Fig. 4: Compound 89 was identified as a potent tubulin polymerization inhibitor binding to the colchicine site.

A HeLa cells were lysed using liquid nitrogen and three repeated cycles of freeze-thaw, and the cell lysate was treated with 89 (10 μM) or DMSO for 30 min at r.t. The cell suspension was heated for 3 min to 40, 44, 48, 52, 56 and 60 °C, cooled at 25 °C for 3 min, and then centrifuged at 20,000 × g for 30 min. Finally, the supernatant was collected for Western blot analysis. B In vitro tubulin polymerization assay was performed. β-tubulin was exposed to DMSO, colchicine (10 µM), paclitaxel (10 µM) or the indicated concentrations of 89. GTP was added to initiate the reaction. The tubulin polymerization rate was monitored for 60 min at 37 °C and the absorbance at 340 nm was measured. C Six-well plates were seeded with HeLa or HCT116 cells (2 × 10⁵) for 24 h. The tumor cells were incubated with 89, colchicine or DMSO for 2 h and afterward treated with EBI (100 μM) for 1.5 h. Cells were finally harvested and lysed, and the cell extracts were used for Western blotting analysis. D Cut-away view of the ligand-binding pocket at the colchicine site in the docking complex of 89 with tubulin. E Diagrammatic illustration of the interactions between tubulin protein and 89.