Grayanotoxin III Shows Microtubule-Targeted Activity

Grayanotoxins are diterpenes responsible for plant toxicity [1]. They occur in species belonging to the Ericaceae family. [2]. Grayanotoxins share structural similarities with paclitaxel a known antineoplastic drug which stabilizes microtubules by tubulin polymerization [3]. Therefore, we studied whether grayanotoxin interacts with microtubules. We used immunofluorescence to visualize alpha- and beta-tubulin in MCF7 breast cancer cells, after exposure to grayanotoxin III. Paclitaxel and colcemid were used as control drugs. While paclitaxel induced the formation of tubulin circumferential bundles and colcemid disassembled the tubulin polymer, grayanotoxin III showed a disorganized microtubule structure, with curled filaments forming a denser reticular network towards the nucleus. Because of the different effects on microtubules, we hypothesized that the binding domain of grayanotoxin III is different from the one of paclitaxel and colcemid. We used the crystal structure of alpha- and beta-tubulin for molecular „blind docking“ with grayanotoxin III and paclitaxel. We found that grayanotoxin does not bind to the binding site of paclitaxel on beta-tubulin, but on alpha-tubulin. Subsequently, we took the binding sites of grayanotoxins and paclitaxel for „defined docking“ studies. Defined docking provides detailed information on docking properties. Using this approach, we validated that grayanotoxin III binds with high affinity to its own pharmacophore on alpha-tubulin, but not to the paclitaxel pharmacophore on beta-tubulin, and vice versa paclitaxel does not bind to the grayanotoxin binding site. In conclusion, the effect of grayanotoxin III on microtubule organization in immunofluorescence correlated with the molecular docking data indicating the alpha-tubulin as a binding site.

References: [1] Kakisawa, H., et al. (1965) Tetrahedron 21: 3091. [2] Comroly, J.D., Hill, R.A., (1991) Dictionary of Terpenoids, Vol. 2, Chapman & Hall, Cambridge. [3] Alberts, B., Johnson, A., (2002) Molecular Biology of the Cell. Garland Science. New York.