Movement on Demand: Pharmacological and Protein-based Inhibition of Mitotic Kinesins

Abstract

Mitosis is the process by which the cell’s duplicated genome is divided into two daughter cells. Accuracy of chromosome segregation is essential for maintaining healthy cells, and thus numerous cellular networks and signaling pathways are charged with ensuring mitotic fidelity. As deregulated cell division and chromosome instability are two hallmarks and drivers of cancer, a leading cause of death worldwide, it is imperative to continue improving our understanding of the molecular mechanisms underpinning mitosis. Kinesins comprise a superfamily of molecular motor proteins whose functions include organizing the mitotic spindle, the microtubule-based machine that powers chromosome segregation. As they are essential for mitotic progression, mitotic kinesins have been recognized as promising therapeutic targets for the development of anticancer agents. Numerous chemical inhibitors of the Kinesin-5 Eg5, a motor critical for building bipolar spindles, have been designed and tested against a range of cancers; unfortunately, while Kinesin-5 inhibitors (K5Is) perform well in preclinical studies, they fail to induce tumor regression in patients. One potential mechanism behind this discrepancy is the existence of another motor protein that is capable of driving spindle assembly in an Eg5-independent manner. Indeed, several functional redundancies exist among the kinesin superfamily, and it has been shown that Eg5-independent mitosis relies on the Kinesin-12 KIF15. KIF15 is non-essential for mammalian cells; however, under the selective pressure of K5I treatment, some cells acquire genetic alterations that make KIF15 essential for spindle assembly. We hypothesize that K5Is may not reach their clinical potential unless paired with an inhibitor of KIF15. Unfortunately, the field lacks a well-characterized, selective KIF15 inhibitor with which to test this theory. Additionally, much remains unknown about how KIF15 is able to drive Eg5-independent spindle assembly, including the mechanisms by which KIF15 is regulated in cells. In this thesis, I present the discovery of two chemical inhibitors of KIF15 identified from a library of 24,000 small molecules (Chapter 3). These inhibitors potently and selectively inhibit KIF15 activity both in vitro and in cells and have distinct mechanisms of inhibition. The findings of this screen represent a useful resource that can be harnessed to study the role of KIF15 in spindle organization. Additionally, I show how this work can be extended to the identification of other kinesin inhibitors through adaptation of our screening pipeline (Chapter 2). I then focus in on KIF15’s physiological role in cells and present work characterizing a novel mechanism of kinesin regulation via kinesin-binding protein (KIFBP) (Chapter 4). A combination of structural biology, biochemistry, and cell-based assays shows that KIFBP binds to the motor domain of KIF15 and remodels its conformation to enable complex formation and prevent interaction with microtubules. Our findings identify several regions of KIFBP that are essential for kinesin-binding and suggest a conformational basis for how KIFBP selectively binds a subset of kinesins in cells. Collectively, this work advances our understanding of how cellular reorganization is regulated each cell cycle, and provides a valuable tool with which to continue advancing our knowledge of the role of KIF15 in cell division.