Abstract
BackgroundProtein assemblies named kinetochores bind sister chromatids to the mitotic spindle and orchestrate sister chromatid segregation. Interference with kinetochore activity triggers a spindle checkpoint mediated arrest in mitosis, which frequently ends in cell death. We set out to identify small compounds that inhibit kinetochore-microtubule binding for use in kinetochore-spindle interaction studies and to develop them into novel anticancer drugs.Methodology/Principal FindingsA fluorescence microscopy-based in vitro assay was developed to screen compound libraries for molecules that prevented the binding of a recombinant human Ndc80 kinetochore complex to taxol-stabilized microtubules. An active compound was identified that acted at the microtubule level. More specifically, by localizing to the colchicine-binding site in αβ-tubulin the hit compound prevented the Ndc80 complex from binding to the microtubule surface. Next, structure-activity analyses distinguished active regions in the compound and led to the identification of highly potent analogs that killed cancer cells with an efficacy equaling that of established spindle drugs.Conclusions/SignificanceThe compound identified in our screen and its subsequently identified analogs represent new antitubulin chemotypes that can be synthetically developed into a novel class of antimitotic spindle drugs. In addition, they are stereochemically unique as their R- and S-isomers mimic binding of colchicine and podophyllotoxin, respectively, two antitubulin drugs that interact differently with the tubulin interface. Model-driven manipulation of our compounds promises to advance insight into how antitubulin drugs act upon tubulin. These advances in turn may lead to tailor-made colchicine site agents which would be valuable new assets to fight a variety of tumors, including those that have become resistant to the (antispindle) drugs used today.
Highlights
Many anticancer drugs used in the clinic inhibit cell division as tumors are characterized by uncontrolled proliferation [1]
The complexity of kinetochores, the lack of insight into 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
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 [12] we focused on this complex to screen for inhibitors of kinetochoreMT binding
Summary
Many anticancer drugs used in the clinic inhibit cell division as tumors are characterized by uncontrolled proliferation [1]. In S phase, maternal chromosomes replicate and form sister chromatid pairs. During the subsequent M phase, protein assemblies called kinetochores form on the centromere of each chromatid and attach the sister chromatids in a bipolar manner to the microtubules (MTs) of the mitotic spindle. At the metaphase-anaphase transition, the sister chromatids are first separated and segregated into the daughter cells. During the final cell cycle stage named cytokinesis, the daughters divide, each containing an identical set of chromosomes [2]. Protein assemblies named kinetochores bind sister chromatids to the mitotic spindle and orchestrate sister chromatid segregation. We set out to identify small compounds that inhibit kinetochore-microtubule binding for use in kinetochore-spindle interaction studies and to develop them into novel anticancer drugs
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