Abstract B184: Design, synthesis, and evaluation of novel mitotic centromere-associated kinesin (MCAK) inhibitors.

Abstract

Abstract 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.