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

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. EC 50 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 ( R 2 = 0.998) was found for EC 50 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.

Discovery of Selective Inhibitors for In Vitro and In Vivo Interrogation of Skeletal Myosin II

“…4 Therefore, we utilized cytokinesis as a cell-based assay primarily to determine potential cytotoxicity and cell membrane penetrance of our most selective skeletostatins (MT-114, -115, -134). 34 The results also informed NMII activity. COS7 cells, which primarily express the NMIIB isoform, 35 were treated with a concentration series of skeletostatins or blebb on 96-well plates.…”

“…Cytotoxicity was calculated as the ratio of dead nuclei to total nuclei. 34 None of the skeletostatins or blebb showed any sign of cytotoxicity, even when applied at saturation levels (slightly above solubility; Figure 2). The nuclei-to-cell ratio, a measure of polyploidy that reflects cytokinesis defects, 34 was then calculated to determine compound potencies (EC 50 ).…”

“…25 Previously, we have also observed the strong fluorescence of blebb crystals in live-cell imaging experiments using λ ex = 405 nm and λ em = 455 nm. 34 Interestingly, these crystals showed much weaker fluorescence when excited at 488 nm (λ em = 525 nm). Nevertheless, the 3D fluorescence spectra presented here clearly show that blebb's fluorescence will not interfere with fluorescence-based imaging applications at these wavelengths when the compound is in solution.…”

“…Blebb interferes with cytokinesis through NMII inhibition, resulting in the accumulation of multinucleated cells in cell cultures . Therefore, we utilized cytokinesis as a cell-based assay primarily to determine potential cytotoxicity and cell membrane penetrance of our most selective skeletostatins (MT-114, -115, -134) . The results also informed NMII activity.…”

“…The cytokinesis assay was performed as previously described. 34 Further experimental details are provided in the Supporting Information.…”