Design, synthesis, and biological evaluation of novel MCAK and 14-3-3 protein family inhibitors.

Allison Ann Arrendale, Purdue University

Abstract

The Borch lab is interested in the synthesis and biological evaluation of phosphopeptidomimetic prodrugs in order to disrupt proteins that are critical for cancer cell growth and proliferation. Biological evaluation of a phosphotyrosine mimetic prodrug, 1 (IC50=7µM) suggested defects in both mitosis and cytokinesis consistent with MCAK inhibition. To better understand the location of the protein-ligand interaction, a competitive ATP binding assay was run to confirm the likely binding site. Concentrations of ATP as low as 3 μM can selectively elute MCAK from the immobilized 3 ligand. To determine if the free ligand could disrupt the ATPase activity of the MCAK, a radioactive ATPase assay was run. At low micromolar concentrations, the compound inhibits the ATPase activity of the MCAK motor domain. A focused library of analogs was synthesized and analyzed for growth inhibitory activity in DG75 cells. This library resulted in compounds with IC50 values from 4-50 μM. 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. Docking experiments in silico exploring variations to the non-phosphate portion of 1 provided compounds with significant improvements in predicted binding affinity. The highest affinity compounds identified in silico were chosen for synthesis and evaluation in a growth inhibition assay in DG75 cells and a cell-free radioactive ATPase assay. These new compounds exhibited sub-micromolar growth inhibitory activity. To create small molecules that inhibit phosphoserine dependent interactions, we developed methodology for the synthesis of a prodrug that generates a phosphoserine peptidomimetic in cells. As a proof of principle, a small molecule inhibitor of 14-3-3 proteins was synthesized that incorporates a nonhydrolyzable difluoromethylenephosphoserine prodrug moiety. The small molecule prodrug 58 and the corresponding free phosphonate ligand 57 were evaluated for their growth inhibitory activity in DG75 leukemia cells. Compound 58 proved to be a potent inhibitor with a low mircromolar IC50 while the free ligand showed no meaningful growth inhibition at much higher concentrations. The potential cytotoxic effects of the prodrug were investigated by monitoring the cleavage of PARP, a well known caspase substrate. Significant PARP cleavage was observed by Western blot at 12 µM, suggesting 58 induces apoptosis at low micromolar concentrations. To explore a direct involvement of 14-3-3 proteins in the actions of 58 in cells, we developed an assay using a luciferase readout to monitor the 14-3-3-dependent inhibition of FOXO transcription factors. To demonstrate this disruption on a molecular level, a competitive binding assay was run using immobilized 14-3-3 tao/FOXO3a complex and free ligand 57. These experiments validated that the activity of prodrug 58 is due to 14-3-3 protein inhibition.

Degree

Ph.D.

Advisors

Borch, Purdue University.

Subject Area

Biochemistry|Organic chemistry|Medicine

Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server
.

Share

COinS