeptide mimics targeted to the SH2 domain of p85 have potential applications both in blocking PI3K signaling as well as in modulating non-catalytic functions.

Citation Format: Pijus K. Mandal, Yanhua Yao, Anne R. Bresnick, Jonathan M. Backer, John S. McMurray. Peptide prodrugs targeting the SH2 domains of p85 block PI3K signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4838.