Abstract A124: Synthesis of a phosphoserine mimetic prodrug with potent 14-3-3 protein inhibitory activity.
Authors: Arrendale Allison A. , Kim Keunho , Geahlen Robert , Borch Richard F. 
Published: June 2012

Background. Many protein-protein interactions in cells are mediated by domains that bind to motifs that contain phosphorylated serine and threonine residues. To create small molecules that inhibit these interactions, we developed methodology for the synthesis of a prodrug that generates a phosphoserine peptidomimetic in cells. As a proof of principle, we synthesized a small molecule inhibitor of 14-3-3 proteins that incorporates a nonhydrolyzable difluoromethylenephosphoserine prodrug moiety. 14-3-3 is a highly conserved and ubiquitously expressed family of proteins that is involved in many vital cellular processes including signal transduction, apoptosis, cell-cycle regulation, growth, and development. 14-3-3 proteins bind to many targets including the tumor suppressor p53, Raf family kinases, FOXO transcription factors, the pro-apoptotic protein BAD, and Cdc25 phosphatases which makes the 14-3-3 family an interesting target for cancer therapeutics.

Methods. The small molecule prodrug KK7–83 and the corresponding free phosphonate ligand KK8–81 were synthesized. The growth inhibitory activity of both compounds was measured in DG75 leukemia cells. The potential cytotoxic effects of the prodrug were investigated by monitoring the cleavage of PARP, a well known caspase substrate. To explore a direct involvement of 14-3-3 proteins in the actions of KK7–83, we developed an assay using a luciferase readout to monitor the 14-3-3-dependent inhibition of FOXO transcription factors.

Results. In growth inhibition studies, prodrug KK7–83 was a potent inhibitor with an IC50 = 5 M, while the free phosphonate KK8–81 showed no meaningful growth inhibition up to 100 M. Significant PARP cleavage was observed by Western blot following prodrug treatment at 12 M, suggesting that KK7–83 induces apoptosis at low micromolar concentrations. In the luciferase assay, the ectopic expression of FOXO3a led to a substantial increase in FOXO3a-mediated gene transcription compared to control, which was inhibited upon co-transfection of Akt1 as expected. FOXO3a-stimulated transcription was recovered in a dose dependent manner by treatment of cells with increasing concentrations of KK7–83, consistent with the prodrug's inhibition of 14-3-3-mediated retention of FOXO3a in the cytoplasm. The concentration of KK7–83 required to recover 50% of the FOXO3a induced gene expression was consistent with the IC50 value for growth inhibition, further suggesting that the cytotoxicity of KK7–83 is a function of its ability to bind 14-3-3.

Conclusions. KK7–83 is a potent inhibitor of cancer cell growth leading to apoptosis at low micromolar concentrations through direct interactions with the 14-3-3 family of proteins. Development of this small molecule validates our prodrug strategy and the further exploration of the 14-3-3 family for the treatment of cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A124.