To verify association modes of the ligands identified by the C-MST assay as the colchicine site binders, we used the coumarin-30 fluorescence enhancement assay described in Fig. . This analysis confirmed that all predicted colchicine site binders indeed displaced coumarin-30 from its binding site, while the non-interacting compounds, such as compound 4e, displayed no competition with coumarin-30 for binding to tubulin (Fig. ).