The goal of this proposed research is to determine whether the drug colchicine undergoes a conformational transformation upon binding to the protein tubulin and, if so, to define the precise structural nature of the conformational change induced in the drug. The colchicine-tubulin interaction has been intensively investigated since its discovered by Taylor and co-workers in 1967, and it has been shown to be the source of the potent antimitotic and antiinflammatory effects attributed to the drug. Several central features of this interaction remain unresolved. We recently suggested that colchicine may possess fluxional conformational character and that a less thermodynamically preferred conformer, not the ground state conformer, may undergo binding to tubulin. This suggestion enables for the first time rationalization of several diverse aspects of the important tubulin-colchicine interaction. I propose here an integrated set of investigations which intend to confirm and extend this hypothesis. These investigations include the development of a direct synthetic entry into the colchicine alkaloids capable of generating colchicinoid analogs with rationally defined conformations, the synthesis of labeled colchicine derivatives capable of directly monitoring the colchicine-tubulin interaction via NMR spectroscopy, and the rationalization of the puzzling antimitotic behaviors of two colchicine derivatives--isocolchicine and desacetamidocolchicine. All new compounds will be evalulated for their ability to inhibit tubulin polymerization in vitro. These results will directly enlarge our understanding of the pharmacological applications of colchicine and will indirectly contribute to understanding of the fundamental mechanism of polymerization of microtubules. The postulation of a conformational transformation of colchicine prior to tubulin binding may have powerful and far-reaching consequences for the employment of colchicinoids as antitumor agents. It may ultimately allow design and synthesis of analogs with defined conformations which would possess enahanced selectivity for interaction with tubulin (or even a select tubulin dimer isozyme) in preference to other biological receptors.
| Hastie, S B; Macdonald, T L (1990) Binding of colchiceine to tubulin. Mechanisms of ligand association with tubulin. Biochem Pharmacol 39:1271-6 |
| Hastie, S B; Williams Jr, R C; Puett, D et al. (1989) The binding of isocolchicine to tubulin. Mechanisms of ligand association with tubulin. J Biol Chem 264:6682-8 |
| Humphreys, W G; Macdonald, T L (1988) The effects on tubulin polymerization and associated guanosine triphosphate hydrolysis of aluminum ion, fluoride and fluoroaluminate species. Biochem Biophys Res Commun 151:1025-32 |
