The
antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural
fungal diseases. In this paper, we investigated the interaction of
benomyl with mammalian brain
tubulin and microtubules. Using the hydrophobic
fluorescent probe 1-anilinonaphthalene-8-sulfonic acid,
benomyl was found to bind to brain
tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further,
benomyl bound to at a novel site, distinct from the well-characterized
colchicine and
vinblastine binding sites.
Benomyl altered the far-UV circular dichroism spectrum of
tubulin and reduced the accessibility of its
cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic
acid, indicating that
benomyl binding to
tubulin induces a conformational change in the
tubulin.
Benomyl inhibited the polymerization of brain
tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of
benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of
benomyl on HeLa cell proliferation and mitosis.
Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of
benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in
cancer chemotherapy.