The antitubulin hit compound and lead analogs identified in this study are chemotypically unique colchicine site agents. In addition, they interact with the colchicinebinding pocket in a unique manner: our docking studies suggest that the R-isomers interact with tubulin via their furan ring, while the S-isomers localize to the colchicine pocket via their ester side chain. Future analysis and modification of our compounds will advance insight into the colchicine site-drug interaction and promise to result in new anticancer compounds with optimal performance and, possibly, minimal toxicity. During the last twenty-five years antispindle drugs have been used with great success in the fight against cancer. However, as cancer cells are developing resistance against these drugs, there is an urgent need for compounds targeting alternative mitotic targets. As kinetochores orchestrate chromosome segregation and comprise.100 proteins, they are appealing mitosis-specific drug targets. The high antitumor activity of compounds inhibiting LY294002 154447-36-6 kinetochore regulators and the kinetochore-associated kinesin CENP-E supports the concept of targeting kinetochore function to eradicate proliferating cells. The complexity of kinetochores, the lack of insight into GANT61 intrakinetochore protein-protein contacts and protein-activity relationships, as well as the difficulty to produce kinetochore subunits in large quantities for use in in vitro screens has long hampered the conversion of structural kinetochore components into anticancer drug targets. Arguably the most intensely studied kinetochore subunit, both from a functional and structural point of view, is the outer kinetochore Ndc80 complex, which recruits the SAC and attaches the kinetochore structure to the MTs of the mitotic spindle. As the Ndc80 complex can be produced recombinantly in high quantity and because the recombinant complex is fully active as shown following injection in cells we focused on this complex to screen for inhibitors of kinetochoreMT binding. Such inhibitors would leave sister chromatids detached from the spindle, leading to a robust SAC mediated arrest of the cells in mitosis. As mitotically arrested cells frequently undergo apoptotic death these drug would be potent eradicators of cancer cells characterized by uncurbed proliferation. In addition, we��d like to use these inhibitors to study how detached kinetochores prepare for kinetochore-spindle contact. Out of the 10,200 compounds that were screened, one molecule prevented binding of the Ndc80 complex to taxol-stabilized MTs by acting at the MT level. Indeed, the compound prevented MT binding not only of the Ndc80 complex but also of the MT plus-end tracking CLIP-170 protein, suggesting that it acted specifically towards the MTs. We confirmed this hypothesis and showed that the compound localized to the colchicine site 
at the ab-tubulin interface. We believe that a conformational change in the MT polymers caused by binding of compound B to the colchicine pocket in the ab-tubulin dimer may have prevented the association of the proteins with the MT surface. Importantly, colchicine-site agent nocodazole did not prevent the Ndc80 complex from binding to taxol-stabilized MTs, further arguing that compound B affects MT integrity in a unique manner. Unfortunately, our study of the interaction between compound C and the Ndc80 complex has been complicated by the inability of the compound to enter cells. However, injecting the compound into HeLa cells significantly reduced the ability of the cells to align.