During the subsequent M phase in a bipolar manner to the microtubules of the mitotic spindle. The spindle MTs are a dynamic array of ab-tubulin fibers that extend from two oppositely localized centrosomes. At the metaphase-anaphase transition, the sister chromatids are first separated and then segregated into the daughter cells. During the final cell cycle stage named cytokinesis, the daughters divide, each containing an identical set of chromosomes. Antiproliferative drugs used in the clinic include agents that target mitotic spindle integrity or dynamics. In response to the spindle defects caused by these drugs, the spindle assembly checkpoint delays mitosis allowing cells to reverse the druginduced damage. Cells that do not recover and satisfy the SAC either Rapamycin 53123-88-9 undergo cell death or adapt. Adapting cells may continue to cycle, undergo senescence or die in the subsequent interphase. Almost all antispindle drugs suppress MT integrity and dynamics by stabilizing MTs and stimulating tubulin polymerization, or by destabilizing MTs and inhibiting tubulin polymerization. MT stabilizing drugs including taxanes and ixabepilone, or MT destabilizing agents including vinca alkaloids and estramustine, are very effective against a broad range of tumors. However, resistance to antitubulin drugs has become a significant problem due to P-glycoprotein overVE-821 expression and, perhaps, to mutations in genes encoding the tubulin subunits, changes in tubulin isotype composition of MTs, altered expression or binding of MT-regulatory proteins including Tau, mutations in or reduced levels of c-actin, and/or a reduced apoptotic response. To deal with resistance, structurally diverse antiMT drugs are being developed while alternative mitosis-specific drug targets are being evaluated. A mitosis-specific structure that has recently been focused on for development into a drug target is the kinetochore, the protein complex that coordinates chromosome segregation. Interfering with kinetochore activities, including MT binding, triggers a SACmediated arrest of mitosis, which frequently leads to cell death. As kinetochores assemble from.100 proteins, they are, in principle, almost inexhaustible drug targets. We wished to identify compounds that inhibit kinetochore-MT binding to develop them into new antimitotic agents. We also wanted to use these compounds as chemobiological tools to study the mechanisms that drive kinetochore-MT binding. To identify such compounds we focused on the outer kinetochore Ndc80 complex, which attaches the kinetochore structure to the MTs of the mitotic spindle. To screen chemical libraries for active molecules we developed an in vitro fluorescence microscopy-based binding assay using a recombinant Ndc80 complex and taxolstabilized MTs. Of 10,200 compounds screened, one compound prevented the Ndc80 complex from binding to the MTs by acting at the MT level. 
More specifically, the compound localized to the colchicine-binding site at the ab-tubulin interface. Using a computational approach, the antitubulin compound was structurally dissected and analogs were identified containing a 20-fold higher antitubulin activity. Of these, the most potent compound mitotically arrested and killed adenocarcinoma cells with an IC50 value of 25 nmol/l. The classic colchicine site agents, most of which are structurally similar and rather complex in nature, are not used in the clinic because they are systemically toxic. This is unfortunate as colchicine site agents would represent powerful alternatives to the clinically used taxaneor vinca-site drugs against which tumor cells have been developing resistance.