The overall goal of this research is to determine the sites of interaction between tubulin and microtubule-stabilizing anti-mitotic agents. Taxol is the best known of the microtubule-stabilizing compounds but others such as the epothilones show great potential as anti-tumor agents. Knowledge of the binding site shared by taxol and other structurally diverse anti-mitotic compounds with similar taxol-like activity wilt be a great aid in the rational design of anti-tumor agents that bind to the taxol binding region of tubulin. Our approach is to use mutagenesis of yeast tubulin. Although tubulin from the budding yeast Saccharomyces cerevisiae does not bind taxol, we have been able to create strong taxol binding to yeast tubulin by mutating just five of the 124 amino acids that are different between yeast and mammalian beta-tubulin. We now plan a more comprehensive mutational analysis of the taxol-binding region to provide a thorough understanding of the interactions required for taxol, the epothilones, and other compounds to bind to this region of beta-tubulin. We plan to determine which of the five mutations we made are most responsible for taxol binding, and what other amino acids that have also been implicated in taxol binding, actually make a contribution. We will determine whether the mutations we create affect taxol binding, microtubule stability, or both. Our studies will determine the differences in binding interactions with tubulin amongst the microtubule-stabilizing antimitotic agents taxol, the epothilones, the eleutherobins, and discodermolide. By using taxol photoaffinity analogues we will determine what part of the taxol molecule is likely to make contact with the mutated regions of beta-tubulin. Finally, we will study the taxol binding properties of tubulin from the fission yeast Schizosaccharomyces pombe in which the taxol binding region appears to more closely resemble that of mammalian tubulin.
