SUMMARY 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 difluoromethylene-phosphoserine 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.

SH2 domains are attractive targets for chemotherapeutic agents due to their involvement in the formation of protein-protein interactions critical to many signal transduction cascades. Little is known, however, about how synthetic SH2 domain ligands would influence the growth properties of tumor cells or with which intracellular proteins they would interact due to their highly charged nature and enzymatic lability. In this study, a prodrug delivery strategy was used to introduce an enzymatically stable, phosphotyrosine peptidomimetic into tumor cells. When tested in a human tumor cell panel, the prodrug exhibited a preference for inhibiting the growth of leukemia and lymphoma cells. In these cells, it was largely cytostatic and induced endoreduplication and the appearance of midbodies. Proteomic analyses identified multiple targets that included mitotic centromere-associated kinesin (MCAK). Molecular modeling studies suggested the ATP-binding site on MCAK as the likely site of drug interaction. Consistent with this, ATP inhibited the drug-MCAK interaction and the drug inhibited MCAK ATPase activity. Accordingly, the effects of the prodrug on the assembly of the mitotic spindle and alignment of chromosomes were consistent with the identification of MCAK as an important intracellular target.

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.

Background. The uncontrolled growth that characterizes cancer cells is attributed to aberrations in pathways that regulate progress through the cell cycle. Mitotic centromere-associated kinesin (MCAK) is a microtubule-depolymerizing kinesin that functions in spindle assembly and is critical for regulating kinetochore microtubule (MT) dynamics to control proper attachment of chromosomes to the spindle. The critical role that MCAK plays in mitosis makes it an attractive candidate for further investigation. Several studies suggest that MCAK levels correlate with poor prognosis in certain cancers. Knockdown of MCAK in cells derived from multiple cancers reduced cell proliferation. Thus, inhibitors of MCAK have considerable potential as a novel cancer therapeutic. We have synthesized a phosphotyrosine mimetic prodrug, RH6-22, that is cytostatic at low micromolar concentrations for leukemia cells and delivers a non-hydrolyzable phosphotyrosine peptidomimetic intracellularly. A screen for candidate target proteins using immobilized RH6-22 phosphonate identified MCAK as a potential target for this peptidomimetic. RH6-22 induces polyploidy in DG75 cells with no measureable cell death. Disruption of the microtubule network was seen in WEHI 231 cells treated with RH6-22 and stained with antibodies against α-tubulin. These experiments suggested defects in both mitosis and cytokinesis consistent with MCAK inhibition. We hypothesized that RH6-22 was interacting with MCAK at the ATP-biding site. Methods. A competitive ATP binding assay was run to confirm the interaction of RH6-22 at the ATP binding site. A focused library of RH6-22 analogs was synthesized and analyzed for growth inhibitory activity in DG75 cells. Molecular modeling studies were carried out using the crystal structure of mammalian MCAK (pdb 1V8J) with the goal of developing higher affinity small molecule inhibitors. The highest affinity compounds identified in silico were chosen for synthesis and evaluation in a growth inhibition assay in DG75 cells. Results. Concentrations of ATP as low as 6μM can selectively elute MCAK from the immobilized RH6-22 ligand, indicating that the ligand binds at the ATP binding site. The small library of RH6-22 analogs tested in growth inhibition assays resulted in compounds with IC50 values from 4–50μM. Docking experiments in silico exploring variations to the non-phosphate portion of RH6-22 provided compounds with significant improvements in predicted binding affinity. The best compounds chosen for synthesis and biological evaluation are similar in that they contain a dihydroxybenzofuran moiety that hydrogen bonds with Arg78 and allows a favorable pi-pi stacking interaction with His170. Synthesis of the dihydroxybenzofuran is accomplished through an enzyme-catalyzed oxidation of catechol followed by the Michael addition of methyl 2-chloroacetoacetate and subsequent cyclization. These new compounds exhibited sub-micromolar growth inhibitory activity. Conclusion. We have used our phosphopeptidomimetic prodrug RH6-22 as a lead compound and structure-based drug design to synthesize novel inhibitors targeted to MCAK. The best compounds demonstrate submicromolar growth inhibitory activity in cell-based assays. 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 B184.