Synthesis of a Phosphoserine Mimetic Prodrug with Potent 14-3-3 Protein Inhibitory Activity

Many protein-protein interactions in cells are mediated by functional domains that recognize and bind to motifs containing phosphorylated serine and threonine residues. To create small molecules that inhibit such interactions, we developed methodology for the synthesis of a prodrug that generates a phosphoserine peptidomimetic in cells. For this study, we synthesized a small molecule inhibitor of 14-3-3 proteins that incorporates a nonhydrolyzable difluoromethylenephosphoserine prodrug moiety. The prodrug is cytotoxic at low micromolar concentrations when applied to cancer cells and induces caspase activation resulting in apoptosis. The prodrug reverses the 14-3-3-mediated inhibition of FOXO3a resulting from its phosphorylation by Akt1 in a concentration-dependent manner that correlates well with its ability to inhibit cell growth. This methodology can be applied to target a variety of proteins containing phosphoserine and other phosphoamino acid binding domains. [Display omitted] ► Synthesis of the first nonhydrolyzable phosphoserine mimetic prodrug ► Synthesis of a 14-3-3 protein inhibitor with low micromolar activity ► Inhibitor induces cytotoxicity and disrupts 14-3-3 function in a dose-dependent manner Phosphoserines control number of critical cellular processes. Arrendale et al. report a synthesis of a nonhydrolyzable phosphoserine prodrug and its incorporation into a peptidomimetic that inhibits interactions mediated by phosphoserine residues, using 14-3-3 protein-protein interactions as a proof of principle.