Screening for inhibitors of microtubule-associated motor proteins.
The mitotic spindle is an important target for cancer chemotherapy. The main protein target for drugs in clinical use is tubulin, the building block of microtubules. In recent years, other proteins of the mitotic spindle have been identified as potential targets for the development of more specific drugs with the hope that these will have fewer side effects than known antimitotics (taxanes, vinca alkaloids). The human genome contains more than 40 members of the kinesin superfamily, with at least 12 of these involved in mitosis and cytokinesis. HsEg5 (also called KSP, kinesin spindle protein), a member of the kinesin-5 family, involved in the formation of the bipolar spindle, is a very promising target for cancer chemotherapy with specific inhibitors in Phase I and II clinical trails. Several successful approaches exist today to screen Eg5 for inhibitors, including phenotype-based assays and simple in vitro assays that explore the intrinsic enzymatic ATPase activity of Eg5. Here, we describe a robust and straightforward in vitro method to rapidly screen Eg5 for inhibitors. The assay can easily be adapted to other mitotic kinesins that may be identified in the future as potential drug targets, or simply to obtain specific kinesin inhibitors for use in "chemical genetics" to study the function of this important class of proteins.
Kozielski
Frank
F
Institut de Biologie Structurale, CEA-CNRS-UJF, Grenoble, France.
DeBonis
Salvatore
S
Skoufias
Dimitrios A
DA
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors*; D008856:Microscopy, Fluorescence / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors*; D008938:Mitosis / Q000187:drug effects; D010641:Phenotype; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
D004791:Enzyme Inhibitors; C470243:KIF11 protein, human; D008869:Microtubule-Associated Proteins; D054852:Small Molecule Libraries; D000251:Adenosine Triphosphatases; D016547:Kinesins