Date of Completion

Spring 4-29-2016

Project Advisor(s)

Charles Giardina; Dennis Wright

University Scholar Major

Molecular and Cell Biology

Disciplines

Biochemistry | Molecular Biology | Organic Chemicals

Abstract

The Giardina Laboratory has recently identified AK301 as a novel mitotic arrest agent. This work aimed to characterize the arrest state induced by AK301 (EC50 ~ 150nM) and identify the cellar targets responsible for the arrest. It was found that AK301 arrest is readily reversible upon withdrawal of AK301. Cells that slip from mitosis after removal of AK301 are sensitized to apoptosis. This was found to be unique for AK301 when compared to other mitotic arrest agents like colchicine, vincristine, and BI2536. Arrested cells were found to have increased ATM activity as well as an upregulation of p53 and several apoptotic proteins. The apoptosis was found to be p53 dependent. Studies in YAMC and IMCE cells suggest AK301 is more effective at sensitizing cells with an APC mutation to apoptosis.

A structural activity relationship study led to the development of more potent AK301 derivatives. The most potent derivative, MJB6 (EC50 ~ 75nM) was used to construct several molecular probes, most notably a biotinylated compound. Affinity chromatography experiments using the biotinylated compound identified clathrin as a possible target for the AK301/MJB family of compounds. Western blotting and an endocytosis assay were used to confirm clathrin as a hit. Immunofluorescent staining using a clathrin antibody showed disruption of clathrin during mitosis when cells were treated with MJB6.

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