title: Small Molecule Approach to Study the Function of Mitotic Kinesins.
abstract: Mitotic motor proteins of the kinesin superfamily are critical for the faithful segregation of chromosomes and the formation of the two daughter cells during meiotic and mitotic M-phase. Of the 45 human kinesins, roughly a dozen are involved in the assembly of the bipolar spindle, alignment of chromosomes at the spindle equator, chromosome segregation, and cytokinesis. The functions of kinesins in these processes are highly diverse and include the transport of cargo molecules, sliding and bundling of microtubules, and regulation of microtubule dynamics. In light of this multitude of diverse functions and the complex functional interplay of different kinesins during M-phase, it is not surprising that one of the greatest challenges in cell biology is the functional dissection of individual motor proteins. Reversible and fast acting small molecules are powerful tools to accomplish this challenge. However, the validity of conclusions drawn from small molecule studies strictly depends on compound specificity. In this chapter, we present methods for the identification of small molecule inhibitors of a motor protein of interest. In particular, we focus on a protein-based large throughput screen to identify inhibitors of the ATPase activity of kinesins. Furthermore, we provide protocols and guidelines for secondary screens to validate hits and select for specific inhibitors.
authors.affiliation: Department of Biology, Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstr. 10, Konstanz, 78457, Germany.
authors.forename: Naowras
authors.initials: N
authors.lastname: Al-Obaidi
authors.affiliation: Department of Biology, Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstr. 10, Konstanz, 78457, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Kastl
authors.affiliation: Department of Biology, Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstr. 10, Konstanz, 78457, Germany. thomas.u.mayer@uni-konstanz.de.
authors.forename: Thomas U
authors.initials: TU
authors.lastname: Mayer
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D057205:Automation, Laboratory; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods; D057166:High-Throughput Screening Assays; D006801:Humans; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis / Q000235:genetics; D005810:Multigene Family; D012394:Rosaniline Dyes; D013552:Swine
chemical_list: D012394:Rosaniline Dyes; C005095:malachite green; D000251:Adenosine Triphosphatases; D016547:Kinesins
keywords: Enzyme-coupled assay; Kinesins; Large throughput screening; Malachite green assay; Microtubule; Mitotic motor proteins; Protein-based screen; Small molecule inhibitors