Benomyl, a
tubulin-targeted
antimitotic antifungal agent, belongs to the
benzimidazole group of compounds, which are known to inhibit the binding of
colchicine to
tubulin. Therefore,
benomyl was thought to bind at or near the
colchicine-binding site on
tubulin. However, recent mutational studies in yeast and fluorescence studies involving competitive binding of
benomyl and
colchicine on goat brain
tubulin suggested that
benomyl may bind to
tubulin at a site distinct from the
colchicine-binding site. We set out to examine whether
colchicine and
benomyl bind to
tubulin at distinct sites using a human
cervical cancer (HeLa) cell line with the thinking that these agents should exert either additive or synergistic activity on cell proliferation if their binding sites on
tubulin are different. We found that
benomyl and
colchicine synergistically inhibited the proliferation of HeLa cells and blocked their cell cycle progression at mitosis. The synergistic activity of
benomyl and
colchicine was also apparent from their strong depolymerizing effects on both the spindle and interphase microtubules when used in combinations, providing further evidence that these agents bind to
tubulin at different sites. Using NMR spectroscopy, we finally demonstrated that
benomyl and
colchicine bind to
tubulin at different sites and that the binding of
colchicine seems to positively influence the binding of
benomyl to
tubulin and vice versa. Further, an analysis of the saturation transfer difference NMR data yielded an interesting insight into the
colchicine-
tubulin interaction. The data presented in this study provided a mechanistic understanding of the synergistic effects of
benomyl and
colchicine on HeLa cell proliferation.