A simple and robust cell-based assay for the discovery of novel cytokinesis inhibitors

Laszlo Radnai^1,2, Rebecca F. Stremel^1,2, Thomas Vaissiere², Li Lin¹, Michael Cameron¹, William H. Martin³, Gavin Rumbaugh², Theodore M. Kamenecka¹, Patrick R. Griffin¹, Courtney A. Miller^1,2

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¹ Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL 33458, USA

² Department of Neuroscience, The Scripps Research Institute, Jupiter, FL 33458, USA

³ WHM Consulting, LLC, 111 Sterling City Rd., Lyme, CT 06371, USA

JBM 2020 , 7(3), 1; https://doi.org/10.14440/jbm.2020.335

Published: 17 September 2020

© 2020 by the author. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )

Abstract

Cytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells. Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we present a detailed protocol for a cell-based cytokinesis assay that can be used for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 h. Living cells, nuclei and nuclei of dead cells are identified by a single staining step using three fluorescent dyes, followed by rapid live cell imaging. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio increases over time, as the ratio of multinucleated cells increases in the population. The ratio of dead nuclei to total nuclei provides a simultaneous measure of cytotoxicity. A screening window coefficient (Z`) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC50 values can be reliably determined in a single 96-well plate by using only six different compound concentrations, enabling the testing of 4 compounds per plate. An excellent test-retest reliability (R2 = 0.998) was found for EC50 values covering a ~1500-fold range of potencies. Established small molecule inhibitors of cytokinesis operating via direct action on actin dynamics or nonmuscle myosin II are used to demonstrate the robustness, simplicity and flexibility of the assay.

Keywords

drug discovery

actin cytoskeleton

screening

multinucleated

cytotoxicity

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