Abstract
Mitotic kinesins play essential roles during mitotic spindle assembly and in ensuring proper chromosome segregation. Chemical inhibitors of mitotic kinesins are therefore valuable tools to study kinesin function in vitro and in cells. Because cancer is a disease of unregulated cell division, inhibitors also represent potential chemotherapeutic agents. Here, we present assays that can be used to evaluate the potency and specificity of mitotic kinesin inhibitors identified from high-throughput screening. By evaluating their effects in a variety of in vitro, fixed-cell, and live cell assays, screening hits can be prioritized and optimized to produce effective, on-target inhibitors.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Verhey KJ, Hammond JW (2009) Traffic control: regulation of kinesin motors. Nat Rev Mol Cell Biol 10(11):765–777
Drummond DR (2011) Regulation of microtubule dynamics by kinesins. Semin Cell Dev Biol 22(9):927–934
Akhmanova A, Steinmetz MO (2015) Control of microtubule organization and dynamics: two ends in the limelight. Nat Rev Mol Cell Biol 16(12):711–716
Hirokawa N, Pfister KK, Yorifuji H, Wagner MC, Brady ST, Bloom GS (1989) Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration. Cell 56(5):867–878
Sawin KE, Leguellect K, Philippet M, Mitchison TJ (1992) Mitotic spindle organization by a plus-end-directed microtubule motor. Nature 359(6395):540–543
Mayer TU, Kapoor TM, Haggarty SJ, King RW, Schreiber SL, Mitchison TJ (1999) Smart molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286(5441):971–974
Blangy A, Lane HA, d’Hérin P, Harper M, Kress M, Niggt EA (1995) Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell 83(7):1159–1169
Ferenz NP, Gable A, Wadsworth P (2010) Mitotic functions of kinesin-5. Semin Cell Dev Biol 21(3):255–259
Sawin KE, Mitchison TJ (1995) Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle. Proc Natl Acad Sci 92(10):4289–4293
Kapoor TM, Mayer TU, Coughlin ML, Mitchison TJ (2000) Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5. J Cell Biol 150(5):975–988
Jordan MA, Wilson L (2004) Microtubules as a target for anticancer drugs. Nat Rev Cancer 4(4):253–265
Tuxen MK, Hansen SW (1994) Neurotoxicity secondary to antineoplastic drugs. Cancer Treat Rev 20(2):191–214
Rowinsky EK (1997) The development and clinical utility of the taxane class of antimicrotubule chemotherapy agents. Annu Rev Med 48(1):353–374
Lipton RB, Apfel SC, Dutcher JP, Rosenberg R, Kaplan J, Berger A, Einzig AI, Wiernik P, Schaumburg HH (1989) Taxol produces a predominantly sensory neuropathy. Neurology 39(3):368–373
Dumas ME, Chen G-Y, Kendrick ND, Xu G, Larsen SD, Jana S, Waterson AG, Bauer JA, Hancock W, Sulikowski GA, Ohi R (2019) Dual inhibition of Kif15 by oxindole and quinazolinedione chemical probes. Bioorg Med Chem Lett 29(2):148–154
Zhang JH, Chung TDY, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4(2):67–73
Sturgill EG, Das DK, Takizawa Y, Shin Y, Collier SE, Ohi MD, Hwang W, Lang MJ, Ohi R (2014) Report kinesin-12 Kif15 targets kinetochore fibers through an intrinsic two-step mechanism. Curr Biol 24:2307–2313
Tanenbaum ME, Macůrek L, Janssen A, Geers EF, Alvarez-Fernández M, Medema RH (2009) Kif15 cooperates with Eg5 to promote bipolar spindle assembly. Curr Biol 19(20):1703–1711
Debonis S, Skoufias DA, Lebeau L, Lopez R, Robin G, Margolis RL, Wade RH, Kozielski F (2004) In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities. Mol Cancer Ther 3(9):1079–1090
Sturgill EG, Norris SR, Guo Y, Ohi R (2016) Kinesin-5 inhibitor resistance is driven by kinesin-12. J Cell Biol 213(2):213–227
Hobro AJ, Smith NI (2017) An evaluation of fixation methods: spatial and compositional cellular changes observed by Raman imaging. Vib Spectrosc 91:31–45
Acknowledgements
We would like to thank the Ohi lab that made this work possible, as well as Emma Sturgill, Megan Dumas, and George Xu for doing extensive optimization of these assays. We would also like to acknowledge NIH grant R01 GM086610, pilot project funding from Michigan Drug Discovery, and start-up funds from the University of Michigan.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Solon, A., Ohi, R. (2022). Chemical Biology of Mitotic Spindle Assembly Motors. In: Hinchcliffe, E.H. (eds) Mitosis. Methods in Molecular Biology, vol 2415. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1904-9_11
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1904-9_11
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1903-2
Online ISBN: 978-1-0716-1904-9
eBook Packages: Springer Protocols