In vitro tubulin polymerization assay

NOV202 (1.0–10 µM) was tested in a tubulin polymerization assay kit purchased from Cytoskeleton Inc. (Denver, CO, USA). Polymerization is followed by fluorescence enhancement due to the incorporation of a fluorescent reporter into microtubules as polymerization occurs. Vincristine and paclitaxel at 3.0 µM were used as positive controls for tubulin polymerization inhibition and stabilization, respectively. All compounds were dissolved in DMSO and further diluted with sterile water to obtain a maximum DMSO concentration of 0.1%, which was used as solvent control. NOV202 and control compounds were incubated with bovine tubulin protein in a cell-free environment and the fluorescence was then measured continuously using Fluostar Optima (BMG Labtech GmbH, Ortenberg, Germany) at 360/450 nm for 60 min.